Bioinformatician Book

my "ORF finder project" java code

2011-08-05T05:17:00.000-07:00

i did ma project of java made tool of ORF FINDER with all graphics...if u wana it ....write ur email address below in comment section ..i ll send u in 2-3 days...

computation

2011-08-04T00:50:00.001-07:00

go 2 thz address
http://www.slideshare.net/xulkifal/computation

source code of"DNA to protein converter tool"made in JAVA

2011-08-03T03:41:00.001-07:00

i did my project in JAVA on "DNA to protein converter tool" if u need source code.write down ur email below in comments .i ll mail u in 2-3 days. cheers :)

viral molecular genetics

2011-07-30T05:40:00.000-07:00

click on here
http://www.slideshare.net/xulkifal/viral-molecular-genetics

chromosomal aberrations

2011-07-30T04:21:00.000-07:00

CLICK HERE TO SEE PRESENTATION on chromosomal aberrations

cytogenetics

2011-07-30T04:19:00.000-07:00

presentation is in thz link
http://www.slideshare.net/xulkifal/cytogenetics

dna viruses

2011-07-28T06:45:00.000-07:00

Dna Viruses - Presentation Transcript

DNA VIRUSES-Dnavirus is a virus that has DNA (deoxyribonucleic acid) as its genetic material. -They are usually Large, Icosahedral, enveloped in Lipoproteins-Do not have polymerase enzymes, and cause Latent infection-Examples include Poxviruses, Herpesviruses, hepadnaviruses, Hepatitis b.
HERPESVIRUSES -Larger virions than adenoviruses(180 - 200nm)-Larger genome (three to five times) than adenoviruses. -Linear, double-stranded DNA, Enveloped, icosahedral virus (this means that lipid solvents readily inactivate these viruses)
Adsorption and penetration-Many herpesviruses, including herpes simplex virus, can fuse directly with the plasma membrane (results in partial uncoating). -Such fusion with the plasma membrane has implications for both the virus and the host cell.
Earlyphase-Early transcription (the mRNAs made during this phase are the alpha and beta mRNAs). -Herpes viruses use host RNA polymerase-The virus uses host mRNA modification enzymes.-Initially, alpha-mRNAs are transcribed-Beta-mRNAs are transcribed by the host RNA polymerase again-Beta proteins are involved in gene expression regulation-They decrease alpha-gene expression and are needed for gamma gene expression.
Late phase-Herpesvirusescode for several proteins, in addition to the DNA polymerase, that are needed for DNA replication. -DNA replication is accompanied by a lot of recombination-The replicated DNA is present as long concatamericmolecules-These are cleaved to genome-size lengths when DNA is packaged into the virion.
Late transcription: -Late transcription occurs after DNA replication. -Gamma mRNAs are made and are translated in the cytoplasm. -Gamma proteins are predominantly structural. -There is decreased expression of beta genes in the late stage. -In herpes viruses there is no apparent organization of the genome into blocks for either early or late transcription.
Assembly-Assembly occurs in the nucleus. -A capsid is formed and the DNA enters the capsid. -The capsids acquire an envelope by budding through areas of the inner nuclear membrane which have viral membrane proteins inserted into them. -The virus envelope then fuses with the outer nuclear membrane and the de-enveloped nucleocapisid is delivered into the cytoplasm -It then becomes re-enveloped by budding into Golgi-derived vesicles and is then released.-The late protein required for transcription of immediate early mRNAs in the next round of infection is packaged in the virion.
Evolution of Viruses:
- Viral genomes undergo genetic change by mutation, either spontaneous or induced mutation, and by recombination.
- Recombination may be either intramolecular or, among viruses with divided genomes, by reassortment.
1.Mutations:
These arise naturally during viral replication
2. Exchange of Genetic material:
This involves breaking of covalent bonds within the nucleic acid, exchange of genetic information, and reforming of covalent bonds.
This kind of break/join recombination is common in DNA viruses or those RNA viruses which have a DNA phase (retroviruses).
The host cell has recombination systems for DNA. If a virus has a segmented genome and if two variants of that virus infect a single cell, progeny virions can result with some segments from one parent, some from the other.

FERTILIZATION

2011-07-28T06:43:00.000-07:00

Xulkifal yousaf [me_the_charlie@hotmail.com] Fertilization
FERTILIZATION
- Recognition of Egg and Sperm: 5 steps, (1). Chemo attraction (2). Release of acrosomal enzymes (3). Binding of sperm to extra cellular envelopes (4). Passage through extra cellular envelopes (5). Fusion of sperm and egg nuclei (2 & 3 can be reversed).
Fertilization May 21, 2010
FERTILIZATION
- Sperm attraction…..species specific in many invertebrates, chemo taxis, also timings in these animals. In sea urchin a peptide resact is such chemo tactic molecule. It is also sperm activating molecule.
- Egg jelly has other compounds as well, which bind with specific receptors on sperm, which inturn opens calcium ion channels, leading to exocytosis of acrosome.
Fertilization May 21, 2010
FERTILIZATION
- Acrosome reaction: In marine invertebrates, fusion of acrosomal membrane with sperm membrane & the formation of acrosomal process. Release of proteolytic enzymes ->-> go through jelly coats ->->egg membrane.
- Transportation of sperm and its capacitation. Active, also passive through contraction, species specific recognition (as in sea urchin).
Fertilization May 21, 2010
FERTILIZATION
- Acrosomal protein is Bindin….present on acrosomal process…which is species specific. Its specific receptors not all over the egg membrane……so limited sites of contact and penetration (first step towards block to polyspermy).
- (In mammals, zona pellucida has many glycoproteins ….ZP-1, ZP-2, ZP-3….it is ZP-3 which affects binding & it also triggers acrosomal reaction.
Fertilization May 21, 2010
FERTILIZATION
- ZP-3 of ZP of egg crosslinks with a sperm protein called galactosyltransferase-Ι(which binds with carbohydrate residue of ZP-3)
- Exocytosis of acrosome (its breakdown) releases many proteases----which make a hole in ZP; secondary binding of spermatozoa is through ZP-2).
Fertilization May 21, 2010
FERTILIZATION
- Gamete Fusion and Prevention Of Polyspermy : Fusion of sperm and egg membranes ->->polymerization of actin in egg->-> fertilization cone, as does the sperm actin which forms the acrosomal process.
- These two projections meet so as to form a continuous bridge through which sperm nucleus & centiole pass.
Fertilization May 21, 2010
FERTILIZATION
- (In mammals sperms attach to egg membrane at a side of head (equatorial domain)….of head).
- Prevention of polyspermy: In sea urchin …fast & slow processes……fast by changing of the electric potential of egg membrane, high Na contents outside egg (in sea water), inside high K.
Fertilization May 21, 2010
FERTILIZATION
- Resting membrane potential -70 mv (inside of egg negatively charged)…within 1-3 sec. potential changes to positive, if experimentally this potential is changed polyspermy will be duly affected (i.e. by creating negative charge polyspermy can occur OR no fertilization by keeping the charge positive).
Fertilization May 21, 2010
FERTILIZATION
- Slow block, sea urchin, cortical granule reaction, 15000 granules,1 µm in diameter, fuse with plasma membrane of egg and release contents into space between it & vitelline membrane, i.e. exocytosis, several proteins released.
- First is cortical granule serine protease, it dissolves protein part that connect vitelline envelope protein to cell membrane, also clips off bindin receptors and any sperm attached to them.
Fertilization May 21, 2010
FERTILIZATION
- Another mucopolysaccharide causes formation of fertilization envelope (by causing water get into the space between plasmalemma & vitelline membrane).
- A third protein released by cortical granules is peroxidase enzyme, which hardens fertilization envelope by cross linking tyrosine residues on adjacent proteins. This starts at sperm entry & spreads all over---- starts within 20 seconds and is finished by 60 seconds.
Fertilization May 21, 2010
FERTILIZATION
- A 4th protein, hyalin, forms a coating around egg i.e. a hyalin layer, similar in mammalian egg but no fertilization membrane is formed, some other peculiar proteins in mouse i.e. ZP-3 & ZP-2 are knocked out.
- Calcium as initiator of cortical reaction : Free calcium increases a lot. No influx but release from within egg i.e. intra cellular storage.
- This can be monitored visually by ca-activated luminescent dye such as aequorin or fura-2.
Fertilization May 21, 2010
FERTILIZATION
- These emit light when they bind with calcium.If injected with these dyes then fertilized, a wave of light starts from sperm entry point & go to other end ---completed within 30 sec. An ionophore A23187 (i.e. it transports calcium across membrane from outside), can cause cortical granules reaction & elevation of fertilization membrane if egg is immersed in this medium.
Fertilization May 21, 2010
FERTILIZATION
- If no calcium in surrounding water even then this reaction occurs, so it must be activating ca++ already present.
- Activation Of Egg Metabolism: Early & late responses.
Fertilization May 21, 2010
FERTILIZATION
- Early; Increase in calcium conc.->-> cell division, protein synthesis (calcium increases from 0.1 to 1.0 µM).Several waves of calcium release….. Several activities in egg related to these waves. If ca-chelating agent such as EGTA is injected ->->no cortical granule reaction, no change in membrane potential ->->no action potential.
Fertilization May 21, 2010
FERTILIZATION
- Conversely injection of calcium ionophore (A23187) ->->all activities i.e. elevation of fertilization membrane, rise in intra cellular pH, increase in O2 consumption, increase in the protein synthesis ,increase in DNA synthesis ,activation of enzyme NAD+ kinase which coverts NAD+ to NADP which is important for construction of new cell membranes , needed at cleavage.
Fertilization May 21, 2010
FERTILIZATION
- Late Responses; DNA & protein synthesis, if pH increased artificially, DNA & protein synthesis ensues, nuclear envelope breakdown. Protein synthesis…. uses mRNA ….. already synthesized, especially encoding for histone, tubulin, actins, and morphogenetic proteins. The mRNA that was masked is now released (by maskin protein).
Fertilization May 21, 2010
FERTILIZATION Fusion Of Genetic Material: In sea urchin ----♀pronucleus already haploid, ♂ pronucleus decondenses, fuse to form zygote nuclei (diploid). In mammals at fertilization ♀ nucleus at metaphase of 2nd meiotic division, calcium inactivates MAP kinase & DNA synthesis ensues. Two pronucei move towards each other: no envelope break down & chromosomes come on spindle (so true diploid nucleus is NOT formed in zygote but after first division) Fertilization May 21, 2010
FERTILIZATION
- Rearrangement Of Egg Cytoplasm: Fertilization leads to a lot of cytoplasmic movement, crucial for later cell differentiation. Rearrangement can be seen in tunicate ( Styla)…. various colors.
- Also in amphibian cortex moves by about 30 degree towards sperm entry ->-> grey crescent exposed, this rotation is brought about by microtubules localized between cortex & inner cytoplasm.
Fertilization May 21, 2010
FERTILIZATION
- Sperm Capacitation: Newly ejaculated sperms need to remain in ♀ reproductive tract to be able to undergo acrosomal reaction.
- The set of physiological changes through which sperms become competent to fertilize an egg, is called capacitation.
Fertilization May 21, 2010
FERTILIZATION
- Such changes involve (1). Removal of cholesterol from the sperm cell membrane. (2). Some particular proteins &/or carbohydrates are also lost during capacitation. (3). The membrane potential becomes more negative, as K ions leave the sperm. (4). Protein phosphorylation also takes place during capacitation.
- Capacitation can be achieved in vitro , by incubating sperm in media containing calcium, bicarbonate and serum albumin.

