Biology Topics

ANGIOSPERMS

2020-09-22T18:24:00.001+05:30

ANGIOSPERMS

1. The term "angiosperms" derives from the two Greek words : angeion meaning "vessels" and sperma meaning "seeds".

2. Angiosperms (flowering plants) are the largest group of plants on earth.

3. Approximately 270,000 known species alive today.

4. Angiosperms include all plants that have flowers and account for approximately 80% of all known living plants.

5. Angiosperms means " covered seeds" have flowers, have fruits with seeds.

Live everywhere- dominants plants in the world.

6. 260,000 species (88% of plant kingdom).

Angiosperms are most successful and advanced plant on earth.

Evolutionary Development of Angiosperms:-

1. Angiosperms evolved during the late Cretaceous period, about 125-100 million years ago.

2. This leaf imprint shows a ficus speciosissiva an Angiosperms that flourished during the Cretaceous period.

3. A large number of pollinating insects also appeared during this same time.

4. Advancements over gymnosperm: angiosperms have flowers- many use pollinators.

5. Fruits and seeds adapted for dispersal.

6. Double fertilization of endosperm in the seed.

Characteristics of Angiosperms

1. Angiosperms have developed flowers and fruits.

2. Flowers serves as the reproductive organs for the plants.

3. Flowers have a wide array of colors, shapes and smells all of the which are for the purpose of attracting pollinators.

1. Roots, stems and leaves.

2. Xylem and phloem.

4. Xylem- conveys water and dissolved minerals from the roots to the shoots.

5. Pholem- transports food made in the leaves to the roots and developing leaves and fruits.

6. They are Heterosporous.

Terminologies:-

a. Androecium- bearing one or more stamens.

b. Gynoecium- bearing one or more carpels.

c. Bisexual (perfect) flower- containing both stamens and carpels.

d. Unisexual (imperfect) flower- having only stamen or carpels.

Distribution of Angiosperms:-

1. One reason for this dominance is the relatively high photosynthetic capacity of their leaves.

2. They occupy every habitat on earth except extreme environment such as the highest mountain tops, the regions immediately surrounding the poles and deepest oceans.

3. They lives as Epiphytes, (i.e. living on other plants).

4. They occur abundantly in-

Shallows of rivers and fresh water lakes.

To a lesser extent, in Salt lakes and in the sea.

Angiosperms life cycle:-

Flower has male and female sex organs.

Flower structure and it's part:-

1. Male sex organs: stamens, composed of anther- organ that produces pollen (male gametophyte).

2. Female sex organs- the carpel.

3. Ovary is the enlarged basal portion of carpel that contains the ovules (females gametophyte).

4. The stigma is the receptive portionof the carpel for pollen grains to adhere.

5. Non- reproductive parts; sepals (green) are the outermost whorl of leaf like bracts.

6. Petals (usually colored) are the inner whorl of leaf-like bracts.

7. Both can have various shaped and colors.

SEEDS:-

Endosperm is stored food tissues for the embryo to grow. Mature ovule becomes the seed coat and fruits.

Monocot vs Dicot

1. Angiosperms are divided into monocots and dicots.

2. As the zygote grows into the embryo, the first leaves of the young sporophyte develop and are called as cotyledons (seed leaves).

3. Monocots have one cotyledons (corns, lily etc).

4. Dicots have two cotyledons (beans, oak etc).

Comparing Monocots vs Dicots

Economic Importance of Angiosperms

The flowering plants have a number of uses as food, specifically as grains, sugars, vegetables, fruits, oils, nuts and spices.

In addition, plants and their products serve a number of other need such as dyes, fibres, timber, fuel, medicines, and ornaments.

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BASIC TERMS OF GENETIC

2020-09-16T11:49:00.000+05:30

GENETIC

"Genetic is collective study of heredity and variations".

"Genetics is a field of biology that studies how traits are passed from parents to their offspring".

Genetic term is given by William Bateson in the year 1905.

Heredity- Transformation of genetic character from one generation to another.

Variation- Individual of same species have same differences.

There are two types of Variation

1. Somatic variation- they are non inheritable.

2. Germinal/blastogenic variation- they transfer from one generation to another. i.e. inheritable

a. Continuous (occurs due to crossing over)

b. Discontinuous (occurs due to mutations)

G. J. Mendel is father of genetics. He gave basic principles of genetics in the year 1866.

W. Bateson is father of morden genetics.

Morgan is father of experimental genetics. He did experiments on Drosophila and proposed concept like linkage, sex linkage, crosiing over, criss-cross inheritance, linkage map of Drosophila.

A. Garrod is father of human genetics and Biochemical genetics. He discovered first human metabolic Disorder, i.e. Alkaptonuria ( Balck urine disease). He gave concept of one-mutant gene one-metabolic block.

TERMS OF GENETICS:-

Gene, allele, locus, homologous chromosomes, Dominant, Recessive, homozygous, heterozygous, gamete formation, phenotype, genotype.

Cell

GENE- A gene is the basic physical and functional unit of heredity that controls a particular trait of an organism.

HOMOLOGOUS CHROMOSOME- At the same locus , same character of gene is present are called homologous chromosomes.

Example- In human 46 chromosomes are present in 23 pairs.

maternal and paternal having same character of gene, at the same location; means both have same gene for eye colour.

LOCUS- Particular location of gene on chromosomes.

ALLELE- An allele is an alternate form of same gene, located on same locus on the homologous chromosomes.

Allele are the gene which is coding for the same character but not for same trait. (Alternate form of gene)

Character- eye colour.

Trait- Blue, black, brown etc.

Alleles were first defined by Gregor Mendel in the law of segregation.

DOMINANT- A genetic trait is considered dominant if it is expressed in a person who has only one copy of that gene. A dominant trait is opposed to a Recessive trait which is expressed only when two copies of the gene are present.

RECESSIVE- A recessive gene is a gene whose effects are masked in the presence of a Dominant gene. Every organism that has DNA packed into chromosomes has two alleles, or forms of a gene , for each gene ; one inherited from their mother and one inherited from their father. A Recessive gene is only expressed when an organism has two recessive alleles for that gene.

This is also known as Being homozygous recessive. If an organism has one dominant and one recessive allele, it will show the dominant trait.

HOMOZYGOUS- Homozygous is a word that refers to a particular gene that has identical alleles on both homologous chromosome. It is referred to by two capital letters (XX) for a dominant trait, and two small letters (xx) for a Recessive trait.

HETEROZYGOUS- Gene come in pairs, called alleles and each pair is located in a specific position (locus) on a chromosome. If the two alleles at a locus are identical to eachother, they are Homozygous; if they are different from one another, they are Heterozygous.

GENOTYPE- Genetic makeup.

An organism genotype is the set of gene in its DNA responsible for the particular trait.

PHENOTYPE- physical features/apperance.

An organism phenotype is the physical expression of those gene.

All the observable characteristics of an organism that results from the interaction of its genotype with the environment. Example- colour, shape, size.

GAMETE FORMATION

Gametes are formed through a process of cell division called meiosis. This two-step division process produces four haploid daughter cells.

Haploid cells contain only one set of chromosomes. When the haploid male and female gametes unite in a process called fertilization, they form zygote. The zygote is diploid and contain two sets of chromosomes.

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GENE THERAPY

2020-09-14T21:25:00.000+05:30

GENE THERAPY

Gene therapy is when DNA is introduced into a patient to treat a genetic disease. The new DNA usually contains a functioning gene to correct the effects of a disease-causing mutation.In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including:

Replacing a mutated gene that causes disease with a healthy copy of the gene.
Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective. Gene therapy is currently being tested only for diseases that have no other cures.

TYPES OF GENE THERAPY

There are two different types of gene therapy depending on which types of cells are treated:

Somatic gene therapy: transfer of a section of DNA to any cell of the body that doesn’t produce sperm or eggs. Effects of gene therapy will not be passed onto the patient’s children.
Germline gene therapy: transfer of a section of DNA to cells that produce eggs or sperm. Effects of gene therapy will be passed onto the patient’s children and subsequent generations.

GENE THERAPY WORK

Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.

A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein.

Researchers must overcome many technical challenges before gene therapy will be a practical approach to treating disease. For example, scientists must find better ways to deliver genes and target them to particular cells. They must also ensure that new genes are precisely controlled by the body.

A new gene is inserted directly into a cell. A carrier called a vector is genetically engineered to deliver the gene. An adenovirus introduces the DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

IMPORTANCE OF GENE THERAPY

Genes that are flawed and do not work properly can cause disease. Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:

A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.
An abnormal gene could be swapped for a normal gene through homologous recombination.
The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.
The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

GENE THERAPY FOR SPECIFIC DISORDERS

Gene therapy holds promise for treating a wide range of diseases, such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Researchers are still studying how and when to use gene therapy. Currently, in the United States, gene therapy is available only as part of a clinical trial.

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GENE CLONING AND ITS SIGNIFICANCE

2020-09-13T22:00:00.001+05:30

DNA cloning

“DNA cloning is a molecular biology technique which is used for the creation of exact copies or clones of a particular gene or DNA.”
The production of exact copies of a particular gene or DNA sequence using genetic engineering techniques is called gene cloning.

The term “gene cloning,” “DNA cloning,” “molecular cloning,” and “recombinant DNA technology” all refer to same technique.
When DNA is extracted from an organism, all its genes are obtained. In gene (DNA) cloning a particular gene is copied forming “clones”.
Cloning is one method used for isolation and amplification of gene of interest.

DNA cloning can be achieved by two different methods:

Cell based DNA cloning
Cell-free DNA cloning (PCR)

Requirements of Gene Cloning:-

DNA fragment containing the desired genes to be cloned.
Restriction enzymes and ligase enzymes.
Vectors – to carry, maintain and replicate cloned gene in host cell.
Host cell– in which recombinant DNA can replicate.
Steps of gene Cloning:
The basic 7 steps involved in gene cloning are:

Isolation of DNA [gene of interest] fragments to be cloned.
Insertion of isolated DNA into a suitable vector to form recombinant DNA.
Introduction of recombinant DNA into a suitable organism known as host.
Selection of transformed host cells and identification of the clone containing the gene of interest.
Multiplication/Expression of the introduced Gene in the host.
Isolation of multiple gene copies/Protein expressed by the gene.
Purification of the isolated gene copy/protein.

A. Isolation of the DNA fragment or gene

The target DNA or gene to be cloned must be first isolated. A gene of interest is a fragment of gene whose product (a protein, enzyme or a hormone) interests us. For example, gene encoding for the hormone insulin.
The desired gene may be isolated by using restriction endonuclease (RE) enzyme, which cut DNA at specific recognition nucleotide sequences known as restriction sites towards the inner region (hence endonuclease) producing blunt or sticky ends.
Sometimes, reverse transcriptase enzyme may also be used which synthesizes complementary DNA strand of the desired gene using its mRNA.

B. Selection of suitable cloning vector

The vector is a carrier molecule which can carry the gene of interest (GI) into a host, replicate there along with the GI making its multiple copies.
The cloning vectors are limited to the size of insert that they can carry. Depending on the size and the application of the insert the suitable vector is selected.
The different types of vectors available for cloning are plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian artificial chromosomes (MACs).
However, the most commonly used cloning vectors include plasmids and bacteriophages (phage λ) beside all the other available vectors.

C. Essential Characteristics of Cloning Vectors

All cloning vectors are carrier DNA molecules. These carrier molecules should have few common features in general such as:

It must be self-replicating inside host cell.
It must possess a unique restriction site for RE enzymes.
Introduction of donor DNA fragment must not interfere with replication property of the vector.
It must possess some marker gene such that it can be used for later identification of recombinant cell (usually an antibiotic resistance gene that is absent in the host cell).
They should be easily isolated from host cell.

D. Formation of Recombinant DNA

The plasmid vector is cut open by the same RE enzyme used for isolation of donor DNA fragment.
The mixture of donor DNA fragment and plasmid vector are mixed together.
In the presence of DNA ligase, base pairing of donor DNA fragment and plasmid vector occurs.
The resulting DNA molecule is a hybrid of two DNA molecules – the GI and the vector. In the terminology of genetics this intermixing of different DNA strands is called recombination.
Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is referred to as the recombinant DNA technology.

E. Transformation of recombinant vector into suitable host

The recombinant vector is transformed into suitable host cell mostly, a bacterial cell.
This is done either for one or both of the following reasons:
- To replicate the recombinant DNA molecule in order to get the multiple copies of the GI.
- To allow the expression of the GI such that it produces its needed protein product.

Some bacteria are naturally transformable; they take up the recombinant vector automatically.

For example: Bacillus, Haemophillus, Helicobacter pylori, which are naturally competent.

Some other bacteria, on the other hand require the incorporation by artificial methods such as Ca⁺⁺ ion treatment, electroporation, etc.

F. Isolation of Recombinant Cells

The transformation process generates a mixed population of transformed and non-trans- formed host cells.
The selection process involves filtering the transformed host cells only.
For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed.
For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant gene from the plasmid, so that the recombinant cell become sensitive to Ampicillin.

G. Multiplication of Selected Host Cells

Once transformed host cells are separated by the screening process; becomes necessary to provide them optimum parameters to grow and multiply.
In this step the transformed host cells are introduced into fresh culture media .
At this stage the host cells divide and re-divide along with the replication of the recombinant DNA carried by them.
If the aim is obtaining numerous copies of GI, then simply replication of the host cell is allowed. But for obtaining the product of interest, favourable conditions must be provided such that the GI in the vector expresses the product of interest.

H. Isolation and Purification of the Product

The next step involves isolation of the multiplied GI attached with the vector or of the protein encoded by it.
This is followed by purification of the isolated gene copy/protein.

ITS SIGNIFICANCE

A particular gene can be isolated and its nucleotide sequence determined
Control sequences of DNA can be identified & analyzed
Protein/enzyme/RNA function can be investigated
Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.
DNA cloning can be used to make proteins such as insulin with biomedical techniques.
It is used to develop recombinant versions of the non-functional gene to understand the functioning of the normal gene. This is applied in gene therapies also.
It helps to analyse the effect of mutation on a particular gene.

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Albinism Disorder

2020-09-12T21:23:00.000+05:30

Principal of Inheritance and Variations

HUMAN GENETICS DISORDER

An inherited medical condition caused by a DNA abnormality.

Basically two types:-

1. Chromosomal abnormalities

2. Gene Related human disorder

a. Autosomal disorder

1. Dominant

2. Recessive

b. Sex (gene disorder)

ALBINISM

1. It is autosomal recessive genetic disorder.

2. A group of inherited disorders characterised by little or no melanin production.

3. This condition increases the risk of skin cancer.

4. Albinism Disorder also known as achromasia.

5. Tryosinase enzyme is absent.

6. Individual lack dark pigment melanin in skin, hair and Iris.

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Alkaptonuria Disorder

2020-09-12T21:03:00.001+05:30

Principal of Inheritance and variations

ALKAPTONURIA

It is an gene mutation in autosome recessive type. The metabolic defect is the deficiency of homogenticis acid oxidase enzyme. It's an autosomal recessive condition. It is compatible with fairly normal life.

First inborn metabolic disorder described by A. Garrod.

CAUSE- Defect in the enzyme homogentisate oxidase, that catalysed oxidation of homogentisate to maleylactoactate.

Homogentisate accumulates in blood and body tissues and is excreted in large amounts in urine.

CHARACTERISTICS FEATURE-

1. The urine of alkaptonuria patients become Dark after being exposed to air.

2. The alkapton imparts a characteristic black-brown color to urine.

3. Alkaptonuria is a harmless condition.

4. Later in life deposition of dark colored alkapton pigments in connective tissues and bones occur.

5. This results in black pigmentation of the sclera, ear, nose and cheeks and the clinical condition is known as Ochronosis.

6. Ochronosis leads to tissue damage and may develop joint pain, arthritis and backache.

DIAGNOSIS-

1. The urine sample of patients of alkaptonuria turns dark on standing in air.

2. The urine gives positive test with ferric chloride and silver nitrate due to reducing activity of homogentisate.

3. Benedict's test is strongly positive.

TREATMENT-

1. Since alkaptonuria is not considered life threatening, this condition is not treated.

2. Later in life, the Symptoms of arthritis may be treated but the condition itself is not.

3. No specific treatment is required.

4. But minimal protein intake with phenylalanine less than 500gm/day is recommended.

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ETHICAL ISSUES

2020-07-30T22:56:00.001+05:30

ETHICAL ISSUES

The manipulation of living organisms by the human race cannot go on any further, without regulation. Some ethical standards are required to evaluate the morality of all human activities that might help or harm living organisms.

Going beyond the morality of such issues, the biological significance of such things is also important.Genetic modification of organisms can have unpredictable result when such organisms are introduced into the ecosystem.

Therefore, the Indian Government has set up organisations such as GEAC (Genetic Engineering Approval Committee), which will make decisions regarding the validity of GM research and the safety of introducing GM-organisms for public services.

The modification/usage of living organisms for public services (as food and medicine sources, For example) has also created problems with patents granted for the same.

GM crops are already in cultivation in U.S.A. Europe and several other countries. In India, some insect resistant cotton varieties expressing cry genes have reached the farmers, fields. It has been argued that transgenic crops may be harmful to the environment. The two points. Firstly, the transgene may be transferred through pollen from these crops to their wild relatives secondly GM crops may pollute the environment.

BIO-PATENT

A patent is a right granted by a government to an inventor to prevent others from commercial use of his invention A patent is granted for -

1. An invention [including product].

2. An improvement in an earlier invention.

3. The process of generating products and

4. A concept or design.

There is growing public anger that certain

Companies are being granted patents for products and technologies that make use of the genetic material, plants and other biological resources that have long been identified, developed and used by farmers and indigenous people of specific region/country.

Rice is an important food grain, the presence of which goes back thousands of years in Asia's agricultural history. There are an estimated 200,000 varieties of tice in lndia alone. The diversity of rice in India is one of the richest in the world, Basmati Rice is distinct for its unique aroma atd flavour and 27 documented varieties of Basmati grown in India. There is reterence to Basmati in ancient texts,folklore and poetry, as it has been grown for centuries. In 1997, an American company got patent rights on Basmati rice through the US Patent and Trademark Office. This allowed abroad This the company to sell a 'new' variety of Basmati in, the US and abroad. The 'New' variety of Basmati had actually been derived from Indian farmers varieties. Indian Basmati was crossed with semi-dwarf varieties and claimed as an invention or a novelty. The patent extends to functional equivalents, implying that other people selling Basmati rice could be restricted by the patent.

Several attempts have also been made to patent uses, products and processes based on Indian traditional herbal e

medicines e.g. Turmeric neem. If we are not vigilant and we do not immediately counter these patent applications, other countries/individuals may encash on our rich legacy and we may not be able to do anything about it.

BIOPIRACY

Bio-piracy is the term used to refer to the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concemed without compensatory payment.

Most of the industrialised nations are rich financially but poor in biodiversity and traditional knowledge. In contrast the developing and the underdeveloped world is rich in biodiversity and traditional knowledge related to bio-resources. Taditional knowledge related to bio-resources can be exploited to develop modern application and can also be used to save time, effort and expenditure during their commercialisation.

There has been growing realisation of the injustice, inadequate compensation and benefit sharing between developed and developing countries. Therefore, some nations are developing laws to prevent such unauthorised exploitation of their bio-resources and traditional knowledge.

The Indian Parliament has recently cleared the second amendment the Indian Patents Bill, that takes issues into consideration, including patent terms emergency provision and research and development initiatives.

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BIOTECHNOLOGY AND ITS APPLICATION

2020-07-26T19:35:00.000+05:30

BIOTECHNOLOGY AND ITS APPLICATION

Biotechnology, as you would have learnt essentially deals with industrial scale production of biopharmaceuticals and biological using genetically modified microbes, fungi, plants and animals. The applications of biotechnology include therapeutics, diagnostics, genetically modified crops for agriculture, processed food, bioremediation, waste treatment and energy production.

(A) BIOTECHNOLOGICAL APPLICATION IN AGRICULTURE :

Let us take a look at the three options that can be thought for increasing food production

i. agro-chemical based agriculture

ii. organic agriculture and

iii. genetically engineered crop-based agriculture.

The Green Revolution succeeded in tripling the food supply but yet it was not enough to feed the growing human population. Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of batter management practices and use of agrochemicals (fertilisers and pesticides). However, for farmers in the developing world, agrochemicals are often too expensive and further increases in yield with existing varieties are not possible using conventional breeding is there any alternative path that our undestanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Use of genetically modified crops is a possible solution.

The Green Revolution succeeded in tripling the food supply but yet it was not enough to feed the growing human population. Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of better management practices and use of agrochemicals (fertilisers and pesticides). However, for farmers in the developing world, agrochemicals are often too expensive, and further increases in yield with existing varieties are not possible using conventional breeding. Is there any alternative path that our understanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Uses genetically modified crops is possible solution.

Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO).

GM has been used to create tailor made plants to supply alternative resources to industries, in the form of starches, fuels and pharmaceuticals.

Use of genetically modified (GM) plant:

1. To enhance nutritional quality of food

eg. Golden rice - Vitamin A enriched rice (In this rice gene of B-carotene is transfered).

2. Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).

3. Helped to reduce post harvest losses eg. Flavr Savr Tomato : Transgenic variety of Tomato - Flavr Savr due to the inhibition of polygalacturonase enzyme which degrades pectin. So that tomato variety remains fresh and retain flavour much longer. Flavr Savr Tomato develop by anti-sense technology.

4. Increased efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil).

5. To produce pharmaceutical product

eg. Production of Hirudin : Hirudin is a protein that prevents blood clotting. The gene incoding hirudin wes chemically synthesized and transfered into Brassica napus. Where hirudin accumulates in seeds. The hirudin is purified and used as medicine.

6. To produce herbicide resistant plant eg. First transgenic plant was tobacco. It contains resistant gene against weedicide (Glyphosate).

7. Pest-resistant crops reduced reliance on chemical pesticides.

i. Insect resistant plant

eg. Bt cotton : Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). B.thuringiensis forms protein crystals during a particular phase of their growth. These crystals contain a toxic insecticidal protein.

The Bt toxin protein exist as inactive protoxins but once an insect ingest the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilise the crystals. The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of the insect.

Bacillus thuringiensis, produces crystal [Cry] protein. This Cry protein is toxic to Larvae of certain insects. Each Cry protein is toxic to a different group of insects. The gene encoding cry protein is called "cry gene". This Cry protein isolated and transferred into several crops. A crop expressing a cry gene is usually resistant to the group of insects for which the concerned Cry protein is toxic. There are a number of them, for example, the proteins encoded by the genes crylAc and cryllAb control the cotton bollworms, that of crylAb controls corn borer.

However, gene symbol italics, e.g., cry. The first letter or the protein symbol, on the other hand is always capital and the symbol is always written in roman letters. e.g., Cry.

ii. Nematode resistant plant:

Several nematodes parasitise a wide variety of plants and animals including human beings. A nematode Meloidogyne incognitia infects the roots of tobacco plants and causes a great reduction in yield. A novel strategy was adopted to prevent this infestation which was based on the process of RNA Interference (RNAi). RNAi takes place in all eukaryotic organisms as a method of cellular defense.

This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing). The source of this complementary RNA could be from an infection by viruses having RNA genomes or mobile genetic elements (transposons) that replicate via an RNA intermediate.

Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant. The introduction of DNA was such that it produced both sense and antisense RNA in the host cells. These two RNA's being complementary to each other formed a double stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of the nematode. The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA. The transgenic plant therefore got itself protected from the parasite.

BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE

A. Genetically Engineered Insulin

It is a proteinaceous hormone having 51 Amino acids arranged in two polypeptides A and B having 21 and 30 Amino Acids respectively and joined by S-S disulphide

bridges.

Sir Edward sharpy-Shafer (1916) was the first to note that diabetes of some persons was because of failure of some islands of pancreas to produce a substance which he called insulin (Derived from the latin, insula, meanning island).

Banting and best (1921) were the first to isolate insulin from dog's pancreas and used it to cure diabetes in man. The first genetically engineered insulin obtained by recombinant DNA technique with the help of E-Coli was produced by the American firms, Eli-Lilly on July 5, 1983. It has been given the trade name humulin and has been approved for clinical use.

Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs. Insulin from an animal source, though caused some patients to develop allergy or other types of reactions to the foreign protein. Insulin consists of two short polypeptide chains: chain A and chain B, that are linked together by disulphide bridges. In mammals, including humans, insulin is synthesised as a prohormone (like a pro-enzyme,the pro-hormone also needs to be processed before it becomes a fully mature and functional hormone) which contains an extra stretch called the C peptide.

This C peptide is not present in the mature insulin and is removed during maturation into insulin. The main challenge for production of insulin using rDNA techniques was getting insulin assembled into a mature form. In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E.coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.

maturation of pro-insulin after removal of C-peptide (to be simplified)

B.Gene Therapy:

A new system of medicine gene therapy, may develop to treat some hereditary diseases such as SCID, haemophilia etc.

Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child/embryo. Here genes are inserted into a person's cells and tissues to treat a disease. Correction of a genetic defect involved delivery of a normal gene into the individual or embryo to take over the function of and compensate for the non-functional gene.

The first clinical gene therapy was given in 1990 to a 4-year old girl with adenosine deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function. The disorder is caused due to the deletion of the gene for adenosine deaminase. In some children ADA deficiency can be cured by bone marrow transplantation, in others it can be treated by enzyme replacement therapy, in which functional ADA is given to the patient by injection. But the problem with both of these approaches that they are not completely curative. As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture Outside the body. A functional ADA cDNA (using a retroviral vector) is then introduced into these lymphocytes.

which are subsequently returned to the patient. However, as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. However, if the gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

C. Medical Diagnosis of Disease (Molecular diagnosis)

You know that for effective treatment of a disease, early diagnosis and understanding its pathophysiology is very important. Using conventional methods of diagnosis (serum and urine analysis, etc.) early detection is not possible. Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno- sorbent Assay (ELISA) are some of the techniques that serve the purpose of early diagnosis.

Presence of a pathogen (bacteria, viruses, etc) is normally suspected only when the pathogen has produced a disease symptom. By this time the concentration of pathogen is already very high in the body. However, very low concentration of a bacteria or virus (at a time when the symptoms of the disease are not yet visible) can be detected by amplification of their nucleic acid by PCR. PCR is now routinely used to detect HIV in suspected AIDS patients. It is being used to detect mutations in genes in suspected cancer patients too. It is a powerful technique to identify any other genetic disorders.

A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using autoradiography. The clone having the mutated gene will hence not appear on the photographic film, because the probe will not have complimentarity with the mutated gene.

ELISA is based on the principle of antigen-antibody interaction. Infection by pathogen can be detected by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesised against the pathogen.

TRANSGENIC ANIMALS

Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although over 95 percent of all existing transgenic animals are mice.

1. Normal physiology and development: Transgenic animals can be specifically designed to allow the study of how genes are regulated, and how they affect the normal functions of the body and its development e.g. study of complex factors involved in growth such as insulin-like growth factor. By intoducing genes from other species that alter the formation of this factor and studying the biological effects that result, information is obtained about the biological role of the factor in the body.

2. Study of disease: Many transgenic animals are designed to increase our understanding of how genes contribute to the development of disease. These are specially made to serve as models for humane diseases so that investigation of new treatments for diseases is made possible. Today transgenic models exist for many human diseases, such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's.

3. Biological products : Medicines required to treat certain human diseases can contain biological products, but such products are often expensive to make. Transgenic animals that produce useful biological products can be created by the introduction of the portion of DNA (or genes) which codes for a particular product such as human protein ( α-1-antitrypsin) used to treat emphysema, Similar attempts are being made for treatment of phenylketonuria (PKU) and cystic fibrosis. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (2.4 grams per litre). The milk contained the human alpha-lactalbumin and was nutritionally a more balanced product for human babies that natural cow-milk.

4. Vaccine safety : Transgenic mice are being developed for use in testing the safety of vaccines before they are used on humans. Transgenic mice are being used to test the safety of the polio vaccine. If Successful and found to be reliable, they could replace the use of monkeys to test the safety of batches of the vaccine.

5. Chemical safety testing: This is known as toxicity/safety testing. The procedure is the same as that used for testing toxicity of drugs. Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals. They are then exposed to the toxic substances and the effects studied. Toxicity testing in such animals will allow us to obtain results in less time.

Note:-

1. First transgenic animal was mouse contain gene for growth hormone. This enlarged mouse was known as supermouse.

2. First introduced transgenic crop in India (2002) is Bt-cotton.

3. Charles weismann of University of Zurich, obteined interferon through recombinant E. Coli (1980).

4. Microbe have been engineered to produce Human growth Hormone (HGH) for curing dwarfism.

5. Vaccines which are produced by genetic engineering e.g., for Hepatitis-B and Herpes virus.

6. Nitrogen fixation genes may be transferred from bacteria to the major food crops to boost food production without using expensive fertilizers.

7. Bioremediation: in pollution control, microbes have been engineered to break up the crude oil spills. Dr. Ananda Mohan chakarborthi introduced plasmids from different strains into a single cell of Pseudomonas putida. The result was new genetically engineered bacterium which would clean the oil spills called "Superbug" (Oil eating bug). He transferred four types of genes/ plasmids in this bacteria. These are OCT, XYL, CAM and NAH.

8. Genetic modified food- the food is prepared from genetically modified crop (transgenic) is called genetically modified food or G.M. Food.

Thank you👍

BIOTECHNOLOGY- PRINCIPLES AND PROCESSES

2020-07-24T12:22:00.001+05:30

BIOTECHNOLOGY - PRINCIPLES & PROCESSES

Biotechnology term given by Karl Ereky. Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and process useful to humans.

Types of Biotechnology Two types-

(1) Old/traditional-

Old biotechnology are based on the natural capabilities of micro organisms.

e.g. formation of Citric acid, production of penicillin by Penicillium notatum, making curd bread or wine, which are all microbe mediated processes a form of biotechnology.

(2) New/modern-

Based on Recombinant DNA technology.

e.g Human gene producing Insulin has been transferred and expressed in bacteria like E.coli.

According to the European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modem molecular biotechnology. The definition given by EFB is as follows: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Paul berg (Father of genetic engineering)- He transferred gene of SV-40 virus (simian virus) in to E.coli with the help of -phage (Nobel prize 1980).

Stanley Cohen and Herbert Bover: First made recombinant DNA by linking an antibiotic resistance gene with a native plasmid of Salmonella typhimurium.

Principles of Biotechnology

Among many, the two core techniques that enabled birth of modern biotechnology are:-

1. Genetic Engineering/Recombinant DNA Technology:

Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism. (i.e. formation of genetically modified organism).

2. Bioprocess Technology:

Maintenance of sterile (microbial contamination free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture biotechnological products like antibiotics, vaccines.enzymen, etc.

The concept of genetic engineering was the outcome of two very significant discoveries made in bacterial research. These were-

1. presence of extrachromosomal DNA fragments called plasmids in the bacterial cell, which replicate along with chromosomal DNA of the bacterium.

2. Presence of enzymes restriction endonucleases which cut DNA at specific sites. These enzymes are, Therefore, called 'molecular scissors'.

The main basis of Recombinant DNA Technology is DNA cloning:- it is making multiple identical copies of any template DNA.

There are three basic steps of DNA clonning-

i) identification of DNA with desirable genes.

ii) introduction of the identified DNA into the host.

iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY :

Genetic engineering involves cutting of desired segments of DNA and pasting of this D.N.A in a vector to produce a recombinant DNA (rDNA). The 'biological tools' used in the synthesis of recombinant DNA include enzymes, vehicle or vector DNA, desired DNA and host cells.

1. Enzymes :- A number of specific kinds of enzymes are employed in genetic engineering.

These include lysing enzymes, cleaving enzymes, synthesising enzymes and joining enzymes.

Lysing enzymes - These enzymes are used for opening the cells to get DNA for genetic experiment.

Bacterial cell : is commonly digested with the help of lysozyme.

Plant cell : is commonly digested with the help of cellulase and pectinase

• Fungal cell : is commonly digested with the help of chitinase.

Cleaving enzymes - These enzymes are used for DNA molecules. Cleaving enzymes are of two types: exonucleases and endonucleases.

Exonuclease remove nucleotides from the ends of the DNA.

Endonucleases make cuts at specific positions within the DNA.

eg. Restriction endonucleases

Restriction Endonuclease Enzymes (Molecular scissors or molecular knife) Bestriction enzymes belong to a larger class of enzymes called endonucleases.

Restriction enzymes are used in recombinant DNA Technology because they can be used in vitro to recognize and cleave within specefic DNA sequence typically consisting of 4 to 8 nucleotides. This specie 4 to 8 nucleotide sequence is called restriction site and is usually palindromic, this means that the DNA sequence is the same when read in a 5'-3' direction on both DNA strand.

Restriction enzymes are obtained from bacteria.

What is function of restriction enzymes in bacteria?

They are useful to bacteria because the enzyme bring about fragmentation of viral DNA without affecting the bacterial genome. This is an adaptation against bacteriophages. These enzymes exist in many bacteria beside cleavage some restriction on endonuclease, also have capability of modification.

Modification in the form of methylation, by methylation the bacterial DNA modifies and therefore protects its an own chromosomal DNA from cleavage by these restriction enzymes.

The first restriction endonuclease-Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later. It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for Hind II.

Restriction enzyme (Eco R-I) was discovered by Arter Smith & Nathans (1978 Nobel prize).

Besides Hind II, today we know more thán 900 restriction enzyme that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.

Nomenclature of enzyme -

The first letter- indicates bacterial genus (In italic).

Second and third letter- indicate species of bacteria (in italics).

Fourth letter- indicates strain of bacteria (optional).

Roman numeraical- signifying the order in which the enzymes were Isolated from that strain of bacteria.

eg. EcoRI comes from Escherichia coli RY 13. In EcoRL the letter 'R' is derived from the name of strain. Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

Restriction enzymes forms two types of ends on the basis of mode of cutting.

Sticky end (Free end) : Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands.

This leaves single stranded portion at the ends. There are overhanging stretches called sticky ends on each. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

eg. EcoRI, Hind III, Bam HI, Sal l.

Blunt end (non sticky end) : some enzymes cleave both strand of DNA at exactly the same nucleotide position, typically in the center of the recognition sequence resulting in blunt end or flush end.

eg. Sma l, EcoRV, Hae III.