MICROARRAY TECHNOLOGY

2011-07-28T06:36:00.001-07:00

BACKGROUND: NEED FOR MICROARRAY TECHNOLOGY:
A cell functions by using its genes to produce proteins and although each cell within an organism will usually contain the same set of genes, there are significant differences in which genes are activated and how they are controlled because a fraction of these genes are turned on and it is the subset that is "expressed" that confers unique properties to each cell type. "Gene expression" is the term used to describe the transcription of the information contained within the DNA. Scientists study the kinds and amounts of mRNA produced by a cell to learn which genes are expressed, which in turn provides insights into how the cell responds to its changing needs. Gene expression acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. For many years, study of a gene in this manner had to be done individually—looking at whether a specific gene is turned on (upregulated) or turned off (downregulated) under certain conditions. MICROARRAY is a technological advancement that has allowed scientists to study many, if not all, the genes of an organism’s at once. This high throughput method allows for the global study of changes in gene expression, giving us a complete cellular snapshot.
INTRODUCTION
Microarrays are small, solid supports onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations. The supports themselves are usually glass microscope slides, but can also be silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support. It is a high throughput technology that allows simultaneous detection of thousands of genes.
In formal terms a microarray can be defined as:
A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.
The American Heritage Dictionary defines "array" as "to place in an orderly arrangement". It is important that the gene sequences in a microarray are attached to their support in a fixed way, because a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cDNA, or oligonucleotides. Microarrays are also referred to as DNA arrays, DNA chips, biochips and GeneChips.
Microarrays come up with the advantageous features as:
– Multiplexing: handling multiple samples at a time
– Parallelism: Studying Thousands of genes simultaneously
– Miniaturization: small sized chip

PRINCIPLE:

The principle of microarrays is based upon base paired hybridization probing, a technique that uses fluorescently labeled nucleic acid molecules as "mobile probes" to identify complementary molecules, and sequences that are able to base-pair with one another.
PARAMETERS DETERMINING THE NATURE OF A MICROARRAY TECHNOLOGY

PARAMETER OPTION
Probes: features arrayed on the microarray substrate that have known identity or sequence cDNA, oligonucleotides, proteins, peptide nucleic acids, small molecules, cells, tissues, and organisms
Fabrication: techniques to array probes on the microarray substrate In situ synthesis, robotic deposition
Targets: samples to be analyzed against the probes DNA, mRNA, proteins, enzymes, small molecules
Assays: principles based on which the targets are being analyzed Hybridization, electrophoresis, flow cytometry, ELISA
Signal readout: principles based on which the assay results can be detected Fluorescence , chemiluminescence, mass spectrometry, radioactivity, electrochemistry
Image processing: signal intensities of hybridized array spots are quantified from the scanning image Software for image processing
Informatics: computational tools with which the huge amount of data generated from a microarray experiment can be effectively stored and interpreted Database management system, data mining and visualization, interpretation of biological meaning

There are various types of microarrays, and to name a few, they include: DNA Microarrays, Protein Microarrays, Tissue Microarrays, Cellular Microarrays and Antibody Microarrays. Among the various types, DNA microarrays, protein microarrays and tissue microarrays are most widely used. The focus in the following sections will be on DNA microarrays.

DNA MICROARRAYS

DNA Microarray is the most widely used microarray technology. It is a type of nucleic acid-based multiplex technique involving high-density arrays of nucleic acids on glass that allows evaluating mRNA abundance of up to tens of thousands of genes simultaneously.
A DNA microarray consists of an orderly arrangement of DNA fragments representing the genes of an organism. Each DNA fragment representing a gene is assigned a specific location on the array, usually a glass slide, and then microscopically spotted (less than 1 mm) to that location. Through the use of highly accurate robotic spotters, over 30,000 spots can be placed on one slide, allowing molecular biologists to analyze virtually every gene present in a genome. The main advantage of microarrays is that the spots are single stranded DNA fragments that are strongly attached to the slide, allowing cellular DNA or RNA to be fluorescently labeled and laid overtop of the array. DNA or RNA in the overlaid sample will stick (through hybridization) to a complementary spot on the array, that is, Gene-A will stick to a spot composed of a Gene-A fragment. By exposing the microarray to a fluorescently labeled sample the DNA that hybridizes will be identifiable as glowing spots on the array, while the spots that have nothing hybridized to them will not be visible.
TYPES OF DNA MICROARRAYS
Two main types of commercial DNA microarrays are: oligonucleotide arrays and cDNA arrays.
1. OLIGONUCLEOTIDE ARRAYS
Oligonucleotide arrays (trademarked as a GeneChip by Affymetrix) use small 25 base pair gene fragments as the DNA probes to be spotted onto an array. These oligonucleotide probes are synthesized either in situ (on-chip) using photolithography or by conventional synthesis followed by on-chip immobilization.
APPLICATIONS OF OLIGONUCLEOTIDE ARRAYS
Oligonucelotide arrays are used for quantification of the amount of mRNA in a single sample (e.g. to determine the amount of mutated vs. non-mutated mRNA) or the comparison of two different samples hybridized to two separate arrays.
OLIGONUCLEOTIDE ARRAY TARGETS
Most array experimenters do not have access to micrograms of total RNA from their biological sample. A linear amplification strategy is highly recommended to obtain sample to be used for hybridization (targets). This technique consists of the following steps:

• First strand cDNA synthesis using reverse transcriptases;
• Generation of second strand cDNA using DNA polymerases; and
• In vitro transcription of the second strand cDNA to generate antisense RNA (aRNA) or complementary RNA (cRNA).
The probes are selected to have little cross-reactivity with other genes (targets) so that non-specific hybridization will be minimized.
2. cDNA ARRAYS
A cDNA array (also known as DNA microarray) is a different technology using the same principle; the probes in this case are larger pieces of DNA that are complementary to the genes one is interested in studying. With the cDNA probes prepared they can be mechanically spotted (robot spotting) onto a glass slide, normally in duplicate to serve as controls.
cDNA PROBES
• cDNA probes for making the array can also be generated from a commercially available cDNA library ensuring a close representation of the entire genome of an organism on the array.
• In an alternate target synthesis protocol, the target is generated by reverse transcription of the total RNA or the aRNA.
• Alternatively, PCR using specific primers can be used to amplify specific genes from genomic DNA to generate the cDNA probes.
When using cDNA spotted microarrays, two pools of RNA can be compared on the same array simultaneously. This is unlike Affymetrix GeneChip arrays, where RNA isolated from a single source is hybridized to an array or multiple arrays are required for comparison between more than one RNA samples.

DESIGN AND WORKING OF MICROARRAYS

DESIGN OF MICROARRAYS
Micro-array probes are synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization.
• Micro-arrays are printed in situ (on chip) using a combination of photolithography and combinatorial chemistry.
• For on-chip immobilization, probes can be mechanically spotted onto a glass slide by using robot.
PHOTOLITHOGRAPHY:
The production begins with a 5-inch square of quartz wafer.
This wafer is washed with a blocking compound that can be removed by exposure to light. What is needed next is referred to as a ‘mask’, which is designed with 18-20 micron square windows that allow light to pass through areas where a specific nucleotide is needed.
EXAMPLE:
If the mask is designed for the addition of thymine to the probe, you will find a window at that location, while probe locations where another base is required are blocked. Such a mask is laid on top of the array and ultraviolet light is shone onto it.
Probe locations that have windows are exposed to the light and the blocking compound removed from that probe, while covered locations still have the blocking compound present.
The quartz wafer is then washed with a solution of the desired nucleotide (e.g. thymine) that is linked to the same blocking compound, causing the nucleotide to attach to the probes that were exposed to light, while the nucleotide-attached blocking compound ensures that all of the probes are protected again. A capping step is added so that probes that did not attach their appropriate nucleotide are not incorrectly synthesized. The array is now ready for a new mask and the addition of a new nucleotide (cytosine in the figure). The process is repeated until all of the probes are complete (25 nucleotides each). Computer algorithms can calculate the optimal design of the masks that will minimize the number of reactions needed.
Once constructed, a microarray is ready for use in the laboratory.
WORKING OF MICROARRAYS
1. ISOLATION OF RNA FROM BIOLOGICAL SAMPLES

For microarray analysis, RNA can be isolated from a diverse set of biological samples using a combined phenol and glass-filter purification protocol. Each sample will contain thousands of different mRNA sequences representing all of the genes expressed in those cells.

2. GENERATION OF LABELED TARGETS:

Microarrays require 10-30 micrograms of labeled target per hybridization. Either labeled cDNA or aRNA (antisense) can be used for microarray experiments. In the Affymetrix system, the dNTPs are biotinylated, and later detection is performed with fluorescent streptavidin staining. In the simplest case, reverse transcription is performed using fluorescently tagged (e.g., Cy3 or Cy5), dinucleotide triphosphates (dNTPs), resulting in the generation of fluorescent cDNA (cDNA arrays).

2. TARGET HYBRIDIZATION.
The fluorescently labeled “targets,” are hybridized to gene-specific “probes.” Each target anneals to its corresponding probe spot on the microarray. The probes can be spotted cDNAs or oligonucleotides, or oligonucleotides that were synthesized directly on the micro-array surface. Stringent conditions are used to ensure that the probe sequences are entirely complementary to the micro-array spot sequences.
3. WASHING OF UNBOUND TARGETS
The slide is washed off to remove excess fluorescent targets not bound to spots.
4. DETECTION OF SIGNAL AND ANALYSIS OF DATA
After hybridization, a laser scanner is used to detect the specific fluorescence at each spot at two different wavelengths for green Cy3 or red Cy5. The colors of the spots are as follows:
Green = bound to Cy3-labeled cDNA, Red = bound to Cy5-labeled cDNA and Yellow = bound to both Cy3 and Cy5-labeled cDNA (these represent genes such as “housekeeping genes” that are required by all cells)
Software associated with the scanner allows identification of individual spots and measures each spot intensity. If all goes well, fluorescence intensity is proportional to the concentration of the relevant mRNA in the original sample.

DIAGRAMATIC REPRESENTATION OF MICROARRAY EXPERIMENT

The microarray is scanned with a laser beam, first at one wavelength to collect fluorescence data representing one probe, and then is scanned at a second wavelength to collect data representing the second probe. A computer compares the amount of fluorescence at each spot on the microarray for each probe. Through the use of computer software, the ratio of fluorescence is obtained and correlated with the clone address so the investigator knows which gene (spot on the slide) was expressed more in treated tissue as compared to control tissue.

OTHER TYPES OF MICROARRAYS

Apart from DNA microarrays, protein microarrays and tissue microarrays are the types most widely used.
1. TISSUE MICROARRAYS
Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time, either at the DNA, RNA or protein level. Tissue microarrays (TMA) consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.
Tissue microarrays are different from DNA microarrays where each spot on an array represents a cloned cDNA or oligonucleotide that binds to the target sequence. With tissue microarrays, each array has patient specific histological samples from cancer infected tissues. The tissue microarray technique is best suited for screening one genetic marker or protein across thousands of samples where as DNA microarrays are best suited to study gene expression across thousands of genes.
2. PROTEIN MICROARRAYS
In the past few years, protein microarray technology has shown its great potential in basic research, diagnostics and drug discovery. It has been applied to analyze antibody–antigen, protein–protein, protein–nucleic-acid, protein–lipid and protein–small-molecule interactions, as well as enzyme–substrate interactions. Recent progress in the field of protein chips includes surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyze entire proteomes.

APPLICATIONS OF MICROARRAYS

Microarrays can be used for large number of applications where high-throughput is needed. The current scope of microarray applications includes sequencing by hybridization, resequencing, mutation detection, assessment of gene copy number, comparative genome hybridization, and drug discovery and expression analysis.
1. SEQUENCE ANALYSIS
One of the earliest applications for microarrays was sequencing by hybridization. It is possible to determine the sequence of a target from the target’s pattern of hybridization with an array of all possible combinations of an oligonucleotide of a particular length. Subsequently, many other sequence-based applications have been developed, including genotyping, detection of point mutations, single nucleotide polymorphisms, and insertions and deletions, as well as the detection, identification, and enumeration of microorganisms.
2. GENE EXPRESSION ANALYSIS: GENE DISCOVERY
One of the most important applications of DNA Microarrays is the monitoring of gene expression where the abundance of the mRNA produced is determined for each gene. Such differences in gene expression are accountable for morphological and phenotypic differences as well as indicative of cellular responses to environmental stimuli and perturbations. Changes in the multi-gene patterns of expression can provide clues about regulatory mechanisms and broader cellular functions and biochemical pathways. In the context of medicine and treatment, such changes in patterns of expression can help to determine the causes and consequences of disease, how drugs and drug candidates work in cells and organisms.
Microarray technology has revolutionized studies of gene expression. A cDNA or oligonucleotide array complementary to expressed mRNA provides a means to simultaneously assess the expression of thousands of genes. Two-color labeling strategies introduce an additional degree of sophistication because they facilitate simultaneous comparison between gene expression in control and test cells on the same array. A goal in expression analysis is to place all of the genes for an organism on a single chip and then use that microarray to monitor changes in gene expression in cells. Already, there are chips with arrays representing all or many of the genes of the human and other species, such as rat, mouse, dog, monkey, honey bee, Arabidopis, Drosophila, and Escherichia coli.
3. DISEASE DIAGNOSIS:
DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.

4. DRUG DISCOVERY:
Drug discovery has evolved to become a large-scale and highly sophisticated endeavor. Microarray technology has extensive application in Pharmacogenomics. Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.
5. TOXICOLOGICAL RESEARCH:
Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants. Microarray technology provides a unique tool for the determination of gene expression at the level of messenger RNA (mRNA). The simultaneous measurement of the entire human genome (thousands of genes) will facilitate the uncovering of specific gene expression patterns that are associated with disease.
6. COMPARATIVE GENOMIC HYBRIDIZATION (CGH) AND GENE COPY NUMBER
CGH is used as a molecular cytogenetic technique that permits quantitative analysis of gains and losses of whole or portions of chromosomes. A comprehensive high-resolution test for small chromosomal imbalances (microduplications or microdeletions) can now be performed in a single test. Investigators have developed a newer method that combines the principles of CGH with the use of microarrays. Instead of using metaphase chromosomes, this method—which is known as array CGH, or simply aCGH—uses slides arrayed with small segments of DNA as the targets for analysis. Microarray CGH works by comparing the levels of patient DNA to levels of a control (normal) DNA sample.In traditional CGH, DNAs from a "test" and a "reference" genome are differentially labeled with fluorophores and hybridized to normal metaphase chromosomes. In array CGH arrays of genomic BAC, P1, cosmid or cDNA clones are used for hybridization. CGH can not detect genomic changes involving <10–20 megabases. The use of microarray CGH overcomes many of these limitations, with improvement in resolution and dynamic range and improved throughput.