Sma l (Serratia marcescens)

Synthesizing enzymes- These enzymes are used to synthesize new strands of DNA, complementary to existing DNA or RNA template. They are of two types; reverse transcriptases and DNA polymerases.

Reverse transcriptases help in the synthesis of complementary DNA strands on RNA templates.

DNA polymerases- help in the synthesis of complementary DNA strands on DNA templates.

Joining enzymes- These enzymes help in joining the DNA fragments. For example DNA ligase from Escherichia coli is used to join DNA fragments. Joining enzymes are, therefore, called molecular glues.

2. Vehicle DNA or Vector DNA- The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vehicle or vector DNA.

You know that plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.

The following are the features that are required to facilitate cloning into a vector.

A. Origin of replication (ori) : This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

B. Selectable marker: In addition to 'ori', the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.

• Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc. are considered useful selectable markers for E.coli

• Enzyme forming gene also act as selectable marker gene, eg Lac. Z gene Note: The normal E. coli cells do not carry resistance against any of these antibiotics.

C. Restriction sites/Cloning sites : In order to link the alien DNA, the vector needs, recognition sites for the commonly used restriction enzymes. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

A vector should have restriction site for many enzyme but only one restriction site for each enzyme, Otherwise vector will get fragmented.

Selection of vector depends on :-

1. Size of desired gene

2. Type of host

Some examples of vectors:-

a. Plasmids- They are double stranded, circular and extra chromosomal DNA segments found in bacteria which can replicate independently. Plasmids can be taken out of bacteria and made to combine with desired DNA segments by means of restriction enzymes and DNA ligase. A plasmid carrying DNA of another organism integrated with it, is known as recombinant plasmid or hybrid plasmid or Chimeric plasmid.

eg. pBR 322, PUC 18 (used for gene transfer in bacteria).

Ti plasmid, Ri Plasmid (used for gene transfer in dicot plant).

b. Viruses- The DNA of certain viruses is also suitable for use as a vehicle DNA.

Bacteriophage DNA also used as a vector DNA for gene transfer.

Lambda phage (λ phage) has been used for transferring lac genes of E. coli into haploid callus of tomato.

Retro virus - useful for gene transfer in animal cell.

3. Desired DNA/ Alien DNA/ Foreign DNA/ Passenger DNA:-

It is the DNA which is transferred from one organism into another by combining it with the vehicle DNA. The passenger DNA can be complementary, synthetic or random.

i. Complementary DNA (cDNA)- It is synthesized on mRNA template with the help of reverse transcriptase and necessary nucleotides. cDNA formed through reverse transcription is shorter than the actual or in vivo gene because of the absence of introns or non-coding regions.

ii. Synthetic DNA (sDNA)- It is synthesized with the help of DNA polymerase on DNA template.

Kornberg (1961) synthesized first synthetic DNA.

Khorana (1968) synthesized first artificial gene (DNA) without a template. They synthesized the gene coding for yeast alanine t-RNA, which contained only 77 base pairs. However, it did not function in the living system. In 1979, Khorana was able to synthesis a functional tyrosine t-RNA gene of E. coli with 207 nucleotide pairs. Since then a number of genes have been synthesized artificially.

4. Host organism :-

This organism is used for DNA cloning. Host may be plant, animal, bacteria (E.col. fungi (Yeast).

PROCESSES OF RECOMBINANT DNA TECHNOLOGY:

1. Isolation of DNA: The DNAs which are to be used as passenger DNA and the vehicle DNA are extracted out of their cells by lysing the cells with the suitable enzyme.

The DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacterial), cellulose (plant cells), chitinase (fungus). You know that genes are located on long molecules of DNA intertwined with proteins such as histones.

The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed ty appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension DNA that separates out can be removed by spooling method.

2. Fragmentation of DNA by restriction endonucleases : Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme. Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.

Both the passenger and vehicle DNAs are then, cleaved by using the same restriction endonuclease so that they have complementary sticky ends.

Note : The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation (you cannot see pure DNA fragments in the visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

3. Amplification of gene of interest using PCR

Polymerase chain reaction technology (PCR-technology)

This technique was invented by Kary mullis (1983).

In 1993 Kary Mullis got nobel prize for PCR (for chemistry).

PCR is a method for amplifying a specific region of DNA molecule without the requirement for time consuming cloning procedures.

PCR reaction takes place in Eppendorf tube.

Using PCR-technique very low content of DNA available from samples of blood or semen or any other tissue or hair cell can be amplified many times and analysed. In this technique Taq-Polymerase is used. Taq polymerase enzyme is used in PCR which is a special type of DNA polymerase enzyme which is resistant to high temperature.

Taq Polymerase is isolated from Thermus aquaticus bacteria.

Some other examples of polymerase which are used in PCR are-

Pflu Polymerase - Isolated from Pyrococcus furiosus bacteria.

Vent Polymerase - Isolated from Thermococcus litoralis bacterium

Main steps in PCR :-

Denaturation (94°) :- In this step a double stranded DNA molecule is placed at 94°C. So double stranded DNA becomes single stranded & each single stranded DNA functions as a template.

Annealing/Cooling (54°) - In this step two primer DNA are attached at 3' end of single stranded DNA.

Extension (72°):- In this process Taq polymerase enzyme synthesize DNA strain over template.

PCR is automatic process because Taq. polymerase enzyme is heat resistant.

4. Ligation of the DNA fragment into a vector- The complementary sticky ends of the passenger and vehicle DNAs are joined with ligase enzyme. This gives rise to a recombinant DNA.

5. Transferring the recombinant DNA into the host (Gene transfer):-

Transfer of desired genes from one organism into another is an important aspect of genetic engineering.

Gene transfer is achieved by two kinds of transfer methods:

1. Indirect method through vectors or carriers and

2. Director vector less transfer method.

Indirect method :-

Gene Transfer in bacterial cell : Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made 'competent' to take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can then be forced into such cells by Incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.

Note: The bacteria to be used as hosts should be without plasmids. The host cells are treated with calcium chloride or lysozyme.

Transformant E.coli with plasmid.

Non-transformant E.coli without plasmid.

Gene Transfer in plant cell

1. A plant pathogenic bacterium-Agrobacterium tumefaciens produces crown galls or plant tumours in almost all dicotyledonous plants.

2. This bacterium infects all broad leaved agricultural crops such as tomato, soyabean, sunflower and cotton but not cereals.

3. Tumour formation is induced by it's plasmid which is therefore called Ti plasmid (TI - tumour inducing) Agrobacterium tumefaciens naturally transfers some part of Ti-Plasmid into host plant DNA without any human effort so it is called natural genetic engineer of plant.

In the transformation process two essential component in Ti-plasmid -

(a) T-DNA - (Transferred DNA)

(b) Vir-region-(Virulence region)

Inside the host plant cell T-DNA is seperated from Ti-plasmid, and integrated into host plant DNA that causes crown gall tumour.

Vir-region contains genes which are essential for T-DNA transfer and integration to host plant DNA.

When we use Ti-plasmid as a vector, first we remove the tumour causing gene from T-DNA region. Then desired gene inserted inplace of it. Now, this plasmid is called disarmed plasmid.

Same as Ri plasmid of A.rhizogenes (causing hairy root disease) also used as vector for gene transfer to plant cell.

Gene transfer in animal

Retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.

Direct method

Foreign genes can also be transferred directly by the following methods:-

a. Electroporation- It creates transient (temporary pores) in the plasma membrane to facilitate entry of foreign DNA.

b. Chemical mediated genetic transformation- It involves certain chemicals such as polyethylene gycol (PEG), that help in the uptake of foreign DNA into host cells.

c. Microinjection- it in the introduction of foreign genes mainly into animal cells using micropipette ar glass needles.

d. Particle gun/Biolistic method- It is a technique in which tungsten or gold particles coated with foreign DNA bombarded into target cells to facilitate entry of the foreign genes.

e. Liposome mediated gene transfer- In this method DNA encloses within lipid begs. Thest lipid begs funed with protoplast.

Selection of Transformed with recombinant cell:-

1. Selection by two antibiotic resistance gene

You can ligate a foreign DNA at the Bam HI site of tetracycline resistance gene in the vector pBR322.

The recombinant plasmids will loss tetracycline resistance due to insertion of foreign DNA (insertional inactivation) now, it can be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium. The transformants (plasmid transferd) growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, non recombinants wil grow on the medium containing both the antibiotics. In This case, one antibiotic resistance gene helps in selecting the transformants.

Note:-

Insertional inactivation- Due to Insertion of desired gene within selectable marker gene of vector, selectable marker gene become inactive or loose their Function. This is called Insertional inactivation.

2. Selection by one Lac Z gene and one antibiotics resistant gene- Selection of recombinants due to inactivation of antibiotics is a cumbersome (troublesome) procedure because it requires simultaneous plating on two plated having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate X-gal gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase (reporter enzyme) and the colonies do not produce any colour, these are identified as recombinant colonies.

Obtaining the Foreign Gene Product :

When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial plant or animal cell the alien DNA gets multiplied. In almost all recombinant technologies, the ultimate aim is to produce a desirable protein. Hence, there is a need for the recombinant DNA to be expressed. The foreign gene gets expressed under appropriate conditions. The expression of foreign genes in host cells involve understanding many technical details.

After having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, one has to consider producing it on a large scale. Can you think of any reason why there is a need for large-scale production? If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein. The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory. The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.

The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein.

Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc. useing microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, Oxygen).

The most commonly used bioreactors are of stirring type -

A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.

The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively, air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

Downstream Processing -

After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product.

The processes include separation and purification, which are collectively referred to as downstream processing.

The product has to be formulated with suitable preservatives. Such formulation has to undergo through clinical trials as in case of drugs. Strict quality control testing for each product is also required. The downstream processing and quality control testing vary from product.to product.

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HUMAN GENOME PROJECT

2020-07-20T01:19:00.001+05:30

HUMAN GENOME PROJECT

Genetic make up of an organism or an individual lies in the DNA sequences. If two individuals differ, then their DNA sequences should also be different, at least at some places. These assumptions led to the quest of finding out the complete DNA sequence of human genome. With the establishment of genetic engineering techniques where it was possible to isolate and clone any piece of DNA and availability of simple and fast techniques for determining DNA sequences, a very ambitious project of sequencing human genome was launched in the year 1990.

Human Genome Project (HGP) was called a mega project. You can imagine the magnitude and the requirements for the project if we simply define the aims of the project as follows:

Human genome is said to have approximately 3 x 10⁹bp, and if the cost of sequencing required is US $3 per bp (the estimated cost in the beginning), the total estimated cost of the project would be approximately 9 billion US dollars. Further, if the obtained sequences were to be stored in typed form in books, and if each page of the book contained 1000 letters and each book contained 1000 pages, then 3300 such books would be required to store the information of DNA sequence from a single human cell. HGP was closely associated with the rapid development of a new area in biology called as Bioinformatics.

Goals of HGP

Some of the important goals of HGP are as follows :

1. Identify all the genes in human DNA.

2. Determine the sequences of the 3 billion chemical base pairs that make up human DNA.

3. Store this information in databases

4. Improve tools for data analysis.

5. Transfer related technologies to other sectors, such as industries

6. Address the ethical, legal and social issues (ELSI) that may arise from the project.

The project was completed in 2003. Knowledge about the effects of DNA variations among individuals can lead to revolutionary new ways to diagnose, treat and someday prevent the thousands of disorders that affect human beings. Besides providing clues to understanding human biology, learning about non-human organisms, DNA sequences can lead to an understanding of their natural capabilities that can be applied toward solving challenges in health care, agriculture energy production, environmental remediation. Many non-human model organisms, such as bacteria, yeast, Caenorhabditis elegans (a freeliving non-pathogenic nematode), Drosophila (the fruit fly), plants (rice and Arabidopsis), etc., have also been sequenced.

Methodologies : The methods involved two major approaches (1) Expressed Sequence Tags (ESTS) - Identifying all the genes that expressed as RNA (2) Sequence Annotation - The blind approach of simply sequencing the whole set of genome that contained all the coding and non-coding sequence, and later assigning different regions in the sequence with functions. For sequencing, the total DNA from a cell is isolated and converted into random fragments of relatively smaller sizes (recall DNA is a very long polymer, and there are technical limitations in sequencing very long pieces of DNA) and cloned in suitable host using specialised vectors.

The cloning resulted into amplification of each piece of DNA fragment so, that is subsequently could be sequenced with ease. The commonly used hosts were bacteria and yeast, and the vectors were called as BAC (bacterial artificial chromosomes), and YAC (yeast artificial chromosomes).

The fragments were sequenced using automated DNA sequencers that worked on the principle of a method developed by Frederick Sanger (Remember, Sanger is also credited for developing method for determination of amino acid sequences in proteins). These sequences were then arranged based on some overlapping regions present in them. This required generation of overlapping fragments for sequencing. Alignment of these sequences was humanly not possible. Therefore, specialised computer based programmes were developed. These sequences were subsequently annotated and were assigned to each chromosome. The Sequence of chromosome I was completed only in May 2006 (this was the last of the 24 human chromosomes 22 autosomes and X and Y to be sequenced). Another challenging task was assigning the genetic and physical maps on the genome. This was generated using information on polymorphism of restriction endonuclease recognition sites, and some repetitive DNA sequences known as microsatellites.

Salient Features of Human Genome-

Some of the salient observations drawn from human genome project are as follows :

1. The human genome contains 3164.7 million nucleotide bases.

2. The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million bases.

3. The total number of genes is estimated at 30,000 much lower than previous estimates of 80,000 to 1,40,000 genes. Almost all (99.9 per cent) nucleotide bases are exactly the same in all people.

5. The functions are unknown for over 50 percent of discovered genes.

6. Less than 2 percent of the genome codes for proteins.

7. Repeated sequences make up very large portion of the human genome.

8. Repetitive sequences are stretches of DNA sequences that are repeated many times, sometimes hundred to thousand times. They are thought to have no direct coding functions, but they shed light on chromosomes structure, dynamics and evolution.

9. Chromosome 1 has most genes (2968). and the Y has the fewest (231).

10. Scientists have identified about 1.4 million locations where single base DNA differences (SNPS- single nucleotide polymorphism, pronounced as 'snips) Occur in humans. This information promises to revolutionise the processes of finding chromosomal locations for disease-associated sequences and tracing human history.

(a) First prokaryotes in which complete genome was sequenced is Haemophilus influenzae.

(b) First Eukaryote in which complete genome was sequenced is Saccharomyces cerviceae (Yeast).

(d) First animal in which complete genome was sequenced is Caenorhabditis elegans (Nematode).

β-globin and insulin gene are less than 10 kilo base pair T.D.F. gene is the smallest gene (14 base pair) and Duchenne muscular Dystrophy gene is made up of 2400 kilo base pair.(Longest gene)

Thank you....🤞

DNA FINGERPRINTING

2020-07-19T12:27:00.000+05:30

DNA fingerprinting/ DNA typing/ DNA profiling/ DNA test

It is technique to identify a person on the basis of his/her DNA specificity.

This technique was invented by sir Alec. Jeffery (1984).

In India DNA Finger printing has been started by Dr. V.K. Kashyap & Dr. Lal Ji Singh.

DNA of human is almost the same for all individuals but very small amount that differs from person to person that forensic scientists analyze to identify people.

These differences are called Polymorphism (many forms) and are the key of DNA typing. Polymorphisms are

most useful to forensic scientist. It is consist of variation in the length of DNA at specific loci is called Restricted fragment. It is most important segment for DNA test made up of short repetitive nucleotide sequences, these are called VNTRs (variable number of tandem repeat).

VNTRs also called minisatellites were discovered by Alec Jeffery. Restricted fragment consist of hypervariable repeat region of DNA having a basic repeat sequence of 11-60 bp and flanked on both sites by restriction site.

The number and position of minisatellites or VNTR in restriction fragment is different for each DNA and length of restriction fragment is depend on number of VNTR.

Therefore, when the genome of two people are cut using the same restriction enzyme the length of fragments obtained is different for both the people.

These variations in length of restricted fragment is called RFLP or Restriction fragment length polymorphism.

Restriction Fragment Length Polymorphism distributed throughout human genomes are useful for DNA Fingerprinting.

DNA Fingerprint can be prepared from extremely minute amount of blood, semen, hair bulb or any other cell of the body.

DNA content of 1 - Microgram is sufficient.

Technique of DNA Fingerprinting involves the following major steps.

Extraction - DNA extracted from the cell by cell lysis. If the content of DNA is limited then DNA can be amplified by Polymerase chain reaction (PCR). This process is amplification.

Restriction Enzyme Digestion : Restriction enzyme cuts DNA at specific 4 or 6 base pair sequences called restriction site.

Hae III (Haemophilus aegyptius) is most commonly used enzyme. It cuts the DNA, every where the bases are arranged in the sequence GGCC. These restriction fragment transferred to Agarose Polymer gel.

Gel Electrophoresis :

Gel electrophoresis is a method that separates macromolecules either nucleic acid or proteins-on the basis of size, electric charge.

Gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provides the driving force. A molecule's properties determine, how rapidly an electric field can move the molecule through a gelatinous medium.

Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel.

Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids posses ionisable groups and therefore, at any given pH, exist in solution as electrically charged species either as cation (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode.

By the gel electrophoresis these restricted fragments move towards the positive electrode (anode) because DNA has -ve electric charge (PO₄³⁻).