ROLE OF BIOINFORMATICS IN MICROARRAY TECHNOLOGY

Bioinformatics allows the researchers to tackle tasks easily by using Internet based tools without having to consider technical problems like local installation, maintenance or financial aspects. It aids in analysis of microarray technology by analyzing the expression levels, detecting anomalies, detecting SNP’s, finding GO terms, clustering, classification and then generating trees and processing fluorescent images.
TOOLS
1. Cryoscopy: Viewing a CGH (Comparative genomic hybridization) or expression data in a whole-genome context i.e. analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA. CGH will detect only unbalanced chromosomal changes. Structural chromosome aberrations such as balanced reciprocal translocations or inversions cannot be detected, as they do not change the copy number.

2. Array Viewer: Identification of statistically significant hybridization signals.

3. Cluster: Perform hierarchical clustering and self-organizing maps.

4. BAM array: Bayesian Analysis of Variance for Microarrays detect differentially expressed genes from multigroup microarray data.

5. Expression Profiler: Analysis & clustering of gene expression data.

6. TreeView: Graphically browse results of clustering and other analyses from Cluster.

ROOM FOR IMPROVEMENT
AND
CONCLUSION

ROOM FOR IMPROVEMENT:
A lot of efforts have been made in the industry to improve the construction of the microarrays, as to advance the precision of the detection signals, increased speed of hybridization reactions, less usage of starting material and the cheaper availability of this technology.
"Microarrays will get better over time and a lot of that will be in content as we better understand which genes are important and, specifically, perhaps which splice variants are most important”, Says one of the scientists.
One of the improvements in the development phase is the usage of reflective substrates. It is speculated that printing microarrays on mirrors rather than glass improves the signal-to-noise ratio by as much as 1,000%.
CONCLUSION:
In recent decades, high-throughput scientific methods have been developed to optimize the study of large numbers of molecules, including DNA, proteins and metabolites. Microarray technology has become a crucial tool for large-scale and high-throughput biology. It allows fast, easy and parallel detection of thousands of addressable elements in a single experiment. DNA microarrays in particular have proved valuable in genomic research. They have been used to study gene expression patterns, to locate transcription factor binding sites, and to detect sequence mutations and deletions on a grand scale. However, DNA microarrays tell us only about the genes themselves and provide little information regarding the functions of the proteins they encode. Thus, protein microarrays were developed and were seen as high throughput approaches for the study of protein. On the other hand, tissue microarray is seen to provide a remarkable degree of standardization, speed, and cost efficiency for cancer analysis.

REFERENCES:

http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/basicbio.htm
http://static.msi.umn.edu/tutorial/lifescience/IntroMicroarray.pdf
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
http://www.chipscreen.com/articleFile/Microarrays-Tech%20and%20Application1.pdf
http://www.nature.com/nature/journal/v416/n6883/full/416885a.html
http://php.med.unsw.edu.au/cellbiology/index.php?title=Group_8_Project_-_Microarray
http://www.premierbiosoft.com/tech_notes/tissue-microarray.html
http://en.wikipedia.org/wiki/DNA_microarray
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http://www.ncbi.nlm.nih.gov/geo/info/GEOHandoutFinal.pdf
http://www.sydneygenetics.com/Resources/WhatismicroarrayCGH/tabid/714/Default.aspx
http://www.ncbi.nlm.nih.gov/pubmed/12782098
http://chagall.med.cornell.edu/I2MT/MA-tools.pdf
http://discover.nci.nih.gov/tools.jsp
http://smd.stanford.edu/resources/restech.shtml
http://www.scq.ubc.ca/spot-your-genes-an-overview-of-the-microarray/
http://www.ambion.com/techlib/resources/microarray/basics1.html
http://static.msi.umn.edu/tutorial/lifescience/IntroMicroarray.pdf
http://www.fastol.com/~renkwitz/microarray_chips.htm

Project on Morpholino

2011-07-28T06:15:00.000-07:00

1. Background:
By 1984 a growing concern in the then-emerging antisense field was that antisense therapeutics might never be commercially viable because of their very high production costs.
Two key factors in the high cost of DNA analogs are:
1) The limited availability and high cost of their deoxyribonucleoside precursors;
2) The complexity and expense associated with coupling to hydroxyls, required in forming the phosphoester intersubunit linkages of most nucleic acid analogs.

In 1985 Summerton devised the Morpholino structural type to circumvent both of these cost problems. This is achieved by starting with much less expensive ribonucleosides and introducing an amine.
1. Introduction:
In molecular biology, a Morpholino is a molecule used to modify gene expression.
Morpholino oligonucleotides (oligos) are an antisense technology used to block access of other molecules to specific sequences within nucleic acid. Morpholinos block small (~25 base) regions of the base-pairing surfaces of ribonucleic acid.

Morpholinos are usually used as a research tool for reverse genetics by knocking down gene function. This is achieved by preventing cells from making a targeted protein or by modifying the splicing of pre-mRNA. Knocking down gene expression is a powerful method for learning about the function of a particular protein; similarly, causing a specific exon to be spliced out of a protein can help to determine the function of the protein moiety encoded by that exon. These molecules have been applied to studies in several model organisms, including mice, zebrafish, frogs, and sea urchins.
Morpholinos are also in development as pharmaceutical therapeutics targeted against pathogenic organisms such as bacteria or viruses and for amelioration of genetic diseases. These synthetic oligos were conceived by James E. Summerton (Gene Tools, LLC) and developed in collaboration with Dwight D. Weller ( AVI BioPharma Inc. )
2. Structure:
Morpholinos are synthetic molecules which are the product of a redesign of natural nucleic acid structure. Usually 25 bases in length, they bind to complementary sequences of RNA by standard nucleic acid base-pairing. Structurally, the difference between Morpholinos and DNA is that while Morpholinos have standard nucleic acid bases, those bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates.
Replacement of anionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits.
Morpholinos are most commonly used as single-stranded oligos, though heteroduplexes of a Morpholino strand and a complementary DNA strand may be used in combination with cationic cytosolic delivery reagents.

4. Why Choose Morpholinos?
There is no other gene knockdown reagent (including siRNA, PNA, mPNA, S-DNA, and LNA) that combines the properties of stability, nuclease-resistance, efficacy, long-term activity, water-solubility, low toxicity and exquisite specificity. Only Morpholino oligos provide all of these.
Morpholinos provide much better specificity than RNAi, siRNA and phosphorothioate based oligos, greatly decreasing the chance of catastrophic off-target antisense effects.
The exquisite specificity of Morpholinos contributes to their extremely low toxicity and suitability for use in embryos, while off-target gene modulation makes siRNA unusable in most embryonic systems.
The non-ionic backbone of a Morpholino minimizes interactions with proteins, eliminating this mechanism for inducing non-antisense effects. Morpholinos do not trigger cellular responses by binding to Toll-like receptors, eliminating a confounding artifact associated with knockdowns using siRNA.
As steric blocking oligos, Morpholinos can be used not only to block translation but also to alter mRNA splicing, to bind miRNAs or to block binding of miRNA or regulatory proteins to RNA targets. Splice-blocking Morpholinos allow researchers to delete targeted exons and even analyze specific splice-forms of a gene with multiple splice variants. Morpholinos can target a pri-miRNA or a pre-miRNA to inhibit miRNA maturation or target a miRNA or one of its targets to inhibit miRNA activity. Binding of a splice-regulatory protein can be prevented by protecting its binding site with a Morpholino oligo.
5. Properties:
i. Solubility
An antisense oligo should have good water solubility to assure good access to its target sequence within the cell. Conventional perception in the antisense field is that non-ionic antisense oligos (such as Morpholinos) invariably show poor water solubility. Contrary to this perception, it has been found that if the nucleobases in a non-ionic antisense oligo stack poorly then that oligo has poor water solubility, but if the nucleobases are well stacked in aqueous solution then that oligo can show excellent water solubility.
It is assumed that the low water solubility of non-ionic oligos with poorly stacked bases is a result of the difficulty of inserting the hydrophobic faces of the unstacked bases into an aqueous environment. On the other hand, the high water solubility of non-ionic phosphorodiamidate-linked Morpholinos is likely due to effective shielding of those hydrophobic faces from the polar solvent because of the exceptionally good stacking of the bases.

ii. Stability
For optimal activity, an antisense oligo should be completely resistant to nucleases. DNA antisense oligos are degraded in serum and within cells in a matter of minutes.
Morpholino oligos are completely resistant to nucleases, as well as being resistant to a broad range of other degradative factors in biological systems - even including a liver homogenate. Accordingly, Morpholinos are effective in even long-term experiments and they are free of complications, which could arise from toxic degradation products.
iii. Efficiency: Cell-Free
It has been found that in a cell-free translation system with added RNase H the "old" 25-mer Morpholinos containing uracils typically exhibited slightly higher efficacies than corresponding S-DNAs, while "new" thymine-containing 25-mer Morpholinos generally achieve substantially higher efficacies than corresponding S-DNAs.
iv. Efficiency: In Cells
In experiments with Morpholinos carried out at ANTIVIRALS Inc. it has been found that Morpholino antisense oligos, which exhibit good activity in a cell-free translation system also, exhibit correspondingly good activity when scrape-loaded into cultured animal cells.
v. Binding Affinity
Targeting Morpholino oligos is vastly simpler than targeting DNA-based antisense oligos. Morpholinos usually work on the first attempt if they are successfully delivered to the cytosol. DNA and S-DNA oligos have a low probability of shutting down a target gene on the first attempt; it is common for five or six oligos to be tried before an effective sequence is found.
This is in part because a Morpholino oligo has a higher binding affinity than an equivalent DNA-based antisense oligo. This higher affinity allows Morpholinos to invade RNA secondary structure and substantially increases the probability of designing effective oligos. Precisely targeting a single gene is also simplified because Morpholino oligos act via steric-blocking as opposed to an RNAse H-mediated mRNA degradation mechanism.

6. Function of Morpholinos
Morpholinos do not degrade their target RNA molecules, unlike many antisens structural types (e.g. siRNA). Instead, Morpholinos act by "steric blocking", binding to a target sequence within an RNA and simply getting in the way of molecules that might otherwise interact with the RNA.

Normal gene expression in eukaryotes

Eukaryotic gene expression without intervention by a Morpholinos

In eukaryotic organisms, pre-mRNA is transcribed in the nucleus, introns are spliced out, then the mature mRNA is exported from the nucleus to the cytoplasm. The small subunit of the ribosome usually starts by binding to one end of the mRNA and is joined there by various other eukaryotic initiation factors, forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches a start codon, and then the large subunit of the ribosome attaches to the small subunit and translation of a protein begins. This entire process is referred to as gene expression; it is the process by which the information in a gene, encoded as a sequence of bases in DNA, is converted into the structure of a protein. A morpholino can modify splicing or block translation, depending on the morpholino's base sequence.

i. Blocking translation
Translation blocked by a Morpholino oligo

Bound to the 5'-untranslated region of messenger RNA (mRNA), morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the targeted transcript (called "knocking down" gene expression). This is useful experimentally when an investigator wishes to know the function of a particular protein.Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism.

ii. Modifying pre-mRNA splicing
Splicing blocked by a Morpholino oligo

Morpholinos can interfere with pre-mRNA processing steps by:
• preventing splice-directing small nuclear ribonucleoproteins (snRNP) complexes from binding to their targets at the borders of introns on a strand of pre-mRNA
• by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure
• by interfering with the binding of splice regulatory proteins such as splice silencer and splice enhancers.
Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction.
iii. Blocking other mRNA sites
Morpholinos have been used to block miRNA activity and maturation.They can also block ribozyme activity. Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts.Activities of morpholinos against this variety of targets suggest that morpholinos can be used as a general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA.

7. Applications of Morpholinos:
There are three main applications of morpholinos which are:

a) Blocking mRNA translation
b) Blocking nuclear processing
c) Anti-sense therapeutics
Let us explain them one by one.

a) Blocking translation:
Bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the targeted transcript (called "knocking down" gene expression). This is useful experimentally when an investigator wishes to know the function of a particular protein.Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism. Some Morpholinos knock down expression so effectively that, after degradation of preexisting proteins, the targeted proteins become undetectable by Western blot.

b) Blocking nuclear processing:
Morpholinooligos can block nuclear processing events, pre-mRNA processing in particular. The power of high specificity and steric blocking allows one to specifically and reproducibly delete exons of choice by blocking access of the splicing machinery to the pre-mRNA. This technology, not possible with RNase-dependent or RISCdependentoligos (phosphorothioates, RNAi and others), not only allows characterizing specific exon function and creating loss-of-function deletions or insertions but it also allows researchers to eliminate a specific splice variant while leaving anothersplice variant of the same gene intact.

c) Anti-sense therapeutics:
Highly complementary antisense morpholino oligonucleotides (AMOs) can bind to pre-mRNA and modulate splicing site selection. This offers a powerful tool to regulate the splicing process,
such as correcting subtypes of splicing mutations and nonsense mutations and reprogramming alternative splicing processes. Therefore, AMO-mediated splicing modulation represents an attractive therapeutic strategy for genetic disorders. Primary immunodeficiency diseases (PIDs) are a heterogeneous group of genetic disorders that result from mutations in genes involved in development and maintenance of the immune system. Many of these mutations are splicing mutations and nonsense mutations that can be manipulated by AMOs.

8. DELIVERY OF MORPHOLINOS

To make a morpholino effective we have to effectively deliver it to the cytosol through the cell membrane. In cytosol they can freely diffuse and start functioning. Different methods are used for delivering morpholinos, some of which are mentioned below:

i. Delivery to Embryo:

Microinjection:
A microinjection apparatus is usually used for delivery into an embryo, with injections most commonly performed at the single-cell or few-cell stage.

Electroporation:
An alternative method for embryonic delivery is electroporation, which can deliver oligos into tissues of later embryonic stages.

“Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.”

ii. Delivery to Cultured cells:

One of the many features that make morpholino oligos unique among the antisense structural types is an uncharged backbone. While this feature eliminates the nonspecific interactions of traditional S-oligos, it also renders the morpholino undeliverable via the traditional lipid-based delivery systems.
So as a solution a nonionic morpholino oligo is paired to a complementary DNA "carrier." The DNA is then bound electro statically to a partially ionized, weakly-basic ethoxylated polyethylenimine (EPEI). This morpholino/DNA/EPEI complex is efficiently endocytosed, and when the pH drops within the endosome, the EPEI more fully ionizes, resulting in permeabilization of the endosomal membrane and release of the morpholino into the cytosol.

iii. Delivery to Adult cells:

Use of cell-penetrating peptides to help Morpholinos into cells, primarily through an endocytotic pathway followed by release from the endosome.

Systemic delivery into many cells in adult organisms can be accomplished by using covalent conjugates of Morpholino oligos with cell-penetrating peptides, and, while toxicity has been associated with moderate doses of the peptide conjugates, they have been used in vivo for effective oligo delivery at doses below those causing observed toxicity. An octa-guanidinium dendrimer attached to the end of a Morpholino can deliver the modified oligo (called a Vivo-Morpholino) from the blood to the cytosol. Delivery-enabled Morpholinos, such as peptide conjugates and Vivo-Morpholinos, show promise as therapeutics for viral and genetic diseases.

9. Vivo morpholinos

Vivo-Morpholinos are the knockdown, exon-skipping or miRNA blocking reagent of choice for in vivo experiments. Outstanding results can be achieved systemically with intravenous injection, and modest systemic delivery achieved with Vivo-Morpholinos by intraperitoneal (I.P.) injection. Efficient localized delivery can be achieved by injecting the Vivo-Morpholinos directly into the area of interest. If you wish to initially validate the oligo in cultured cells you can use the same Vivo-Morpholinos in cultures. However, unmodified Morpholinos, delivered with either electroporation or Endo-Porter, are more efficient in cell cultures than Vivo-Morpholinos

i. Composition:

A Morpholino molecule consists of a Morpholino oligo with a unique covalently linked delivery moiety, which is comprised of an octa-guanidine dendrimer. It uses the active component of arginine rich delivery peptides (the guanidinium group) with improved stability, low toxicity and reduced cost.
The Vivo-Morpholino is assembled by coupling the vivo-delivery group to a Morpholino while the oligo is still bound to its synthesis resin, allowing excellent purification by washing the solid-phase resin. Vivo-Morpholinos must be chosen prior to synthesis and cannot be added later because the vivo-delivery group is added to a Morpholino prior to cleavage from its synthesis resin.

ii. Delivery and dosage:

For best systemic delivery results the injection method of choice is I.V. although I.P. can also be used. The maximum suggested dosage in mammals is 12.5 mg/kg in a 24 hour period. This can be repeated daily. The dosage for short term and long term experiment differ. For short term the experiment lasts 2-3 days the dosage can be 12.5 mg/kg. While for the long term experiments the dosage is preferably started at dosage equivalent to the short term. The dosage and duration can be increased to optimize the results.
One can expect quantifiable knockdown or exon-skipping in liver, small intestine, colon, muscle, lung, and stomach tissues with lesser but quantifiable delivery in the spleen, heart, skin and brain.
It has been found that younger or older mice do not tolerate Vivo-Morpholinos as well and may require substantially lower doses. In addition the use of compromised mice, such as those with less robust genetic backgrounds, may require further dose limitations.