Smaller Fragment more move towards the positive pole due to less molecular weight. So after the gel electrophoresis DNA fragment arranged according to molecular weight.

These separated fragments can be visualized by staining them with a dye that fluoresces ultraviolet radiation.

Southern transfer / Southern blotting :

The gel is fragile. It is necessary to remove the DNA from the gel and permanently attaches it to a solid support. This is accomplished by the process of Southern blotting. The first step is to denature the DNA in the gel which means that the double-stranded restriction fragments are chemically separated into the single stranded form.

The DNA then is transferred by the process of blotting to a sheet of nylon. The nylon acts like an ink blotter and "blots" up the separated DNA fragments, the restriction fragments, invisible at this stage are irreversibly attached to the nylon membrane the "blot".

This process is called Southern blot by the name of Edward Southern (1970).

Hybridization: To detect VNTR locus on restricted fragment, we use single stranded Radioactive Phosphorus-32 (P32) DNA probe which have the base pair sequences complimentary to the DNA sequences at the VNTR locus. Commonly we use a combination of at least 4 to 6 separate DNA probes.

Labelled Probes are attached with the VNTR loci of restricted DNA Fragments, this process is called Hybridization.

Autoradiography: Nylon membrane containing radio active probe exposed to X-ray. Specific bands appear on X-ray film. These bands are the areas where the radioactive probe bind with the VNTR.

This appears the specific restriction fragment length pattern. This length pattern is different in different individual. This is called Restriction Fragment length Polymorphism (RFLP).

These allow analyzer to identify a particular person DNA, the occurrence and frequency of a particular genetic patter contained in this X-ray film. These x-ray film called DNA signature of a person which is specific for each individual.

The probability of two unrelated individual having same pattern of location and repeat number of minisatellite (VNTR) is one in ten billion (world population 6.1 billion).

In India the centre for DNA fingerprinting and diagnosis (CDFD - center for DNA finger printing & diagnosis) located at Hyderabad.

Application of DNA Fingerprinting

1. Paternity tests- The major application of DNA finger printing is in determining family relationships. For identifying the true (biological) father, DNA samples of Child, mother and possible fathers are taken and their DNA finger prints are obtained. The prints of child DNA match to the prints of biological parents.

2. Identification of the criminal- DNA fingerprinting has now become useful technique in forensic (crime detecting) science, specially when serious crimes such as murders and rapes are involved. For identifying a criminal, the DNA fingerprints of the suspects from blood or hair or semen picked up from the scene of crime are prepared and compared. The DNA fingerprint of the person matching the one obtained from sample collected from scene of crime can give a clue to the actual criminal.

schematic representation of DNA fingerprinting: few representative chromosomes have been shown to contain different copy number of VNTR. For the sake of understanding colour schemes have been used to trace the origin of each band in the gel. The two alleles (parental and maternal) of chromosome also contain different copy numbers of VNTR. It is clear that the banding pattern of DNA from crime sceme matches with individual B, and not with A.

Thank you👍

MUTATION

2020-07-18T16:49:00.000+05:30

MUTATION

Sudden heritable change in genetic material of an organism is called as Mutation.

Mutation are source of discontinuous variation.

Only those mutation are heritable which occur in germinal cell of an organism. While somatic mutations are non heritable. Somatic mutations are also heritable in vegatative propagated plants.

Mutation word was given by Hugo De Vries.

De Vries studied mutations in the plant Oenothera lamarckiana (evening primrose).

Mutation was first observed by Seth Wright. He observed some short legged sheep (Ancon) variety in a population of long legged sheep.

Beadle and Tatum induced mutations in Neurospora by the help of U. V. rays. or X-rays.

Wild neurospora (PROTOTROPH)----U.V. Rays----Mutant Neurospora (AUXOTROPH)

Normal Neurospora can be grown in minimal medium because Neurospora can make all essential nutrients required for it. This is known as Prototroph.

Mutant Neurospora doesn't has capability to grow in minimal medium because due to mutation It loses those genes which codes for the enzyme that helps to prepare some special nutrients for it. They gave "one gene one enzyme" concept. This form is known as Auxotroph.

M.S. Swaminathan induced mutations in wheat by the help of γ-rays to obtain good varieties tor eg. Sharbati Sonora, Pusa Lerma. Swaminathan established γ (gamma) garden in IARI-New Delhi (Pusa Institute).

Types of mutation :

i. CHROMOSOMAL MUTATION

ii. GENE MUTATIONS

1. Chromosomal Mutations- Each species of organisms has constant number of chromosomes. There are two sets of homologous chromosomes in the somatic cells of an organisms. The number, type and sequences of genes on chromosomes is fixed and all the genes are oriented at their definite loci. The gene loci retain the same sequence they have had for many generations past.

Sometimes, during cell division, as a results of certain abnormalities, changes in structure and number of chromosomes are produced. Such type of change are called chromosomal aberration. These chromosomal aberration can be classified into following two classes:-

Types of chromosomal mutation :

1.Heteroploidy/Genomatic mutation-change in chromosome number.

2. Chromosomal aberration-change in structure of chromosome.

1. Heteroploidy / Genomatic mutationor ploidy- In different species of all the organism, the number of chromosomes remains fixed and stable throughout their life, but sometimes change in the number of chromosomes occurs. The Change in number of one or few chromosomes in a set or number of entire set of chromosome.

It is of two types:

Euploidy-Change in number of chromosome sits

Aneuploidy - Change in number of chromosome in a set

EUPLOIDY- Change in number of sets of chromosome i.e. either loss or addition of sets of chromosomes.

Monoploidy (x) - Presence of one set of chromosomes.

Diploidy (2x) - Presence of two sets of chromosomes.

Polyploidy - Presence of more than two sets of chromosomes.

It may be : Triploidy (3x) Tetraploidy (4x) Pentaploidy (5x) Hexaploid (6x) Heptaploid (7x) octaploidy (8x)

Polyploid plants with even number of sets are always fertile, reproduce sexually and form seeds.

Polyploid plants with odd number of sets are always sterile don't reproduce by sexual reproduction. They don't produce seeds but they may produce seedless fruits by parthenocarpy. eg. Banana and seedless grapes.

ANEUPLOIDY- Loss or addition of chromosomes in a set of chromosomes.

Types of Aneuploidy:-

1. Hypoaneuploidy (loss)

2n-1=Monosomy - (loss of one chromosome in one set)

2n-1-1=Double monosomy (loss of one chromosome from each set, but these are non homologous).

2n -2=Nullisomy (loss of two homologous chromosome)

2. Hyperaneuploidy (addition)

2n +1=Trisomy : addition of one chromosome in one set.

2n +1 +1=Double Trisomy : addition of one chromosome in each set

2n +2=Tetrasomy: addition of two chromosome in one set.

Cause of aneuploidy is chromosomal nondisjunction means chromosomes fail to separate during meiosis.
Chances of aneuploidy are more in higher age female due to less activity of oocyte, so chances of syndrome increase in children who are bom from higher age female.

2. Chromosomal Aberrations : Change in structure of chromosome. The change in the number or orientation of genes located on chromosomes is called structural chromosomal abberation. Such types of changes are produced mainly as a results of some irregularities occured during meiotic divisons. Such irregularities include breaking, reattachment, and rearrangement etc. Structural changes in the chromosomes are of following four types:-

(i) Deletion : Loss of a part or segment of chromosome which leads to loss of some gene is called as deletion.

It is of 2 types :

(A) Terminal deletion -Loss of chromosomal segment from one or both ends.Eg. The cry-du-chat syndrome is an example of terminal deletion in short arm of 5th chromosome.

(B) Intercalary deletion - Loss of chromosomal part between the ends.

(ii) Inversion : Breakage of chromosomal segment but reunion on same chromosome in reverse orders. It leads to change in distance between genes on chromosome or sequence of genes on chromosome so crossing over is affected

It is of 2 types:-

1. Paracentric -If inversion occur only in one arm and inverted segment does not include centromere.

2. Pericentric - In this type of inversion inverted segment include centromere.

(iii) Duplication : Occurence of a chromosomal segment twice on a chromosome.

Example : In drosophila "Bar eye character" is observed due to duplication in X-chromosome. Bar eye is a character whose eyes are narrower as compared to normal eye shape.

(iv) Translocation : In this, a part of the chromosome is broken and may be joined with non homologous chromosome. This is also known as illegitimate crossing over (illegal crossing over).

Types of translocation

(A) Simple Translocation- When a chromosomal segment breaks and attached to the terminal end of a non homologous chromosome.

(B) Reciprocal Translocation- exchange of segments between two non homologous chromosome.

In man, STRUCTURAL CHROMOSOMAL ABERRATIONS.

In man, many examples of chromosomes breakage with resulting aberration have been found.

For example, a terminal deletion of a part of long arm of chromosome 21 in man causes Chronic granulocytic leujemia. Since this shortened chromosome was discovered in Philadelphia, it is known as the Philadelphia chromosome. Similarly deletion of a part of short arm of chromosome 5 in man causes undeveloped larynx in children as a result of which they produce mewing sound. Because of this it is called as Cri-du-chat or cat cry syndrome.

Chronic myeloid leukemia (C M L) is a type of blood cancer. This disease is a result of reciprocal transiocation between 22 and 9 chromosome.

Note: If exchange of segments take place in between homologous chromosomes then it is called crossing over.

(2) GENE MUTATION OR POINT MUTATION

Two types:-

1. Substitution

2. Frameshift mutation.

A. Substitution:

Replacement of one nitrogenous base by another nitrogenous base is called as substitution.

It causes change in one codon in genetic code which leads to change in one amino acid in structure of protein. eg. Sickel cell anaemia.

Change may not occur some time because for one amino acid more than one type of codons are present

Substitution is of two types

1. Transition- Replacement of one purine by another purine or replacement pyrimidine by another pyrimidines.

2. Transversion- Replacement of purine by pyrimidine or pyrimidine by purine is called transversion.

B. Frame shift mutation/Gibberish mutation:

Loss or addition of one or rarely more than one nitrogenous bases in structure of DNA.

Frame shift mutation is of two types.

1. Addition-Addition of one or rarely more than one nitrogenous bases in structure of DNA.

2. Deletion- loss of ons or rarely more than one nitrogenous bases in structure of DNA.

Due to frame shift mutation complete reading of genetic code changed. It leads to change in all amino acids in structure of proterin so a new protein is formed which is completely different from previous protein.

So frameshift mutations are more harmful as compared to substitution.

eg: Thalassemia (lethal genetic disorder)

MUTAGENS-

Mutagens are those substances which cause mutations.

Non ionising:- U.V. Rays.

Thank you👍

CANOPY ARCHITECTURE

2020-07-16T10:07:00.003+05:30

Canopy architecture

" The overall profile of the tree top is known as canopy architecture".

Vegetation canopy structure describes the three-dimensional arrangement of leaves and includes such canopy attributes as canopy architecture, leaf angle distribution, ground cover fraction, leaf morphology, vegetation spatial heterogeneity, and shadows.

Canopy architectures in plants:-

Canopy architecture is the overall profile of tree the tops. The trees gets grouped together leading to the formation of canopy and reduces the amount of light beneath them.

Factors controlling the canopy architecture:-

1. Pattern of growth in plant axis.

2. Arrangements and distribution of branches on main shoot.

3. Angle of branches on shoot.

4. Direction of branches.

5. Size, arrangement, distribution and shapes of leaves.

6. Genetic, environmental and nutritional factors.

Heliophytes or shade-introlerant plants- those plants which grows in full sunlight. eg. Sunflower, poplars, pines etc.

Sciophytes or shade-tolerant plants- certain plants which survive only under shade. eg. Fir, spruce etc.

However some plants can be facultative sciophytes as well as facultative heliophytes.

fig : Heliophytes and Sciophytes

The forest is made up of some individual tall plants which protrudes above normal level of canopy and are called as emergents.

The process of stratification i.e. arrangements of different plants or animals in different layer of substratum takes place in canopy.

Eg. Stratification in a tropical rain forest.

Based upon branching a tree, it may classified as:-

1. Culm- The stem is unbranched showing distinct nodes and internodes imparting a jointed apperance. Eg. Bambusa arundinacea (Bamboo).

2. Columnar (caudex)- In such case trunk is unbranched and bears a crown of leaves at its apex. Eg. Fan plam, areca catechu (supari) , Cocos nucifera (coconut) etc.

3. Excurrent- In such case lateral branches do not complete with the growth of main stem. Branches appear in acropetal succession. Thus, tree gives cone-like apperance. Eg. Pinus, Eucalyptus etc.

4. Decurrent or Deliquescent- When some of the lateral branches grows vigorously, the trunk Disappears after some distance due to the suppression of growth of terminal bud, the form called Decurrent. Eg. Magnifera indica (mango), Dalbergia(Shisham), Ficus benghalensis etc.

SHAPE OF CANOPY ARCHITECTURE

a. Round canopy- Trees which shows Decurrent or Deliquescent branching shows round canopy. Branch appears in alternate pattern. Branches present on upper side grows towards upper and outer sides. However, lower branches spread out. Minimum overlapping of leaves in such cases. Eg. Magnifera indica , lagerstromia, Dalbergia, Ficus etc.

b. Conical canopy- In such case lateral branches do not complete with the growth of main stem. Branches appears acropetally (Monopodial/Racemose).

Branches appear either horizontally (eg. Araucaria) or ascent at a particular angle (eg. Thuja). Shape of tree is conical.

Eg. Pinus, Eucalyptus etc.

c. Oblong or cylindrical canopy- Branching in these trees can be Racemose (Gnetum) or cymose type (peltophorum). Main trunk remains upright. Lateral branches are short and grows towards upper side providing cylindrical shape to trees.

Eg. Artocorpus (jack fruit).

d. Flat topped canopy- Growth of apical bud on main trunk stops after attaining a certain height. It bends on upper side, a twig appears which further give rise to Obliquely rising branches. Thus, tree imparts umbrella shape. Eg. Cassia, Terminalia (imli), Thespesia actifolia, Enterolobium saman (rain tree) etc.

e. Weeping tree- Due to weak branching or due to drooping branching pattern, trees impact weeping appearance.

Stem is Monopodial (Polyalthia) or stem is sympodial (Zizyphus jujuba).

Eg. Salix (weeping willow), Acacia arabica (Kikar).

f. Pagoda trees- Trees bear spirally arranged compacts, simple or palmate type of leaves. The branches on trunk arises in wholed fashion. The pagoda shape is very distinct when the tree is young. As the whorls ascent from the base to tip, length internode shortens and gives a typical pagoda shape to trees. Eg. Salmalia, Achras sapota (Chiku), Terminalis etc.

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REGULATION OF GENE EXPRESSION ( Lac Operon & Trp Operon)

2020-07-16T10:07:00.002+05:30

REGULATION OF GENE EXPRESSION

Constitutive genes (House-keeping genes) - These genes are expressed constantly because their products are constant needed for cellular activity, eg. genes for glycolysis, gene of ATPase enzyme.

Non-constitutive genes (Smart gene or Luxury gene) - These genes remain silent and are expressed only when the gene product is needed. They are switched 'on' or 'off' according to the requirement of cellular activities. Non-constitutive genes are of two types; inducible and repressible. The Inducible genes are switched on in presence of a chemical substance called inducer, required for the functioning of gene activity. The repressible genes continue to express themselves till a chemical,, often an end product of the metabolism inhibits or represses their activity. Such type of inhibition is called feed back inhibition or feed back repression.

The mechanism which stimulates the expression of certain genes and inhibits that of others is called regulation of gene expression.

It is possible only if the organism has a mechanism of regulating gene activity by allowing some to function and others to restrain their activity through switching on and switching off system. This means, the genes are turned 'on' or 'off' as per requirement.

A set of genes is 'switched on' when enzymes are required to metabolise a new substrate. The enzymes produced by these genes metabolise the substrate.

The molecules of metabolite that come to switch on of the genes are termed as inducers and the phenomenon is called induction.

Similarly, certain genes which are in their 'switch on' state, continue to synthesise a metabolite till the later is produced in amount more than required or else, It is supplied to the cell from outside. In other words, certain genes continue to express themselves till the end product of inhibits or repress their expression. Inhibition by an end product is known as 'feed back repression'.

Regulation of gene expression refers to a very broad term that may occur at various levels. Considering that gene expression results in the formation of a polypeptide, it can be regulated at several levels. In eukaryotes the regulation could be exerted at:-

1. transcriptional level (formation of primary transcript).

2. processing less (regulation of splicing).

3. transport of mRNA from nucleus to the cytoplasm.

4. translational level.

OPERON CONCEPT

In 1961, two French microbiologist Francis Jocob and Jacques Monad at the Pasteur Institute in Paris, proposed a mechanism called operon model for the regulation of gene action in E coli.

An operon is a part of genetic material or DNA, which acts as a single regulated unit having one or more structural genes-an operator gene, a promoter gene, a regulator gene.

Operons are of two types:-

(i) inducible

(ii) repressible

1. Inducible System (Lac operon of E. coli)

An Inducible operon system normally remains in switched off condition and begins to work only when the substance to be metabolised by it is present in the cell. Inducible operon system generally occurs in catabolic pathways. e.g. Lac operon of E. coli.

Active repressor + inducer = inactive repressor

An Inducible operon system consists of four types of genes.