10. Problem Assosiated with Morpholinos:

Occasionally Morpholino oligos can fail and there are many factors which can contribute to oligo failure, some correctable ones determined.

1) Morpholino Oligos can lose activity if improperly stored or stored in anything other than sterile pure water. It needs to be stay fluffy and smooth, and should not turn hard palette like. To avoid this, if the oligo is to be stored long term keep it in a desiccator at room temperature. If the oligo was fluffy and there is still problems resuspending it, it may be due to high G content or reduced solubility due to an added moiety such as lissamine fluorescent tag. In these cases, try heating to 65C for 10 minutes and vortexing. Leaving the oligo overnight on a vigorous shaker might also help dissolution and Continue until completely resuspended.

2) Stock Morpholino oligo should be dissolved in sterile water without Diethylpyrocarbonate (DEPC) because DEPC forms bond with morphiline ring in the oligos backbone. It is possible for an oligo to lose activity if they have undergone long-term exposure to acid or DEPC.

3) Some highly expressed transcripts, like actin, may be difficult to shut down irrespective of oligo concentration because of high expressivity.

4) Specificity is reduced as concentration is increased. For this reason it is important to deliver only enough oligo to achieve near-quantitative shutdown without affecting non-specific targets.

5) There are other factors that can contribute to reduced activity which include potential secondary oligo targets and secondary structure within an oligo. The secondary targets can come from homologous genes. Secondary structure of an oligo can have a significant impact on activity. However, the concern is much greater for inter-strand pairing between oligos than intra-strand pairing within an oligo since single-molecule stem-loops do not readily form due to the oligo's limited flexibility, but inter-strand pairing can tie-up oligo and thus eliminate it from the pool of oligo that can pair with target sequence.

6) We cannot target point mutations with morpholinos. The property which gives Morpholino oligos their spectacular sequence specificity also largely precludes their use for effectively targeting point mutations. However, Morpholinos are the best tools for studying point mutations, polymorphisms, and a host of other genetic variations using the gene switch strategy or splice-blocking.

7) we cannot target the amino acid coding region of mRNA with morpholinos, because Morpholino oligos functions solely by a steric blocking mechanism, only the leader sequence and the first 25 bases of the amino acid coding region can be targeted for translation blocking applications.

8) Morpholino oligos enter unperturbed tissue culture cells but high concentrations (tens of µM) and long incubation times (multiple days) are required to achieve even very modest amounts of delivery in most cell types.

9) The following cells can NOT be delivered with the Morpholinos
a. All T-cell derived lines (apparently do not endocytose)
b. All differentiated colon carcinoma cell lines (also apparently do not endocytose)

10) Non-ionic Morpholinos will not complex with cationic lipids for delivery
11. Alternates of morpholino:

i. Small interfering RNA (siRNA):
Also known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants".

ii. MicroRNAs (miRNAs):
MicroRNAs are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long and are found in all eukaryotic cells, except fungi, algae, and marine plants. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger (mRNAs), usually resulting in translational repression and silencing. The human genome may encode over 1000 miRNAs, which may target about 60% of mammalian gene and are abundant in many human cell types.

Reference:

http://www.gene-tools.com/node/12

http://network.nature.com/groups/drugdelivery/forum/topics/359

http://nar.oxfordjournals.org/content/early/2007/08/01/nar.gkm478.full.pdf+html

http://www.reference.com/browse/morpholino

http://morpholinos.yuku.com/

http://www.gene-tools.com/node/25

http://www.gene-tools.com/faq

epigenetics

2011-07-27T08:52:00.000-07:00

GO TO THIS LINK
CLICK HERE ON ME

Homozygosity Mapping and LOD Score

2011-07-27T08:02:00.000-07:00

1. HOMOZYGOSITY MAPPING
Homozygosity is the state of possessing two identical forms of a particular gene (alleles), one inherited from each parent.
Homozygosity mapping is a method for mapping the human genome, used to detect genes that cause disease only when both copies in an individual are mutated (the genes are homozygous, or the same). This technique works for genetic disorders that are inherited from both parents, since inheriting a pair of heterozygous (different) genes results in expression of a non-mutated version from one parent, and the absence of disease symptoms. It is the way to map human recessive traits with the DNA of inbred children.
2. HOW IS HOMOZYGOSITY MAPPING DONE
Homozygosity mapping can be done by either of the following methods
1. SNPArrays
2. RFLP
3. Microsatellite markers

2.1. SNP ARRAYS FOR HOMOZYGOSITY MAPPING
It was proposed that a set of SNPs evenly spread across the human genome could be used to screen two populations (typically populations with and without a disorder) and that some SNPs would associate more with the disease group, thus implicating the SNP, or a DNA sequence close by, in the disease state. A massive technological effort followed and whole genome scans, using tens of thousands of SNPs, were made a reality with the advent of array-based technologies.
One recent advance is the development of high-density SNP microarrays for genotyping. The SNP arrays overcome low marker informativity by using a large number of markers to achieve greater coverage at finer resolution.

BARDET-BIEDL SYNDROME
We used SNP microarray genotyping for homozygosity mapping in a small consanguineous Israeli Bedouin family with autosomal recessive Bardet-Biedl syndrome (BBS; obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal and cardiac abnormalities, and cognitive impairment) in which previous linkage studies using short tandem repeat (STR) polymorphisms failed to identify a disease locus.
SNP genotyping revealed a homozygous candidate region. Mutation analysis in the region of homozygosity identified a conserved homozygous mis-sense mutation in the TRIM32 gene, a gene coding for an E3 ubiquitin ligase. Functional analysis of this gene in zebra-fish and expression correlation analyses among other BBS genes in an expression quantitative trait loci data set demonstrate that TRIM32 is a BBS gene. This study shows the value of high-density SNP genotyping for homozygosity mapping and the use of expression correlation data for evaluation of candidate genes and identifies the proteosome degradation pathway as a pathway involved in BBS.
Positional cloning is a powerful approach to identify genes mutated in human and animal models monogenic (single gene case) diseases. The genomic DNA region (and the embedded polymorphisms) harboring the disease-causing mutations segregates with the disease in analyzed pedigrees. However, positional cloning relayed until recently on the availability of large pedigrees to reach a significant linkage.
This protocol describes the use of whole genome genotyping on sporadic consanguineous patients to identify potential disease loci and subsequent positional candidate genes, by homozygosity mapping (autozygosity). It takes advantage of high density single nucleotide polymorphism (SNP) genotyping arrays, and of the assumption that unrelated patients from several consanguineous families are mutated in the same gene.

2.2. RFLP (RESTRICTION FRAGMENT LENGTH POLYMORPHISM)
RFLP is a method used by molecular biologists to follow a particular sequence of DNA as it is passed on to other cells. It is technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites. By cutting two different DNA molecules with the same restriction enzyme, scientists can compare the lengths of the fragments; two identical molecules will have identical fragments, while two similar molecules may be largely alike, with perhaps a few differences in fragment size. These differences in restriction fragment lengths are called polymorphisms and are used in all types of DNA typing.
RFLP PRODUCTION
RFLP methodology involves
• Cutting a particular region of DNA with known variability, with restriction enzymes
• Separating the DNA fragments by agarose gel electrophoresis
• Determining the number of fragments and relative sizes
The pattern of fragment sizes will differ for each individual tested.
APPLICATIONS
RFLP technique has many applications like
DNA fingerprinting in forensic science
Tracing ancestry
Studying evolution and migration of wild life
Detection and diagnosis of certain diseases
Genetic mapping (to calculate the genetic distance between two loci)
2.3. MICROSATELLITE MARKERS
Microsatellites are simple sequence tandem repeats (SSTRs). The repeat units are generally di-, tri- tetra- or pentanucleotides. For example, a common repeat motif in birds is ACn, where the two nucleotides A and C are repeated in bead-like fashion a variable number of times (n could range from 8 to 50). They tend to occur in non-coding regions of the DNA (this should be fairly obvious for long dinucleotide repeats) although a few human genetic disorders are caused by (trinucleotide) microsatellite regions in coding regions. On each side of the repeat unit are flanking regions that consist of "unordered" DNA. The flanking regions are critical because they allow us to develop locus-specific primers to amplify the microsatellites with PCR (polymerase chain reaction). That is, given a stretch of unordered DNA 30-50 base pairs (bp) long, the probability of finding that particular stretch more than once in the genome becomes vanishingly small. In contrast, a given repeat unit (say AC19) may occur in thousands of places in the genome. We use this combination of widely occurring repeat units and locus-specific flanking regions as part of our strategy for finding and developing microsatellite primers. The primers for PCR will be sequences from these unique flanking regions. By having a forward and a reverse primer on each side of the microsatellite, we will be able to amplify a fairly short (100 to 500 bp, where bp means base pairs) locus-specific microsatellite region.
P.S SEE USES IN PRESENTATION.
3. HOMOZYGOSITY MAPPER
It is a web based approach for homozygosity mapping with a store of marker data in a database into which users can upload their SNP genotype files. Database analyses the data in a few minutes, detects homozygous portions (alleles) and provides agraphical interface of the results. Software also provides the option to zoom into single chromosomes anduser-defined chromosomal regions. It is integrated with a gene search engine GeneDistiller which enables users to determine most promising gene. Users can restrict access or make their uploaded sequences public. Hence homozygosity mapper can be used as a data repository for homozygosity mapping based researches.
4. GENETIC LINKAGE
Genetic linkage means that certain genes tend to be inherited together, because they are on the same chromosome. Thus parental combinations of characters are found more frequently in offspring than non parental. Genetic loci that are physically close to one another on the same chromosome tend to stay together during meiosis, and are thus genetically linked.