Structural genes - These genes synthesise mRNAs, which in turn synthesise polypeptide or enzyme over the ribosomes. An operon may have one or more structural genes. Each structural gene of an operon is called cistron. The lac operon (lactose operon) of Escherichia coli contains three structural genes (Z, Y and A). These genes occur adjacent to each other and thus are linked. They transcribe a polycistronic mRNA molecule (a single stretch of mRNA covering all the three genes), that helps in the synthesis of three enzymes-β galactosidase (breaks lactose into glucose and galactose) lactose permease (helps in entry of lactose in cell from outside and transacetylase (transfers an acetyl group from acetyl Co A to β galactosidase).

Operator gene - It lies adjacent to the structural genes and directly controls the synthesis of mRNA over the structural genes. It is switched off by the presence of a repressor. An inducer can take away repressor and switch on the gene that directs the structural genes to transcribe.

Promoter gene - This gene is the site for initial binding of RNA polymerase. When the operator gene is turned on, the enzyme RNA polymerase moves over it and reaches the structural genes to perform transcription.

Regulator gene- It produces a repressor that binds to operator gene and stops the working of the opertor gene.

Repressor - It Is a protein, producd by the regulator gene. It binds to the operator gene so that the transcription of structural gene stops. Repressor has two binding site (1) operator gene (2) effective molecule (inducer/corepressor.

Inducer- It is a chemical (substrate, hormone or some other metabolite) which after coming in contact with the repressor, forms an inducer repressor complex. This complex cannot bind with the operator gene, which is thus switched on. The free operator gene allows the structural gene to transcrip mRNA to synthesise the enzymes.

The inducer for lac operon of Escherichia coli is lactose (in fact allolactose as isomer of lactose). When the sugar lactose is added to the culture of E.coli, a few molecules of lactose gets into the bacterial cells by the action of the enzyme permease, a small amount of this enzyme is present in the cell even when the operon is not working. These few lactose molecules are then converted into an active from which acts as an inducer and binds to the repressor protein. The inducer repressor complex fails to join with the operator, which is turned on. The three genes are expressed as three enzymes to metabolise lactose. Allolactose is real inducer of lac operon.

Repressible system (Tryptophan operon of E.coli)

A repressible operon system is normally in It's switch on state and continue to synthesise a metabolise till the latter is produced in amount more than required, or else it becomes available to the cell from outside. Repressible operon system is commonly found in anabolic pathway, eg. Tryptophan operon of E.coli.

[Inactive repressor + co-repressor =acitve repressor]

Tryptophan operon of Escherichia coli is an example of repressible system. It consists of the following:

Structural genes- These genes are meant for transcription of mRNA, which in turn synthesise enzyme.

Tryptophan operon has five structural genes E, D, C,B and A. They lie in continuation and synthesise enzymes for five steps of tryptophan synthesis.

Operator gene (trp O)- It lies adjacent to the structural genes and controls the functioning of the structural genes. Normally, it is kept switched on, because the apo-repressor produced by the regulator gene does not bind to it. The operator gene is switched off when a co-repressor is available alongwith apo repressor.

Promoter gene (trp P)- It marks the site at which the RNA polymerase enzyme binds. When the operator gene is switched on, it moves from promotor gene to structural genes for transcription.

Regulator gene (trp R)- It produces a regulatory protein called apo-repressor for (Inactive repressor) possible blocking the activity of operator gene.

Apo-repressor- It is a regulatory protein synthesised by regulator gene. When a co-repressor substrate is available in the cell, the apo-repressor combines with the co-repressor to form a apo-repressor co-repressor complex. This complex binds with the operator gene and switches it off. Presence of apo-repressor alone, the operator gene is kept switched on because, by itself the apo-repressor is unable to block the working of operator gene.

Co-repressor- It is an end product of reactions catalysed by enzymes produced by the structural genes.

In the presence of tryptophan some molecules of tryptophan act as co-repressor, co-repressor-bind with inactive repressor. Co-repressor repressor complex bind with operator region and prevent the binding of RNA polymerase to the promoter, the trp-operon is off.

The repressor molecule has key role in regulation of lac-operon. Repressor molecule active or inactive. Active Repressor may be rendered inactive by addition of an inducer while the inactive repressor can be made active by addition of a co-repressor. Because the product of regulator gene the repressor act by shutling off the transcription of structural gene the operon model, as originally proposed by Jacob & Monod is referred as -negative control system.

GENETIC CODE

2020-07-16T10:07:00.001+05:30

GENETIC CODE

Term Given by George Gamow.

Discovered by Nirenberg, Matthaei and Khorana.

The relationship between the sequence of amino acids in a polypeptide chain and nucleotide sequence of DNA or m-RNA is called genetic code.

There occur 20 types of amino acids which participate in protein synthesis. DNA contains information for the synthesis of any types of polypeptide chain. In the process of transcription, information is transfered from DNA to m-RNA in the form of complementary N2- base sequences.

m-RNA contains code for each amino acid and it is called codon. A codon is the nucleotide sequence on m-RNA which codes for particular amino acid; whereas the genetic code is the sequence of nucleotides on m-RNA molecule, which contains information for the synthesis of polypeptide chain.

Triplet Code :-

The main problem of genetic code was to determine the exact number of nucleotide in a codon which codes for one amino acid.

There are four types of N2-bases in m-RNA (A, U, G, C) for 20 types of amino acids.

If genetic code is singlet i.e. codon is the combination of only one nitrogen base, then only four codons are possible A, C, G and U. These are insufficient to code for 20 types of amino acids.

Singlet code - (4^{1 =}4 x1= 4) codons

If genetic code is doublet (i.e. codon is the combination of two nitrogen bases) then 16 codons are formed.

Doublet code - (4²=4x4=16) codons.

16 codons are insufficient for 20 amino acid.

Gamow (1954) pointed out the possibility of three letters code (Triplet code).

Genetic code is triplet i.e. one codon consists of three nitrogen bases.

Triplet code - (4³=4x4x4=64) codons.

In this case there occurs 64 codons in dictionary of genetic code.

64 codons are sufficient to code 20 types of amino acids.

The chemical method developed by Har Gobind Khorana was instrumental in synthesising RNA molecules with defined combinations of bases (homopolymers and copolymers). Marshall Nirenberg's cell-free system for protein synthesis finally helped the code to be deciphered. Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful in polymerising RNA with defined sequences in a template independent manner (enzymatic synthesis of RNA).

Characteristics of Genetic Code:-

i. Triplet in Nature-

A codon is composed of three adjacent nitrogen bases which specific one amino acid in poly chain.

For example:-

In m-RNA if there are total 90 N2-bases.

Then this m-RNA determine 30 amino acids in polypeptide chain.

In above example, number of nitrogen base are 90 so codons = 30 and 30 codons decide 30 amino acid in polypeptide chain.

ii. Universality:-

The genetic code is applicable universally. The same genetic code is present in all kinds of living organism Inicluding viruses, bacteria unicellular and multicellular organisms.

iii. Non-Ambiguous and specific unamfiguous:-

Genetic code is non ambiguous i.e. one codon specifies only one amino acid and not any other.

In this case one codon never code for two different amino acids. Exception GUG codon which codes both valine and methionine amino acids.

iv. Non-Overlapping:-

A nitrogen base is a constituent of only one codon.

v. Comma less:-

There is no punctuation (comma) between the adjacent codon i.e. each codon is immediately followd by the next codon.

If a nucleotide is deleted or added, the whole genetic code read differently.

A polypeptide chain having 50 amino acids shall be specialized by a linear sequence of 150 nucleotides.

If a nucleotide is added in the middle of this sequence, the first 25 amino acids of polypeptide will be same but next 25 amino acids will be different.

vi. Degeneracy of Genetic code :-

There are 64 codons for 20 types of amino acids, so most of the amino acids (except two) can be coded by more than one codon. Single amino acid coded by more than one codon is called "Degeneracy of genetic code". This incident was discovered by Baurnfield and Nirenberg.

Only two amino acids Tryptophan and Methionine are specified by single codon.

UGG for Tryptophan

AUG for Methionine.

All the other amino acids are specified or coded by 2 to 6 codons.

Leucine, serine and arginine are coded or specified by 6-codons.

Leucine = CUU, CUC, CUA, CUG, UUA & UUG.

Serine = UCU, UCC, UCA, UCG, AGU, AGC.

Arginine = CGU, CGC, CGA, CGG, AGA, AGG.

Degeneracy of genetic code is related to third position (3'- end of triplet codon) of codon. The third bases is described as "Wobbly base".

Chain Initiation and Chain Termination Codon -

Polypeptide chain synthesis is signalled by two initiation codon is AUG or GUG.

AUG codes methionine amino acid in eukaryotes and in prokaryotes AUG codes N-formyl methionine.

Some times GUG also functions as start codon it codes for valine amino acid normally but when it is present at starting position it codes for methionine amino acid.

Out of 64-codond 3-codons are stopping or Nonsense Codons or termination codon.

UAA (Ochre)

UAG (Amber)

UGA (Opal)

All these are Non-Sense codons or Stop codons.

So only 61 codons are sense codons which specify 20 amino acid.

WOBBLE HYPOTHESIS

It is propounded by CRICK.

Normally an anticodon recognises only one codon, but sometimes an anticodon recognises more than one codon.

This is known as Wobbling. Wobbling normally occurs for third nucleotide of codon.

For eg. anticodon AAG can recognise two codons i.e. UUU and UUC, both stands for phenyl alanine.

Types of m-RNA- m-RNA is of 2 types:-

1. Monocistronic- The mRNA, in which genetic signal is present for the formation of only one polypeptide chains eg. Eukaryotes.

2. Polycistronic- The m-RNA, in which genetic signal is present for the formation of more than one polypeptide chains eg. Prokaryotes.

Non sense codons are found in middle position in polycistronic m-RNA.

CENTRAL DOGMA

Central dogma was given by Crick.

The formation (production) of m-RNA from DNA and then synthesis of protein from it, is known as Central Dogma.

It means, it includes transcription and translation.

Reverse Transcription:-

The formation of DNA from RNA is known as Reverse - transcription. It was discovered by Temin and Baltimore in Rous - Sarcoma virus. So it is also called Teminism.

ss-RNA of Rous-Sarcoma virus (Retro virus) produces ds-DNA in host's cell with the help of enzyme reverse transcriptase (DNA-polymerase). This DNA is called

c-DNA (Complementary DNA).

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GYMNOSPERMS - EPHEDRA

2020-07-14T11:02:00.003+05:30

EPHEDRA

OCCURRENCE

Ephedra is an arid plant. It is found in Europe, Western and Central Asia and Central and South America. It is also found in arid areas of Pakistan. Ephedra is source of drug Ephedrine. It is used for the treatment of asthma, cough and hay-fever.

GENERAL STRUCTURE

The plant body of Ephedra is sporophyte. It is a bushy plant. Sometimes, it becomes a climber. It height is ranging from I to 25 feet. The body is divided into root stems and leaves.

1. Stem:

Stem and branches are slender and green in colour. It has longitudinal ridges and furrows. Stem becomes woody due to the limited secondary growth. In some cases underground rhizomes are also produced. The branches arise from axillary buds. Their arrangement gives the plants characteristic bushy and broom-like appearance, Apical meristem produces nodes and internodes. Meristematic tissues are also present at the base of each internode. Their activity further increases the length of stem. Sometimes, meristematic tissue becomes hard at the end of the growing season. Thus many branches fall These branches are replaced by axillary shoot. It gives bushy appearance to the plants. The ridges and furrows of the successive internodes alternate with each other.

2. Leaves:

The leaves are scale like. They are opposite and arranged in pairs. The leaves of each pair are joined with each other at their bases. It forms a small sheath around the stem.

3. Roots:

The primary root grows deep in the soil. It develops many secondary roots.

INTERNAL STRUCTURE OF STEM

The stem has ridges and furrows. So it has a wavy outline in transverse section.

1. Epidermis: Stem is covered by heavily cutinized epidermis. The epidermis has stomata. They are present in pits in the region of furrows. A group of sclerenchymatous cells is present below the epidermis in the region of each ridge.

2. Cortex: The cortex is wide. It is formed of parenchymatous cells. These cells have chloroplasts. Thus the cortex of stem is the main photosynthetic tissue of Ephedra. The cells of peripheral region of the cortex are loosely arranged and form palisade layer. The cells in the inner region of the cortex from the spongy tissue.

3. Stele: The central part of the stem is stele. It is surrounded by endodermis. Wide pith is present in the centre of the stele. The vascular bundles are collateral and endarch. They form a ring around the pith. The vascular bundles form a continuous ring due to narrowness of medullary rays. The xylem in Ephedra has true vessels like Angiosperms. Phloem consists of the usual phloem parenchyma and the sieve tubes. But they are without any companion cells. Secondary growth occurs due to the activity of cambium. It produces prominent annual rings.

4. Cork: Phellogen (cork cambium) is produced in the older stem. It forms periderm. The outer tissues of periderm are transformed into the bark.

REPRODUCTION

Ephedra is a dioecious plant. But some monoecious plants are also found.

MALE STROBILUS

Each male cone arises in the axil of a leaf. The male cones are small. They are 1-2 cm in length. Each cone has a central axis. This axis bears 2-12 pairs of thick bracts. Bracts are arranged in an opposite manner. These bracts are closely set on the axis. A single male microsporophyll is present in the axil of each bract. Two small scales are present at the base of each sporophyll. It has a single stamen. Stamen has short stalk (filament). This stalk bears two to six microsporangia (anthers) at the top. The anthers are united in a Each anther has two or three lobes. Each lobe has a pore at its tip for releases of microspore (pollen grain).

GERMINATION OF POLLEN GRAIN

Germination of pollen grain started before the release of pollen grain. Pollen grain divides into two cells. The smaller cell again divides to form two prothallial cells.

The larger cell becomes generative cell.

Generative cell divides into lower stalk and upper body cell. The upper cell divides to form two male gametes. Pollen grain is released at this stage. Two prothallial cells disintegrate just after the release of pollen grains. The pollen grains are dispersed by wind and insects. Pollen grains lodge on the ovule. It grows rapidly.

A pollen tube is produced. This tube carries the two male gametes, the tube nucleus and the nucleus of the stalk cell on its tip. Pollen tube passes through the neck cells and reaches the oosphere. It releases male gametes and one of them enters the oosphere. It fuses with the Ooshere.

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GYMNOSPERMS - CYCAS

2020-07-14T11:02:00.000+05:30

CYCAS

Systematic position

Division: Gymnospermae

Class :Cycadophyta

Order : Cycadales

Family Cycadaceae

Genus : Cycas

Occurrence

Cycas has a limited distribution. It is found in south East Asia, South China, Southern India, South Japan and Australia. The plants grow under xerophytic conditions.

Cycas is also cultivated as an ornamental plant in the gardens and parks. Cycas plants live upto hundred years or more.

Cycas is called a living fossil.

PLANT BODY

The plant body is a sporophyte. It is differentiated into root, stem and leaves.

1. Stem: Stem is unbranched. It is covered by thick, woody, persistent leaf bases. It makes the stem rough.

The apex of the stem is ensheathed by a group of brown scales. The lower of stem is covered by pinnate compound leaves. The growth of the stem is very slow. It produces a cluster of leaves each year. Older leaves fall of after two years.

2. Leaves and scales: The leaves are produced in the axils of the scales near the apex. Each leaf is composed of a petiole, rachis and lateral pinnae.

The young leaves show cincinnati vernation. Scales are also produced each year. Therefore, the clusters of green leaves and scales alternate with each other. Scales are also persistent.

Scales and leaf bases cover the surface of the old stem.

3. Roots: The primary root persists in Cycas. It becomes tuberous. Cycas produces coralloid roots.Coralloid roots are short tufts and dichotomously branched roots. These roots contain an endophytic alga in the inner part of their cortex. Sometimes, bacteria are also present in the cortex, Bacteria fix nitrogen,

INTERNAL STRUCTURE OF STEM

Stem is internally composed of outer epidermis, cortex and vascular bundles.

1. The cortex is very wide. It is composed of parenchymatous cells. Parenchymatous tissue of the cortex

contains numerous mucilaginous canals.

2. Primary vascular bundles are present inside the cortex. They surround the central pith. These bundles are collateral. The primary medullary rays present between the adjacent bundles.

3. Cambium is present in the form of a narrow strip in each vascular bundle.

The xylem is endarch. The strength of the stem is mainly due to the presence of woody leaf bases on the surface.

4. Phellogen is produced in the periphery of the cortex. It produces cork (peridium) under the persistent leaf bases.

INTERNAL STRUCTURE OF THE LEAF

Each pinna of leaf has typical bifacial structure in transverse section.

1. Surface of leaf is covered by a single layered epidermis. The cells of the upper epidermis are slightly thick walled. But those of the lower epidermis are thin walled. Sunken stomata are present in the lower epidermis. A layer of hypodermis is present beneath the upper epidermis.

2. The mesophyll is differentiated into an upper palisade layer a lower spongy parenchyma. The cells of the mesophyll are rich in chloroplasts.

3. Each pinna is supplied by a single undivided vein. Transfusion tissues are present around mid ribs. They cause lateral conduction in the leaf. The vascular bundles are surrounded by pericycle and endodermis.