DISCOVERY
In 1905 the three geneticists William Bateson, Edith Rebecca Saunders, and Reginald C.Punnett discovered an apparent exception to one of Mendel's foundational proposals: the principle of independent assortment.
In their work with pea plants, these researchers noticed that not all of their crosses yielded results that reflected the principle of independent assortment specifically, some phenotypes appeared far more frequently than traditional Mendelian genetics would predict. Based on these findings, they proposed that certain alleles must somehow be coupled with one another, although they weren't sure how this linkage occurred. The answer to this question came just seven years later, when Thomas Hunt Morgan used fruit flies to demonstrate that linked genes must be real physical objects that are located in close proximity on the same chromosome.
In 1910, Morgan discovered a fly with mutant white eyes while normally fruit flies have red eyes, not white eyes. Morgan crossed this white eyed male fly to its red eyed sisters. Later he inbred the heterozygous F1 red-eyed flies, the traits of the F2 progeny did not assort independently. Morgan expected a 1:1:1:1 ratio of red-eyed females, red-eyed males, white-eyed males, and white-eyed females. Instead, he observed the following phenotypes in his F2 generation:
2,459 red-eyed females
1,011 red-eyed males
782 white-eyed males
There were no white-eyed females, and Morgan wondered whether this was because the trait was sex-limited and only expressed in male flies. To test whether this trait was sex limited he completed a second cross between the original white-eyed male fly and some of his F1 daughters. These crosses produced an F2 generation with the following phenotypes:
129 red-eyed females
132 red-eyed males
88 white-eyed females
86 white-eyed males
Thus, the results of this cross did produce white-eyed females, and the groups had approximately equal numbers. Morgan therefore hypothesized that the eye color trait was connected with the sex factor. This in turn led to the idea of genetic linkage, which means that when two genes are closely associated on the same chromosome, they do not assort independently.
LINKAGE MAP
A linkage map is a genetic map of a species or experimental population that shows the position of its known genes or genetic markers relative to each other in terms of recombination frequency, rather than as specific physical distance along each chromosome. Linkage mapping is critical for identifying the location of genes that cause genetic diseases.
A genetic map is a map based on the frequencies of recombination between markers during crossover of homologous chromosomes. The greater the frequency of recombination (segregation) between two genetic markers, the farther apart they are assumed to be. Conversely, the lower the frequency of recombination between the markers, the smaller the physical distance between them. Historically, the markers originally used were detectable phenotypes derived from coding DNA sequences; eventually, confirmed or assumed non-coding DNA sequences such as microsatellites or those generating restriction fragment length polymorphisms (RFLPs) have been used.
Genetic maps help researchers to locate other markers, such as other genes by testing for genetic linkage of the already known markers.
A genetic map is not a physical map (such as a radiation reduced hybrid map) or gene map.
A map of the genes on a chromosome based on linkage analysis. A linkage map does not show the physical distances between genes but rather their relative positions, as determined by how often two gene loci are inherited together. The closer two genes are (the more tightly they are linked), the more often they will be inherited together.
Linkage distance is measured in centimorgans (cM).
CONSTRUCTING A GENETIC LINKAGE MAP
Genetic linkage maps of each chromosome are made by determining how frequently two markers are passed together from parent to child. Because genetic material is sometimes exchanged during the production of sperm and egg cells, groups of traits (or markers) originally together on one chromosome may not be inherited together. Closely linked markers are less likely to be separated by spontaneous chromosome rearrangements. In this diagram, the vertical lines represent chromosome 4 pairs for each individual in a family. The father has two traits that can be detected in any child who inherits them: a short known DNA sequence used as a genetic marker (M) and Huntingtons disease (HD). The fact that one child received only a single trait (M) from that particular chromosome indicates that the fathers genetic material recombined during the process of sperm production. The frequency of this event helps determine the distance between the two DNA sequences on a genetic map.

5. LOD SCORE METHOD FOR ESTIMATING RECOMBINATION FREQUENCY

The LOD score (logarithm (base 10) of odds), developed by Newton E. Morton, is a statistical test often used for linkage analysis in human, animal, and plant populations. The LOD score compares the likelihood of obtaining the test data if the two loci are indeed linked, to the likelihood of observing the same data purely by chance. Positive LOD scores favor the presence of linkage, whereas negative LOD scores indicate that linkage is less likely. Computerized LOD score analysis is a simple way to analyze complex family pedigrees in order to determine the linkage between Mendelian traits (or between a trait and a marker, or two markers).
The method is described in greater detail by Strachan and Read. Briefly, it works as follows:
Establish a pedigree
Make a number of estimates of recombination frequency
Calculate a LOD score for each estimate
The estimate with the highest LOD score will be considered the best estimate
The LOD score is calculated as follows:

Where:
 NR denotes the number of non-recombinant offspring,
 R denotes the number of recombinant offspring.
 Theta is the recombinant fraction, it is equal to R / (NR + R)
The reason 0.5 is used in the denominator is that any alleles that are completely unlinked (e.g. alleles on separate chromosomes) have a 50% chance of recombination, due to independent assortment.
In practice, LOD scores are looked up in a table which lists LOD scores for various standard pedigrees and various values of recombination frequency.
By convention, a LOD score greater than 3.0 is considered evidence for linkage. A LOD score of +3 indicates 1000 to 1 odds that the linkage being observed did not occur by chance. On the other hand, a LOD score less than -2.0 is considered evidence to exclude linkage. Although it is very unlikely that a LOD score of 3 would be obtained from a single pedigree, the mathematical properties of the test allow data from a number of pedigrees to be combined by summing the LOD scores. It is important to keep in mind that this traditional cutoff of LOD>+3 is an arbitrary one and that the difference between certain types of linkage studies, particularly analyses of complex genetic traits with hundreds of markers, these criteria should probably be modified to a somewhat higher cutoff.
5.1. MAPPING GENES WITH THE LOD SCORE METHOD
The dilemma of mapping genes can be overcome through the "lod score method" which involves the estimation of genetic distances in the situations other than simple testcrosses. The data obtained from the pedigree is used to calculate the map distances from the recombination frequencies. It is one of the basic and fundamental human genetics methods used today. The use of spread sheet programs i.e. Lotus 1-2-3 or Microsoft Excel makes the solution of this predicament effortless.
WHAT IS NEEDED?
The fundamental aim of this problem is to determine R, the recombinant fraction (fraction of gametes that are recombinant), using data from relatively small families. R can vary from 0 (2 genes completely linked) to 0.50 (2 genes unlinked).
STEPS INVOLVED
There are 4 basic steps in the method:-
(1) Determine the expected frequencies of F2 phenotypes for every value of R from 0.01 to 0.50
(2) Determine the "likelihood" (L) that the family data observed resulted from a given R value: the maximum likelihood is the best estimate of R for the given data
(3) Determine the Odds Ratio and the logarithm of the odds ratio (lod score) by comparing the Likelihood for each value of R to the Likelihood for unlinked genes (R = 0.50)
(4) Add LOD scores from different families to achieve an acceptably high lod score so a specific most likely R can be assigned.
The following example used for consideration comprises of:-
Two genes showing the complete dominancethe heterozygote is indistinguishable from the dominant homozygote
STEP 1: CALCULATE THE EXPECTED FREQUENCY OF OFFSPRING FOR VALUES OF R FROM 0 TO 0.50
The expected offspring numbers are calculated as follows:
Determine the frequency of each gamete produced by the F1's. For example, if R= 0.20, then 20% of the gametes produced by either parent will be recombinant. Since there are two types of recombinant gamete, A b and a B, the frequency of each will be 0.10. Since 80% of the gametes will be parental, the frequency of the parental types A B and a b will be 0.40 each.
Use a Punnett square to determine the offspring being formed from the union of the gametes. Multiply the gamete frequencies to get the offspring frequency. For instance, one cell of the Punnett square has the A B gamete from the father combining with the A b gamete from the mother. The frequency of the A B gamete is 0.40 and the frequency of the A b gamete is 0.10. Thus the frequency of the offspring in this cell is 0.40 x 0.10 = 0.04.
Determine the phenotype for each cell in the Punnett square and add up the frequencies to get the total frequency for each offspring phenotype.