INTERNAL STRUCTURE OF ROOT

1. Epidermis: the surface of root is covered by a single layered epidermis.

2. Cortex: It is many layered thick and made up of thin walled parenchymatous cells.

3. Endodermis: The innermost layer of cortex is called as endodermis

4. Pericycle: Thin walled cells around the vascular bundle.

5. Stele : The young primary root is Diarch (vascular bundles are arranged in two groups) but in older roots it become Polyarch (vascular bundles are arranged in many groups).

The xylem is exarch (Protoxylem is present towards the outside and metaxylem the towards centre. Phloem alternates with protoxylem elements.

6. Pith: is present in the centre of stele. It is composed thin walled parenchymatous cells. Root cap is present at the tip of root.

CORALLOID ROOTS

The structure is similar to that of primary root. In addition there is a conspicuous broad blue green zone in middle cortex.

This is the algal zone. It lies midway between the vascular bundle and epiblema. The cortical cells in this region get disorganized and are inhibited by blue green algae such as Nostoc and Anabaena.

In coralloid root the cortex is divided into three zones:Outer cortex, Middle cortex and Inner cortex.

The stele is diarch, triarch or tetrarch and

surrounded by an endodermis which is followed by pericycle.

REPRODUCTION

Cycas is dioecious. The male and female plants are separate. Sometimes, Cycas plants also reproduce vegetatively. It produces buds on the stem. These buds grow to form new plant.

MALE CONES

Male cones are produced on the male plants. Number of cones produced each year varies from one to many. Each male cone is fusiform in shape. Each cone has a central axis. It bears a number of spirally arranged microsporophyll. The microsporophylls are woody in texture.

They are wedge-shaped. The microsporangia (Pollen sacs) cover the lower surface of the microsporophylls.

The sporangia form sori. Each sorus has groups of two to six sporangia. Each sporophyll has several hundred sporangia. A large number of spores are produced in Cycas.

DEVELOPMENT OF THE SPORANGIUM

1. Each sporangium develops from a single sporangial initial. This cell arise form the hypodermis of the sporophyll. This sporangial initial divides into an outer primary wall cell and an inner primary sporogenous cell.

2. The primary wall cell divides to produce a several layered wall. The number of layers in the wall varies from four to eight. The primary sporogenous divides to produce a mass of cell called sporogenous tissue or archesporium.

3. The outermost layer of the sporogenous tissue forms the tapetum. Some of the sporogenous cells increase in size and become the spore mother cells. But other cells disintegrate with the tapetum and nourish the spore mother cells.

4. The spore mother cells divide meiotically to form four microspores (pollen grain).

5. The sporangia become boat-shaped.

Each mature microspore has two layered wall, the exine and intine.

Exine is thickest at one end and becomes thinner towards the opposite end. The spores start germination before its liberation from the microsporangium.

GERMINATION OF MICROSPORE (POLLEN GRAIN)

The microspore cut off lateral prothalial cell towards one side of the spore. The larger cell then cuts off a small generative cell adjacent to the prothalial cell. It itself becomes tube cell. The microspore is liberated at this stage Spores are dispersed by wind.

FEMALE CONE

The female cones are produced on the female plants. Female cones of Cycas are very large. It is formed of megasporophylls.

Megasporophylls are loosely arranged to form crowns.

Each megasporophyll is leaf like in form. The upper portion of the sporophyll is pinnate. Ovules (megasporangium) are arranged in two rows in the basal half of the sporophyll. The whole sporophyll and young ovules are covered by a dense mat of hairs. The ovules loose this hairy covering on maturity.

Each ovule is covered by a single massive integument. It has a narrow micropyle at the tip. Integument projects around the micropyle to form a small beak. Nucellus projects into the micropyle. But later the nucellar cells in this region disorganize to form a small cavity called pollen chamber. It plays an important part in early stages of fertilization. One of the nucellar cells increases in size and becomes megaspore mother cell. It undergoes meiosis to form four megaspores. Three megaspores degenerate. Only one becomes functional megaspore.

FEMALE GAMETOPHYTE

The megaspore (embryo sac) enlarges in size. Its nucleus undergoes many nuclear divisions. Thus several nuclei are formed in the megaspore. These nuclei occupy the peripheral region of the cytoplasm in the megaspore. Then the nuclei are surrounded by cell wall. Thus the megaspore becomes multicellular. It gives rise to the female gametophyte. The cells of the gametophyte develop numerous starch grains. The original megaspore wall persists around the prothalial tissue.

1. Three cells enlarge and function as archegonial initials at the micropylar end of the prothallus. Each archegonial initial divides into tipper neck cell and a lower central cell.

2. Neck cell divides to produce neck of the archegonium. The central cell enlarges. It forms the oosphere cell.

The whole interior of oosphere cell is filled with dense granular protoplasm. The wall of the oosphere cell is thick. The cells of the prothallus which surround the oosphere cell form the jacket layer.

3. The nucleus of the oosphere cell divides in two. The small ventral canal nucleus disintegrates. The other large nucleus becomes oosphere nucleus. It increases very much in size. The archegonium is now ready for fertilization.

4. The archegonial necks open in archegonial chamber Original megaspore wall ruptures above the archegonial chamber. The cellar tissue below the pollen chamber disintegrates. A passage is formed between the archegonial chamber and the pollen chamber.

5. A drop of mucilaginous fluid oozes out of the micropyle. The pollen grains are lodged on the micropyle. Pollen grain is trapped this mucilage and the pollen grain moves into the pollen chamber.

MALE GAMETOPHYTE

The pollen grain resumes its development after pollination. Generative cell divides into a stalk cell and a body cell. Both these cells represent an antheridium. A pollen tube grows out of the pollen grain.

It penetrates the nucellus. The pollen grain becomes dormant at this stage. This period lasts for about four months. After that the pollen grain resumes its activity,

Two blepharoplasts appear on the two sides of the nucleus. These blepharoplasts develop cilia. The body cell then divides into two antherozoids. The antherozoid develops thousands of cilia.

The nucleus of each antherozoid enlarged and completely fills the whole of the antherozoid. Antherozoids remain motile for several hours.

Pollen grain reaches the archegonial chamber by pollination. The wall of the pollen grain protrudes towards the archegonial chamber. The pollen grain bursts and release antherozoids into the archegonial chamber. Antherozoid enters the oosphere. Male nucleus unites with the oosphere nucleus. Fertilized oosphere secretes a thick wall and becomes the oospore. Oospore develops embryo.

Development of Embryo and Seed

Oospore divides into 200-300 cells. The cells of the central region disorganize to produce a cavity. This cavity is surrounded by two to three layers of cells. The cells in the lower region elongate very much to form suspensor. The cells at the tip of the suspensor develop into the embryo. The elongating suspensor pushes the developing embryo deep into the prothalial tissue. These tissues provide nutrition to the developing embryo. Whole of the nucellus is consumed during the development of the embryo. Some prothalial tissue persists in the seed and form endosperm. A thick pad of tissue develops near the micropylar end. It functions as coleorhiza. Coleorhiza protects the radical of the embryo. Ovule is transformed into the seed. Embryo has two cotyledons. They occupy the whole seed. The seeds germinate immediately Cotyledons remain within the seed on germination. It absorbs nutrients for the developing embryo.

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GYMNOSPERMS - PINUS

2020-07-14T11:01:00.002+05:30

Pinus

Occurrence

The genus Pinus has about 90 species. It has world wide in distribution. They are mostly present in the temperate regions.

Four species of pinus are found in Pakistan: Pinus wallichiana; Pinus halepensis; Pinus roxburghii; Pinus gerardiana.

GENERAL STRUCTURE

The plant body is sporophyte. It is an evergreen tall tree. Plant body is divided into root, stem and leaves Stem: The main trunk is unbranched. It has branches confined to the apical region. These branches form a characteristic canopy. It is covered with a scaly bark. Its branches

are dimorphic.

(a)Branches of unlimited growth: The main branches have an unlimited growth.

They bear only scale leaves.

(b)Branches of limited growth: Branches of limited growth or dwarf branches are

produced in the axil of the scale leaves on the main branches. These are about

1-2cm long. These are covered by one or two scale leaves. These branches also bear foliage leaves. A dwarf shoot with its foliage leaves is called spur.

Leaf:

Pinus has two types of leaves: scale leaves and foliage leaves.

(a) Scale leaves: The scale leaves are small, membranous and brownish in colour. These are protective in function.

They are present on the main and dwarf shoots.

(b)Foliage leaves: The foliage leaves are

green and needle-like. They are found only on the dwarf shoots forming the spur.

Roots: Pinus has a well developed tap root. It remains short and grows on hard ground or rocks. The lateral roots are well developed. These roots spread over a large area. Young roots are infested with a fungus to form mycorrhizae.

Internal Structure of the Stem

Stem is differentiated internally into epidermis, cortex, vascular tissue and central pith

1. Epidermis:

The surface is covered by an epidermis. It consists of a single layer of cells. Outer walls of these cells are highly cutinized. Below the epidermis is a hypodermis which is formed of layers of lignified cells.

2. Cortex:

The cortex is formed of parenchymatous cells. Cortex has a large number of resin canals. Each resin canal is surrounded by a layer of resin secreting glandular epithelial cells.

3. Vascular bundle:

The vascular bundles are conjoint, collateral and open. They form a ring around the pith. In each vascular bundle the xylem is towards the inner side and phloem towards the outerside.

A narrow strip of cambium is present between xylem and phloem. Pericycle is present outer to the ring of vascular bundles. A single layered thin walled endodermis is present outside the pericycle. Secondary growth takes place by the activity of cambium ring. There are distinct annual growth rings in the wood.

4. Bark:

Phellogen originates in the deeper layers of the cortex. It is present in the form of strips. It produces characteristic scaly bark.

INTERNAL STRUCTURE OF LEAF

A transverse section shows following internal parts of the leaf:

1. Epidermis:

Leaf is covered by thick walled epidermis. Epidermis is covered by a thick layer of cuticle. Sunken stomata are present below the general surface. Two or three layered hypodermis is present underneath the epidermis. Hypodermis is compose of sclerenchymatous tissues. This hypodermis is the main strengthening tissue of the leaf.

2. Mesophyll:

The mesophyll of the leaf is parenchymatous. It is not differentiated into palisade and spongy parenchyma. Resin canals are present below the hypodermis. Each resin canal is lined by a layer of small epithelial cells. Each leaf is supplied by two branched veins.

3. Endodermis and vascular tissues:

Endodermis is present outside the pericycle. Pericycle surrounds the xylem Pericycle is formed of parenchymatous cells. Its cells adjacent to the phloem are called aluminous cells. The cells adjacent to the xylem are called tracheid cells.

These specialized cells form the transfusion tissue. They help in the lateral flow of nutrients.

INTERNAL STRUCTURE OF ROOT

young root is di- to pentarch. Xylem bundles are Y-shaped. Siwtil parenchymatous pith may be present or absent in the centre. The ring of vascular

bundles is surrounded by two to three layered pericycle. Pericycle is surrounded by endodermis. The cortex consists of a few layers of thin walled parenchymatous cells. Root hairs may be present or absent. Roots may be covered by mycorrhizal fungus.

REPRODUCTION

Pinus is monoecious. Plant develops both

male and female strobili on the same

plant. The strobili are monosporous. There is no vegetative reproduction in Pinus.

Male Cone

The male cones are much smaller. They are produced in clusters near the tip of

the long shoots. The male cones are

produced in the spring. Each male cone

has a central axis. It bears a number of

spirally arranged microsporophylls or stamens. Each microsporophyll has sac like microsporangia (pollen sacs) on the ventral side. Each microsporangium produces a large number of microspores (pollen grains). The wall of each microspore (pollen grain) consists of an inner intine and an outer exine. It has balloon like wings. The wings help in the dispersal of spores by wind.

DEVELOPMENT OF MICROSPORANGIUM (STAMEN)

1. A number of hypodermal cells act

sporangial initials. The sporangial initials divides to form outer wall initials and the inner archesporial initial.

2. The wall initials divide to form a many layered wall of the sporangium. The archesporial initials also increase in number by the repeated divisions. The

peripheral cells of the archesporium form the tapetum.

3. Some of the archesporial cells are transformed into microspore mother cells. The remaining archesporial cells and the tapetal layer provide nourishment to the developing microspore mother cells.

4. The microspore mother cell divides by meiosis to form four microspores or pollen grains. The exine of spore forms wings. The pollen grain divides in to smaller and larger cells.

The smaller cell again divides to form two small prothallial cells.

The larger cell becomes antheridial cell. The sporangium splits and microspores are released from the microsporangia at this stage.

MALE PROTHALLUS

1. The pollen grain has two prothalial cells and an antheridial cell. These

cells soon disintegrate. Further development of the pollen grain takes place at the surface of the nucellus.

2. The antheridial cell cut off second prothalial cells.

3. It also cut off generative cell adjacent to the prothallial cells.

4. The remaining large cell is known as the tube cell. Its nucleus is called tube nucleus. Tube nucleus controls the growth of the pollen tube.

5. Exine of the pollen grain ruptures.

6. Intine grows out to form the pollen tube. It grows through the nucellus. But its activity stops till spring.

7. Female cone enlarges very much in size after pollination Outer ends of the ovuliferous scales increase very much. They meet each other to close the gaps in between them. The cone is covered with a lot of resinous secretions.

Female cone

Female cones are produced in the axils of the scale leaves. The production of

female cones is initiated in the winter.

These become ready for pollination during the following spring. Each young female cone has a central axis. It bears spirally arranged scales. The scales are of two types. Some are thin membranous and are directly attached to the central axis. They are called bract scales. Woody ovuliferous scales are present on the ventral surface of each bract scale. The broader end of the ovuliferous scale has projection called the umbo. Each ovuliferous scale bears two ovules. They are situated side by side on upper side.

Each ovule (megasporangium) has a mass of nucellar tissue. They are surrounded by a single integument. The micropylar end

of the ovule is directed towards the

central axis. A single megaspore mother cell is differentiated in the nucellus near

the micropylar end. This megaspore mother cell undergoes meiosis to form four megaspores. Only the lower most megaspore remains functional. The others disintegrate Functional megaspore (embryo sac) increases in size. It occupied the major part of the nucellus. Pollination takes place at this stage.

FEMALE PROTHALLUS

The megaspore divides many times to form female prothallus. Megaspore wall encloses the female prothallus. Three archegonia are produced towards the micropylar end.

Each archegonium develops from a single prothalial cell. Archegonia consist of a large venter and a short neck. The oosphere or egg is very large. It is bounded by the prothallial cells.

There is a small ventral canal cell below the neck.

The neck is without any neck canal cells.

The prothalial tissue enlarges very much in size. It crushes all nucellar tissue except a small amount near the micropylar end.

POLLINATION

Each ovule secretes a mucilaginous drop at the micropylar end. A gap is produced between the ends of the ovuliferous scales. It forms a passage for the entry of pollen grains. Wind carried pollen grains. The mucilage drop entangles the pollen grain. Pollen grain is carried through the micropyle to the surface of the nucellus.

Formation of Embryo and Seed

Diploid nucleus divides thrice to form eight cells. The lower four cells becomes proembryonal cells. The upper four nuclei are separated by incomplete cell walls. Four proembryonal divides to produce three tiers of cells:

1. Embryonal cell: The cells of the lower tier become embryonic cells. The four embryonic cells separate from each other

Each develops into a separate embryo

independently. Each embryonal cell forms secondary suspensor cells. The formation of more than one embryo from a single fertilized oosphere is called polyembryony. Only one embryo reaches maturity. The rest are aborted.

1. Suspensor cells: The cells of the middle

tier become suspensor cells. Suspensor cells elongate very much. it pushes the developing embryos into the prothaliat

tissue for nutrition.

2. Rosette cells: The cells of the upper most tiers are called the rosette cells. These cells do not take part in the development of the embryo.

A fully developed embryo is in the forma short straight axis. Its radicle is present towards the micropylar end. Plumule is present towards the inner side. Plumule is surrounded by ten cotyledons. The unutilized prothalial tissue forms the endosperm. The persistent nucellus tissues near the micropylar end form the perisperm.

The integument becomes hard testa. Some part of the ovuliferous scale fuses with the developing seed. It makes a large wing for dispersal of seed. The axis of the female cone rapidly increases. It produces gaps in ovuliferous scales. The cone becomes woody for the dispersal of seeds.

GERMINATION OF SEED

The radicle grows out. it splits the testa at the micropylar end. This radicle grows down into the soil and forms the primary root. The hypocotyl elongates to form a loop. Then it becomes straight, It carries with it the plumule and the cotyledons. The testa is also carried up with the cotyledons.

GYMNOSPERMS

2020-07-13T12:38:00.001+05:30

GYMNOSPERMS

“Gymnosperms are a group of plants that produce seeds not enclosed within the ovary or fruit. “

The word “Gymnosperm” comes from the Greek words “gymnos”(naked) and “sperma”(seed), hence known as “Naked seeds.” Gymnosperms are the seed-producing plants, but unlike angiosperms, they produce seeds without fruits. These plants develop on the surface of scales or leaves, or at the end of stalks forming a cone-like structure.

Gymnosperms belong to kingdom plantae and sub-kingdom ‘Embryophyta’. The fossil evidence suggested that they originated during the Paleozoic era, about 390 million years ago.

Characteristics of Gymnosperms

Following are the important characteristics of gymnosperms:

They do not produce flowers.
Seeds are not formed inside a fruit. They are naked.
They are found in colder regions where snowfall occurs.
They develop needle-like leaves.
They are perennial or woody, forming trees or bushes.
They are not differentiated into ovary, style and stigma.
Since stigma is absent, they are pollinated directly by the wind.
The male gametophytes produce two gametes, but only one of them is functional.
They form cones with reproductive structures.
The seeds contain endosperm that stores food for the growth and development of the plant.
These plants have vascular tissues which help in transportation of nutrients and water.
Xylem does not have vessels, and the phloem has no companion cells and sieve tubes.

Classification:-

Gymnosperms consist of four main phyla: the Coniferophyta, Cycadophyta, Gingkophyta and Gnetophyta.
Conifers are the dominant plant of the gymnosperms, having needle-like leaves and living in areas where the weather is cold and dry.
Cycads live in warm climates, have large, compound leaves, and are unusual in that they are pollinated by beetles rather than wind.
Ginkgo biloba is the only remaining species of the Gingkophyta and is usually resistant to pollution.
Gnetophytes are the gymnosperms believed to be most closely related to the angiosperms because of the presence of vessel elements within their stems.

Evolution of Gymnosperms

Gymnosperms are believed to have evolved from the paleozoic to the mesozoic eras.
There are 3 groups of extinct plants that played important roles in the evolution of modern gymnosperms
They are progymnosperms, aneurophytales, and a groups of primitive gymnosperms: archaeopteridales.
In middle devonian period, progymnosperms arose from the trimerophytes which were extant until the lower carboniferous period.
The cordaitales were trees and shrubs during the carboniferous and permian periods both in swamp and dry land which had slender leaves.
They also had vascular cambium tissues and ovulate cones.
The cordaitales apparently gave rise to the phylum ginkgophyta, which persists to present day and others which have relatives to cycadophyta, gnetophyta and coniferophyta.
Voltziales is an extinct order of trees that gave rise species related to modern conifers.
Pteridosperms were the first seed plants, with integuments protecting ovules to various degrees
Another extinct group of Pteridosperms are the Bennettitales, which resemble present cycads.
The Archaeopteridales may have given rise to the Cordaitales and the Voltziales.
The Archaeopteridales arose from the Aneurophytales.
Progymnosperms gave rise to aneurophytales that gave rise to pteridosperms and archaepteridales.
Aneurophytales were homosporous, producing many identical spores and had three dimensional branching
Unlike the progymnosperms, the pteridosperms produced seeds appearing in the late devonian period.

Diversity of Gymnosperms

Modern gymnosperms are classified into four phyla. The first three (the Coniferophyta, Cycadophyta, and Gingkophyta) are similar in their production of secondary cambium (cells that generate the vascular system of the trunk or stem and are partially specialized for water transportation) and their pattern of seed development. However, these three phyla are not closely related phylogenetically to each other. The fourth phylum (the Gnetophyta) are considered the closest group to angiosperms because they produce true xylem tissue.

Coniferophyta (conifers - pine, spruce, redwood)

These are the most commonly known species among the gymnosperms family.They are evergreen hence they do not shed their leaves in the winter. These are mainly characterised by male and female cones which form needle-like structures. Coniferous trees are usually found in temperate zones where the average temperature is 10℃. Giant sequoia, pines, cedar and redwood are one of the many examples of Conifers.

Cycadophyta ( cycads)

Cycads are dioecious (meaning: individual plants are either all male or female). Cycads are seed-bearing plants where the majority of the members are now extinct. They had flourished during the Jurassic and late Triassic era. Nowadays, the plants are considered as relics from the past.

These plants usually have large compound leaves, thick trunks and small leaflets which are attached to a single central stem. They range in height anywhere between a few centimetres to several meters.

Cycads are usually found in the tropics and subtropics. Some members have adapted to dry arid conditions, and some also have adapted to oxygen-poor swampy environments.

Ginkgophyta ( ginko bolona)

Another class of Gymnosperms, Ginkgophyta, has only one living species. All other members of this class are now extinct.

The Ginkgo trees are characterised by their large size and their fan-like leaves. Also, Ginkgo trees have a large number of applications ranging from medicine to cooking. Ginkgo leaves are ingested as a remedy for memory-related disorders like Alzheimer’s.

Ginkgo trees are also very resistant to pollution, and they are resilient against diseases and insect infestations. In fact, they are so resilient that after the nuclear bombs fell on Hiroshima, six Ginkgo trees were the only living things to survive within a kilometre or two of the blast radius.

Gnetophyta (Ephedra)

Just like any other member of gymnosperms, Gnetophytes are also relics from the past. Today, only three members of this genus exist.

Gnetophytes usually consist of tropical plants, trees, and shrubs. They are characterised by flowery leaves that have a soft coating. This coating reveals an ancestral connection with the angiosperms.

Gnetophytes differ from other members of this class as they possess vessel elements in their xylem.

Origin of seed habit

Seed habit is known to be the most successful method in the evolution of sexual reproduction in plants.

The first seed was found to be Gymnosperms that was first appeared during the Devonian period in the time line. Soon it started appear on the land plants. As like the Gymnosperms seed habit has been found to be emerged during the Devonian period and now dominating every land plants.

Seeds are specialized structure resultant of gamete fusion. It has several advantages including late rejuvenation, safety besides others.

This means that primitive plants do not have the capacity to produce seed. Hence evolution has taken a direction and all advanced plant now bear seeds.

So seed habit is the ability to produce seed like propagules. This has many intermediary steps including heterospory (which has started in advanced pteridophytes), integument development (started in progymnosperms) and development of seed coat (started in gymnosperms).

GEOLOGICAL TIME SCALE

FOSSILISATION AND FOSSIL GYMNOSPERMS

Gymnosperms means (Greek gymnos = naked; sperma = seed. i.e., the plants with naked seeds. Gymnosperms are phanerogams or spermatophytes without ovary and fruit. Their seeds or ovules are naked or exposed, without a fruit wall. They are therefore considered as fruitless flowering plants and are referred to as "Phanerogams without ovary. Gymnosperm seeds develop either on the surface of scale or leaf-like appendages of cones, or at the end of short stalks (Ginkgo). The word gymnosperm is coined by Theophrastus in 300 B.C. and called them "plants with naked seeds". Palaeobotany is the study of plant fossils preserved in rocks.

The word "Fossil" has been defined as "any evidence of prehistoric life. The first mention of a fossil plant was made by a German scholar Albertus Magnus in the thirteenth century.

Fossils (from Latin fossus, literally having been dug up) are the preserved remains or traces of animals, plants, and other organisms from the remote past. The totality of fossils, both discovered and undiscovered, and their placement in fossiliferous (fossil-containing) rock formations and sedimentary layers (strata) is known as the fossil record.

The process of preservation of living beings or their parts in form of fossils is called fossilization. 'Birbal Sahni' is known as father of Indian Palaeobotany.

(His main contribution is Pentoxifylline of Jurassic gymnosperms from Raj Mahal hills in Bihar).

BirbalSahni Institute of Paleobotany is situated on Trans-Gomati River bank, Lucknow (India).

The essential conditions for fossilization is that whole organism be buried alive soon after death without decay by bacteria, fungi etc. This is the reason why only small number of plants get fossilized.

GYMNOSPERMS LIFE CYCLE

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KAPPA PARTICLES IN PARAMECIUM

2020-07-12T09:04:00.000+05:30

KAPPA PARTICLES IN PARAMECIUM

Sonneborn in 1938 discovered certain strains in paramecium which showed a killer trait due to presence of a cytoplasmic factor called Kappa. The killer strain can destroy the sensitivite strains growing in culture which do not have Kappa by liberating a toxic substance paramecin. Killer strains are not killed by their own paramecin.

Paramecin has two kinds of nuclei, a small micronucleus and a very large macronucleus which is highly polyploid and irregular in shape and behaviour during cell division. Only the micronucleus behave according to mendelian principles. Paramecium has three modes of reproduction. The first is a simple mitotic division called binary fission. The second method is conjugation here two protozoans divide meiotically to form four micronuclei in each cell; out of these, three nuclei degenerates and only one remains which divides by mitosis to produce two genetically identical haploid nuclei in each paramecium. During conjugation only one of the two haploid nuclei is exchanged through a cytoplasmic bridge formed between the two ciliates. The cells than separates as two Exconjugants.

The third method of reproduction is called autogamy. Here a single paramecium divides meiotically and by the same process that occurs in conjugation, two identical haploid nuclei are formed which fuse to form a diploid organism. As there was no genetic exchange, the diploid paramecium is Homozygous.

One noteworthy feature of the sensitive strains is that they are not killed by paramecin while they are in the process of conjugation. This has an advantage because it allows the investigator to perform cross between the killer and sensitive strains. The two strains can be distinguished morphologically as killers have granular cytoplasm and sensitive are clear. When a cross is made between killer and a sensitive paramecium (each made homozygous by autogamy), there is exchange of genetic material through conjugation. This is followed by separation of the two genetically identical Exconjugants. It is found that killer Exconjugants produce only killer paramecia and the sensitivite traits are not controlled by mendelian genes.

If the heterozygous (KK) killer Exconjugants is inbred to another heterozygous killer, it produce three-Quarter killer (1KK and 2kk) and one quarter sensitivites (kk). But if the sensitivite Exconjugant (Kk) is crossed to another heterozygous sensitivite, it results in all sensitivite progeny even through their genotypes are in the ratio of 1KK:2Kk:1kk. The results suggest non-chromosomal inheritance of killer trait.

The final proof regarding inheritance of killer trait was obtained by modifying the experiment in the following way. The cross between killer and sensitivite was prolonged, allowing enough time for exchange of cytoplasm to take place. In this way, some of the Kappa particles could move from killer into the sensitivite strain. Att the resulting progeny of such a cross consisted of killers thus confirming the cytoplasmic inheritance of Kappa particles.

The below experiment can be performed in such a way that paramecia divide very rapidly by controlling the nutrient conditions. Under such conditions, a Homozygous killer strains (kk) containing Kappa particles can produce a few individuals that are sensitivite and without Kappa particles. The explanation is that Kappa particles cannot multiply as rapidly as the cells and become fewer in number in comparison with the number of paramecium cells. Due to their reduced number Kappa particles are not passed on to some members of the progeny at all.

It was found that although Kappa particles are transmitted cytoplasmically, yet they require a dominant K gene for maintenance. The K gene cannot initiate the presence of Kappa particles about 0.2 micron in diameter and have ability to reproduce independent of the nucleus. They have their own DNA, can multiply and produce the substance paramecin.

Fig : Conjugation in Paramecium demonstrating extranuclear inheritance of Killer trait

Fig : exchange of nuclear genes

Fig : exchange of both nuclear genes and cytoplasm

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CYTOPLASMIC INHERITANCE

2020-07-11T09:28:00.000+05:30

CYTOPLASMIC INHERITANCE

Discovered by Correns.
Inheritance of characters which are controlled by cytogene or cytoplasm is called cytoplasmic inheritance.

There are 3 types of cytoplasmic inheritance:-
1. Maternal inheritance.
2. Organellar Inheritance.
3. Inheritance involving infectious particles.

Genes which are present in cytoplasm called 'cytogene' or 'plasmagene' or 'extra nuclear gene'.
Total cytogene present in cytoplasm is called 'plasmon'.
A gene which is located in the nucleus is called 'karyogene'.
Inheritance of cytogene in organisms occurs only through the female. Because female gamete has karyoplasm, simultaneously it has cytogene because of more cytoplasm.
The male gamete of higher plant is called male nucleus. It has very minute [equivelent to nil] cytoplasm. So male gamete only inherited karyogene.
Thus, inheritance of cytogene occurs only through female. (Also called maternal inheritance).
If there is a reciprocal cross in this condition, then results may be affected.
Cytoplasmic inheritance involving essential organelles like, Chroloplast and mitochondria called as organellar inheritance.

Example of Organellar Inheritance: (true examples of cytoplasmic inheritance).

1. plastid inheritance in Mirabilia jalapa- cytoplasmic inheritance was first discovered by Correns in Mirabilia jalapa. In Mirabilis jalapa branch (leaf) colour is decided by type of plastid present in leaf cells. So it is an example of cytoplasmic inheritance.

2. Male sterility in maize plant- Gene of male sterelity is present in mitochondria. If normal male plant crossed with a female plant which has genes of male sterility then all the generation of male become sterile because a particular gene was present with female which has inherited by female.

3. Albinism in plant- Gene of albinism found in chloroplast. Gene of albinism in maize is lethal.

CYTOPLASMIC INHERITANCE- maternal effects in Limnea shell coiling.

Shell Coiling In fresh water Snail (Lymnaea peregra).

In snails (gastropods)the shell is spirally coiled.
Snails exhibit two types of coiling of their shell:

fig : A sinistral (left) and a dextral (right) shell

Shell coiled to right is dextral.
Shell coiled to left is sinistral.
This direction of coiling is genetically controlled. The gene for dextral coiling is dominant 'D' and for sinistral coiling is recessive 'd'. So, that the dextral is 'DD' and sinistral is 'dd'.

Boycott and Driver(1923) showed that the character of coiling is determined by the gene of the mother and not by the individual's own gene.

Shell coiling in the hermaphroditic snail Limnaea peregra may be right-handed (dextral) or left-handed (sinistral).
The coiling depends on the genotype of egg donor parent, regardless of the phenotype of that parent.
Shell coiling in snail is controlled by D (dextral) and d (sinistral) alleles. The genotype of progeny depends on genotype of mother. If mother is "DD", the Dd offspring are dextral but the "Dd" progeny of "dd" mother has sinistral coiling.

CARBON DIOXIDE SENSITIVITY IN DROSOPHILA

Carbon dioxide is used as an anaesthetic for Drosophila and normal flies can with stand high CO2 concentrations without any adverse effect. But some strains of Drosophila are sensitive to CO2 so that within a few minutes they become paralysed and die. Reciprocal crosses between CO2 sensitive and resistance flies gave differing results.

If a CO2 sensitive female is crossed with a normal (resistant) male, almost all the offspring are sensitive. But the reciprocal cross of a sensitive male with a normal female yields almost all normal offspring indicating extra nuclear inheritance of this trait.

There is mostly maternal transmission although some transmission through sperm may also take place. Moreover, normal flies can be induced to become sensitive by injecting a cell free filtrate of haemolyph obtain from sensitive flies.

The casual agent for CO2 sensitivity has been observed in electron micrographs to be a virus-like particle called sigma. In flies that have been induced to become sensitive, sigma enters the egg cytoplasm and is transmitted maternally.

In the naturally occuring sensitive strains, sigma particles become incorporated into the nucleus (without associating with a specific chromosome) of the egg or some of the sperms and are transmitted in a non-mendelian manner. They are self-reproducing independent bodies and can mutate.

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MULTIPLE FACTORS (Blood Group)

2020-07-10T15:54:00.000+05:30

Blood

Blood is a fluid connective tissue. The quantity of blood in the humans body is 7% of total weight. This is a dissolution of base whose pH value 7.4, there is an average of 5-6 litres of blood in the human body. Female contains half a litre of blood less in comparison to male.

History

At the beginning of the 20th century an Austrian scientist, Karl Landsteiner, noted that the RBCs of some individuals were agglutinated by the serum from other individuals. He made a note of the patterns of agglutination and showed that blood could be divided into groups. This marked the discovery of the first blood group system, ABO, and earned Landsteiner a Nobel Prize.

Landsteiner explained that the reactions between the RBCs and serum were related to the presence of markers (antigens) on the RBCs and antibodies in the serum. Agglutination occurred when the RBC antigens were bound by the antibodies in the serum. He called the antigens A and B, and depending upon which antigen the RBC expressed, blood either belonged to blood group A or blood group B. A third blood group contained RBCs that reacted as if they lacked the properties of A and B, and this group was later called "O" after the German word "Ohne", which means "without". The following year the fourth blood group, AB, was added to the ABO blood group system. These RBCs expressed both A and B antigens.

In 1910, scientists proved that the RBCs antigens were inherited, and that the A and B antigens were inherited codominantly over O. There was initially some confusion over how a person's blood type was determined, but the puzzle was solved in 1924 by Bernstein's "three allele model".

The ABO blood group antigens are encoded by one genetic locus, the ABO locus, which has three alternative (allelic) forms—A, B, and O. A child receives one of the three alleles from each parent, giving rise to six possible genotypes and four possible blood types (phenotypes).

Blood contains:-

1. Plasma

2. Blood cells/corpuscles

a. Red blood cells carry oxygen b.white blood cells fight infection c.Platelet stop bleeding in injuries

A. Plasma

Water-90%
Protein, salts, glucose-10%
This is the liquid part of blood 55% of the blood plasma.
It's 90% part is water, 7% Proteins, 0.9% salts and 0.1% is glucose, remaining substances are in a very low quantity.

The function of plasma:-

Transportation of digested food, hormones, excretory products etc. From the body take place through the plasma.

SERUM- when fibrinogen and protein is extracted out of plasma the remaining plasma is called 'Serum'.

B. Blood corpuscles

This is the remaining 45% part of the blood, this is divided into three parts-

1. Red blood corpuscles (RBCs)

2. White blood corpuscles (WBCs)

3. Blood platelets

A.Red blood corpuscles (RBCs) or Erythrocytes-

Red blood corpuscles of a mammal is biconcave.
There is no nucleus in it, exception camel and lama.
RBCs formed in Bone marrow (At the embryonic stage it's formation takes place in the liver).
It's life spanis from 90-120 days.
It's distraction takes place in the liver and spleen, therefore, the liver is called Grave of RBCs.
It's contains Haemoglobin [haemo- iron(Fe2+) and globin- protein (combine to O2 and CO2), in which haemo iron containing compound is found and due to this the colour of iron is red.
Globin is a proteinous compound which is extremely capable of combining with oxygen and carbon dioxide.
The Iron compound found in haemoglobin in haemin.
Anaemia disease is caused due to deficiency of haemoglobin.
At the time of sleeping RBCs reduce by 5% and people who are at the height of 4200 meters RBCs increases by 30% in them.

B. White Blood corpuscles (WBCs) or Leucocytes-

White blood corpuscles in shape and constitution this is similar to an amoeba.
It's formation takes place in Bone marrow, Lymph node and sometimes in liver and spleen.
It's life span is 3-5 days.
It's formation of antibodies which protect from disease/infection.
Nucleus present in it WBCs.

C. Blood platelets or Thrombocytes-

It is found only in the blood of human.
It's formation takes place in Bone marrow.
It dies in blood.
Its main function is to help in clotting of blood.
If the quantity of WBCs is less in blood it causes Dengue.

Function of BLOOD:-

1. To control the temperature of the body and to protect the body from disease.

2. Clotting of blood.

3. Transportation of O2, CO2, Digested food, conduction of hormones etc.

4. To help in establishing coordination among different parts.

MULTIPLE FACTORS

INHERITANCE OF BLOOD GROUP AND RHESUS FACTOR:-

EXAMPLES:-

NOTE:- There are four types blood group and this system is called ABO system. ABO blood group system are determined by three alleles.

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TRANSCRIPTION AND SPLIT GENE

2020-07-10T10:04:00.005+05:30

TRANSCRIPTION

Formation of RNA over DNA template is coled transcription. Out of two strand of DNA only one strand participates in transcription and called "Antisense strand" or "Template strand".

If both strands act as a template during transcription they would code for RNA molecule with different sequence and if they code for protein the sequence of aminoacid in these protein would be different and another reason that if the two RNA molecule produced they would be complementary to each other and form a ds RNA which prevent translation of RNA.

A gene is defined as the functional unit of inheritance. It is difficulty to literally define a gene in terms of DNA sequence, because the DNA sequence coding for tRNA or rRNA molecule is also define a gene (But information of protein is present on the DNA segment which code mRNA).

The segment of DNA which contains signal for the synthesis of one polypeptide is known as "Cistron".
RNA polymerase enzyme is involved in transcription. In eukaryotes there are three types of RNA polymerases.
RNA polymerase-l For 28s RNA, 18s RNA, 5.8 rRNA synthesis.
RNA polymerase-Il for hn-RNA synthesis.(Precursor of mRNA).
RNA polymerase-III for t-RNA, 5s rRNA, SnRNA synthesis.
Prokaryotes have only one type of RNA polymerase which synthesizes all types of RNAs.
RNA polymerase (Core enzyme) of E. Coli has five polypeptide chains β, β', α, α and ω. polypeptide chain is also known as a σ factor (sigma factor).

Core enzyme + Sigma factor = RNA polymerase.

(β, β', α, α and ω) (σ)

A transcription unit in DNA is defined primarily by the regions in the DNA:-

1. A promoter.

2. The structural gene

3. Terminator

Following steps are present in transcription

1. INITIATION :

DNA has a "Promoter site" where RNA polymerase binds and a "Terminator site" Where transcription stops.
Sigma factor (σ) recognises the promoter site of DNA.
With the help of sigma factor RNA polymerase enzyme attached to a specific site of DNA calked “Promoter site".
In prokaryotes before the 10 N2, base from "starting point" a sequence of 6 base pairs (TATAAT) is present on DNA, which is called "Pribnow box".
In eukaryotes before the 20 N2, base from "Starting point" a sequence of 7 base pairs (TATAAAA) or (TATATA) is present on DNA which is called "TATA box" or "Hogness box".
At start point RNA polymerase enzyme breaks H-bonds between two DNA strands and separates them.
One of them strand takes part in transcription. Transcription proceeds in 5'-3' direction.
Ribonucleoside triphosphate come to lie opposite complementary nitrogen bases of anti sense strand.
These Ribonucleotides present in the form of triphosphate ATP, GTP, UTP and CTP. When they are used in transcription, pyrophosphatase hydrolyse two phosphates from each activated nucleotide. This releases energy.
This energy is used in the process of transcription.

2. ELONGATION :

RNA polymerase enzyme establishes phosphodiester bond between adjacent ribonucleotides.
Sigma factor separates and RNA polymerase moves along the anti sense strand till it reaches terminator site.

3. TERMINATION :

When RNA polymerase enzyme reaches at terminator site, it separates from DNA templet.
In most cases RNA polymerase enzyme can recognise the "Terminator site" and stop the synthesis of RNA chain, but in prokaryotes, it recognises the terminator site with the help of Rho factor ( ρ factor).
Rho (ρ) factor is a specific protein which helps RNA polymerase enzyme to recognise the terminator site.

fig : Process of Transcription in Prokaryotes (Bacteria)

SPLIT GENE

Discovered by Phillip A. sharp and Richard J. Roberts. They were awarded Nobel Prize in 1993. Gene which contains non functional part along with functional part is known as Split gene. Non functional part is called intron and functional part is called exon.

By transcription split gene produces RNA which contains coding and non coding sequence and called hn RNA (Hetero genous nuclear RNA). This hn RNA is unstable. Now 7 methyl guanosine is added to its 5' end, and a cap like structure is formed. It is called capping and 200-300 nucleotides of adenylic acid are added to its 3' end, which is called poly 'A' tail. Now it becomes stable. By the process of RNA splicing hn-RNA produces functional m-RNA that is exonic RNA. In RNA splicing non coding parts is removed with the help of spliceosome enzyme and coding part join together with the help of RNA ligase.

Some specific proteins are also helpful in RNA- splicing called 'Small nuclear ribonucleoprotein' or 'SnRNP or 'Snurps'. These SnRNP proteins combine with some other proteins and SnRNA to form spliceosome complex. This spliceosome complex uses energy of ATP to cut the RNA, releases the non-coding part and joins the coding-part to produce functional RNA. Non coding part of hn RNA. remained inside the nucleus and not translated in to protein. Only coding part moves from nucleus to cytoplasm and gets translated into protein.

Mostly Eukaryotic genes are example of split gene, but gene which forms histone and interferon protein are non split gene.
Mostly prokaryotic genes are example of non split gene.
In eukaryotes after transcription splicing process also occured.
The split gene represent an ancient (primitive) feature of gene.
Presence of intron is a primitive character.
The splicing process represent the dominance of RNA world.

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RIBO NUCLEIC ACID (RNA)

2020-07-09T11:48:00.000+05:30

RIBO NUCLEIC ACID (RNA)

Structure of RNA is fundamentally same as DNA, but there are some differences. The differences are as follows:-

1. In place of De-oxyribose sugar in DNA, there is present Ribose sugar in RNA.

2. In place of nitrogen base Thymine in DNA, there is present Uracil in RNA.

3. RNA is made up of only one polynucleotide chain i.e. RNA is single stranded.

Exception:-

RNA found in Reo-virus is double stranded, i.e. it's has two polynucleotide chains.

Types of RNA:

1. Genetic RNA or Genomic RNA- in some viruses RNA works as genetic material and it transfers informatiots form one generation to next generation.

Eg. Reo virus, TMV, QB bacteriophage.

2. Non genetic RNA- mainly of 3 types:-

a. r-RNA

b. t-RNA

c. m-RNA

RNA functions as adapter, structural and in same cases as a catalyst (Ribozyme).

1. Ribosomal RNA (r-RNA)

This RNA is 80% of the cells total RNA.
r-RNA was discovered by Kuntze.
It is found in ribosomes and it is produced in nucleolus.
It is the most stable form of RNA.
These are present in 80s type of ribosomes in Eukaryotic cells. Their subunits are 60s and 40s. In 60s sub unit of ribosome three types of r-RNA are found - 5s,5.8s,28s.
40s sub unit of ribosome has only one type of r-RNA i.e 18s.
So 80s ribosome has total 4 types of r-RNA.
Prokaryotic cells have 70s type of ribosomes and it's subunits are 50s and 30s.
50s Subunit of ribosome contain 2 types of r-RNA i.e 5s and 23s.
30s sub unit of ribosome has 16s type of r-RNA.
So 70s ribosome has total 3 types of r-RNA.

Function:-

At the time of protein synthesis, r-RNA provides attachment site to t-RNA and m-RNA and attaches them on the ribosomes.

It attaches t-RNA to the larger subunit of ribosome and m-RNA to smaller subunit of ribosome.

2. Transfer RNA (t-RNA)

It is 10-15% of total RNA.
It is synthesized in the nucleus by DNA.
It is also known as soluble RNA (sRNA).
It is also known as Adapter RNA.
It is the smallest RNA (4s).

Function:- at the time of protein synthesis it acts as a carrier of amino-acids.

Structure:- the structure of t-RNA is most complicated.

Holley presented Clover leaf model of its structure. In two dimensional structure the t-RNA appears clover leaf like but in three dimensional structure (by Kim) it appears inverted L-shaped.
The structure of tRNA looks like a clover
leaf but in actual structure, the tRNA is compact molecule which looks like inverted "L".
There are present three nucleotides in a particular sequence at 3' end of t- RNA and that sequence is CCA.
All the 5' ends i.e. last ends are having G (guanine).
3' end is known as Acceptor end.
t-RNA accepts amino acids at acceptor points. Amino acid binds to 3' end by its -COOH group.
The molecule of t- RNA is folded and due to folding some complementary nitrogenous bases come across with each other and form hydrogen bonds.
There are some places where hydrogen bonds are not formed, these places are known as loop.

Loops:-

There are some abnormal nitrogenous bases in the loops, that is why hydrogen bonds are not formed.

e.g. 1. Inosine(l)

2. Pseudouracil (ψ)

3. Dihydrouridine (DHU)

(A) T ψ C Loop or Attachment loop -

This loop connects t- RNA to the large subunit of ribosome.

(B) Recognition Loop (Anticodon loop)- This is the most specific loop of t-RNA and different types of tRNA are different due to this loop. There is a specific sequence of three nucleotides called Anticodon, is present at the end of this loop.

t-RNA recognizes its place on m-RNA with the help of Anticodon.

(C) Dihydrouridine (DHU)

It is also known as Amino-acyl synthetase recognition loop. Amino-acyl synthetase is a specific type of enzyme. The function of this enzyme is to activate a specific type of amino acid. after activation this enzyme attaches the amino acid to the 3' end of t-RNA.
There are 20 types of enzymes for 20 types of amino acids.
The function of DHU loop is to recognize this specific Aminoacyl synthetase enzyme.

3. Messenger RNA (mRNA) :-

The m-RNA is 1-5% of the cells total RNA.
The name m-RNA was given by Jacob and Monad.
The m- RNA is produced by genetic DNA in the nucleus.
It is least stable RNA.

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DNA REPLICATION

2020-07-08T09:01:00.000+05:30

RNA WORLD

RNA was the first genetic material. There are evidence to suggest that essential life processes, such as metabolism, translation, splicing etc. evolved around RNA. RNA use to act as a genetic material as well as a catalyst, there are some important biochemical reactions in living system that are catalysed by RNA catalysts and not by Protein enzymes (e.g. splicing) RNA being a catalyst was reactive and hence unstable. Therefore, DNA has evolved from RNA with chemical modification that makes it more stable. DNA being double stranded and having complementary strand further resists changes by evolving a process of repair. RNA is adapter, structural molecule and in some case catalytic. Thus RNA is better material for transmission of information.

DNA REPLICATION

1. While proposing the double helical structure for DNA, Watson and Crick had immediately proposed a scheme for replication of DNA. To quote their original statement that is as follows.

2. "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material" ( Watson and Crick, 1953).

3. The scheme suggested that the two strands would separte and act as a template for the synthesis of new complementary strands. After the completion of replication, each DNA molecule would have one parental and one newly synthesised strand. This scheme was termed as semiconservative DNA replication.

4. DNA capable of self duplication.

5. All living beings have the capacity to reproduce because of this characteristic of DNA.

6. DNA replication takes place in "S-phase" of the cell cycle. At the time of cell division, it divides in equal parts in the daughter cells.

SEMI CONSERVATIVE MODE OF DNA REPLICATION

Semi conservation mode of DNA replication was first proposed by Watson and Crick. Later on it was experimentally proved by Meselson and Stahl (1958) in E.coli and Taylor in Vicia faba (1958). To prove this method, Taylor used Radiotracer Technique in which Radioisotopes (tritiated thymidine = ) were used. Meselon and Stahl used heavy isotope of nitrogen-15.

Matthew Meselson and Franklin Stahl performed the following experiment in 1958:

1. They grew E.coli in a medium containing NH4CL ( N-15 is the heavy isotope of nitrogen) as the only nitrogen source for many generations. The result was that N-15 was incorporated into newly synthesised DNA (as well as other nitrogen containing compounds). This heavy DNA molecule could be distinguished form the normal DNA by centrifugation in as cesium chloride (CsCl) density gradient (please note that N-15 is not a radioactive isotope, and it can be separated from N-14 only based on densities).

2. Then they transferred the cells into a medium with normal NH4CL and took samples at various definite time intervals as the cells multiplied, and extracted the DNA that remained as double stranded helices. The various samples were separated independently on CsCl gradient to measure the densities of DNA.

3. Thus, the DNA that was extracted from the culture one generation after the transfer from N-15 to N-14 medium [that is after 20 minutes; E.coli divides in 20 minutes] had a hybrid or intermediate density. DNA extracted from the culture after another generation [that is after 40 minutes, 2 generation] was composed of equal amounts of this hybrid DNA and of 'light' DNA.

MECHANISM OF DNA REPLICATION

The following steps are included in DNA replication:-

1. Unzipping (Unwinding):-

1. The separation of 2 chains of DNA is termed as unzipping. And it takes place due to the breaking of H-bond. The process of unzipping starts at a certain specific points which is termed as initiation point or origin of replication. In prokaryotes there occurs only one origin of replication but in eukaryotes there occur many origin or replication i.e unzipping starts at many points simultaneously. At the place of origin the enzyme responsible for unzipping (breaking the hydrogen bond) is Helicase (=Swivelase). In the process of unzipping Mg+2 act as cofactor.

2. SSB (Single stranded DNA binding protein) prevents the information of H-bonds.

3. Topoisomerase (in prokaryotes also called as DNA gyrase) release the tension arises due to supercoiling.

Note: The process of DNA replication takes a few minutes in prokaryotes and a few hours in Eukaryotes.

2. Formation of New chain:-

1. To start the synthesis of new chain, special type of RNA is required which is termed as RNA primer. The formation of RNA primer is catalysed by an enzyme- RNA Polymerase (primase). Synthesis of RNA-Primer takes place in 5'-3' direction. After the formation of New chain, this RNA is removed. For the information of new chain Nucleotides are obtained from Nucleoplasm. In the Nucleoplasm, Nucleotides are present in the form of triphosphates like dATP, dGTP, dCTP, dTTP etc.

2. During replication, the 2 phosphate groups of all nucleotides are separated. In this process energy is yeilded which is consumed in DNA replication.

3. Energetically replication is a very expensive process. Deoxyribonucleoside triphosphase serve dual purposes in addition to acting as substrates they provide energy for Polymerisation.

4. The formation of New chain always takes place in 5'-3' direction. As a result of this, one chain of DNA is continuously formed and it is termed as Leading strand. The formation of second chain begins from the centre and not form the terminal points, so this chain is discontinuous and is made up of small segments called Okazaki fragments. This discontinuous chain is termed as Lagging strand. Ultimately all these segments joined together and a complete new chain if formed.

5. The Okazaki fragments are joined together by an enzyme DNA Ligase.

6. The formation of New chains in catalysed by an enzyme DNA Polymerase. In prokaryotes it is of 3 types:

1. DNA-polymerase 1 :- This was discovered by KORNBERG (1957). So it is also called as 'Kornberg's enzyme'. KORNBERG also synthesized DNA first of all, in the laboratory. This enzyme functions as exonuclease. It separates RNA-Primer from DNA and also fills the gap. It is also known as DNA-repair enzyme.

2. DNA-polymerase 2 :- It is least reactive in replication process. It is also helpful in DNA-repairing in absence of DNA-polymerase 1 and DNA-polymerase 3.

3. DNA-polymerase 3 :- This is the main enzyme in DNA-replication. It is most important. The larger chains are formed by this enzyme. This is also known as Replicase.

7. In the semi conservative mode of replication each daughter DNA molecule receives one chain of polynucleotides from the mother DNA- molecule and the second chain is synthesized.

Sepcial points:-

1. All DNA polymerase 1,2 and 3 enzymes have 5'-3' Polymerisation activity and 3'-5' exonuclease activity.

2. DNA polymerase 1 also has 5'-3' exonuclease activity.

3. Any failure in cell division after DNA replication result into polyploidy.

4. Difference between DNAs and DNase is that DNAs means many DNA and DNase means DNA digestive enzymes.

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