A B
0.40 A b
0.10 a B
0.10 a b
0.40
A B
0.40 A B/A B
0.16 A b/A B
0.04 a B/A B
0.04 a b/A B
0.16
A b
0.10 A B/A b
0.04 A b/A b
0.01 a B/A b
0.01 a b/A b
0.04
a B
0.10 A B/a B
0.04 A b/a B
0.01 a B/a B
0.01 a b/a B
0.04
a b
0.40 A B/a b
0.16 A b/a b
0.04 a B/a b
0.04 a b/a b
0.16

F2 Phenotype Cell Sums Expected Freq
A_ B_ 0.16+.04+.04+.16+.04+.01+.04+.01+0.16 0.66
A_ bb 0.01 + 0.04 + 0.04 0.09
aa B_ 0.01 + 0.04 + 0.04 0.09
aa bb 0.16 0.16
Using a Punnett square to determine the genotypes and multiplying the frequencies of the two gametes that go into each type of offspring, then adding up offspring that have the same phenotype.
STEP 2: EXAMINE THE OBSERVED FAMILY DATA IN LIGHT OF THE EXPECTED DISTRIBUTION OF OFFSPRING FOR EACH R VALUE
This is done by determining the likelihood (L) of the observed family for each value of R. The likelihood is simply the probability of the observed family, as determined using the multinomial theorem, an extension of the binomial theorem.

First define the terms for the observed family:
a = number of A_ B_ offspring
b = number of A_ bb offspring
c = number of aa B_ offspring
d = number of aa bb offspring
n = total offspring (= a + b + c + d)
Then define the terms for the expected family proportions (obtained from step 1 above):
p = expected proportion of A _ B _ offspring
q = expected proportion of A_ bb offspring
r = expected proportion of aa B_ offspring
s = expected proportion of aa bb offspring
The term of the multinomial equation that describes the actual family is: pa qb rc sd multiplied by a coefficient.
The coefficient is: n! /(a! b! c! d!), where ! means "factorial".
This is very similar to the coefficient for the binomial.
Thus, the likelihood equation is: L = [n! /(a! b! c! d!)]pa qb rc sd
Above calculated the expected phenotype proportions for R = 0.20 (20 map units between A and B). They are: A_ B_ = 0.66; A_ bb = 0.09; aa B_ = 0.09; aa bb = 0.16. A family of 5 children has 2 with the A_ B_ phenotype, 1 with aa B_, and 2 with aa bb.
L = (5!/2! 0! 1! 2!)(.66)2(.09)0(.09)1(.16)2
L = 30(.4356)(.09)(.0256)
L = 0.0301
The likelihood (L) needs to be calculated for all values of R between 0.01 and 0.50. Note that the coefficient will be the same for all values of R; the coefficient only depends on the observed data. When this is done, the value of R with the highest likelihood is the best estimate of R that can be obtained with data from this particular family.
STEPS 3 AND 4: COMBINING DATA FROM SEVERAL FAMILIES
The data needs to be compared and added from several different families, to get a good estimate of R. To do this, the L values must be standardized by calculating the Odds Ratio (OR), which is the ratio of the L for each R value divided by the L for R = 0.50 (unlinked). Then, the logarithm of the Odds ratio is taken; this is the LOD score. LOD scores from different families can be added. (This is equivalent to multiplying the Odds Ratios, as in the AND rule for two events--family 1 AND family 2--both occurring.) A total LOD score for some R value of 3.0 is considered proof of linkage between the two genes.
For R = 0.20, the Odds Ratio = L0.20 / L0.50. We calculated L0.20 = 0.0301 above; L0.50 = 0.00695. The Odds ratio is thus 4.331 and the LOD score is the base 10 logarithm of this, 0.637. Clearly it would take several families of this size to reach a LOD score of 3.0.

REFRENCES
Discovery and Types of Genetic Linkage By: Ingrid Lobo, Ph.D. (Write Science Right) & Kenna Shaw, Ph.D. (Executive Editor, Nature Education) © 2008 Nature Education Citation: Lobo, I. & Shaw, K. (2008) Discovery and types of genetic linkage. Nature Education 1(1)
http://www.bios.niu.edu/johns/lodprob.htm
http://www.ornl.gov/sci/techresources/Human_Genome/publicat/primer/fig8.html
http://www.bio.davidson.edu/courses/genomics/method/RFLP.html
http://biotech.about.com/od/glossary/g/RFLPdef.htm
http://www.nlm.nih.gov/visibleproofs/education/dna/rflp.pdf
http://www.biology-online.org/dictionary/Genetic_linkage
European Journal of Human Genetics (2007) 15, 362–368. doi:10.1038/sj.ejhg.5201761.
Protocol Exchange (2007) doi:10.1038/nprot.2007.343.

my project on RIBOZYMES

2011-07-27T07:49:00.001-07:00

RIBOZYMES__________________________________________
The structural molecular biology of ribozymes took another great leap forward during the past two years. Before ribozymes were discovered in the early 1980s, all enzymes were thought to be proteins. No detailed structural information on ribozymes became available until 1994. Now, within the past two years, near atomic resolution crystal structures are available for almost all of the known ribozymes. The latest additions include ribonuclease P, group I intron structures, the ribosome (the peptidyl transferase appears to be a ribozyme) and several smaller ribozymes, including a Diels–Alderase, the glmS ribozyme and a new hammerhead ribozyme structure that reconciles 12 years of discord. Although not all ribozymes are metalloenzymes, acid-base catalysis appears to be a universal property shared by all ribozymes as well as many of their protein cousins
Introduction
Ribozymes are enzymes whose catalytic centers are composed entirely of RNA and therefore do not require proteins for catalysis (although many exist naturally as RNA–protein complexes).
All ribozymes were believed originally to be metalloenzymes, requiringMg2+ or other divalentmetal ions for both folding and catalysis. A ‘two-metal mechanism’ had been proposed in which hydratedMg2+ ions played the roles of general acids and bases. This prediction appears to have been correct for the group I intron. Acid-base catalysis appears to be a catalytic strategy so fundamental that it occurs in both protein and RNA enzymes; in many cases, it seems that the RNA itself, rather than acting as a passive scaffold for metal ion binding, is an active participant in acid-base catalysis in the sense that nucleotide functional groups, rather than metal complexes, often mimic the roles that amino acids play in the active sites of protein enzymes. Several of the small self-cleaving RNAs as a consequence do not strictly require divalent metal ions for catalysis and no divalent metal ions have yet been observed in the
active site of the peptidyltransferase, the ribozyme that is embedded in the ribosome.

Ribonuclease P
Ribonuclease P (RNase P) was the ﬁrst true RNA enzyme identiﬁed. An RNA–protein complex, the catalytic subunit of bacterial RNase P is composed entirely of RNA (and it is thought that this is the case with the eukaryotic version as well). It processes precursor tRNAs and other RNAs required for cellular metabolism.

Group I intron
The folds of the various group I introns are quite similar, permitting comparisons between molecular species. The ﬁrst Azoarcus structure was in a pre-catalytic state, in which both exons (the substrate of the reaction in which adjacent exons are spliced as the intron excises itself) were present. The Tetrahymena group I intron structure represents a state in which the 30 -terminal v-guanosine and a metal ion are present in the active site. The newer structures complement these two states with an enzyme– product complex, and a complex in which all substrate, ribozyme functional groups and predicted metal ions are present in the active site.

A Diels–Alderase ribozyme
The protein Diels–Alderase is a catalytic antibody whose structure is known. The structure of a Diels–Alder ribozyme is in both the unbound and enzyme–product complex states, revealing that the ribozyme uses a combination of proximity, spatial complementarity and electronic effects to activate stereoselective catalysis, reminiscent of the protein Diels–Alderase.

The glmS ribozyme
The glmS ribozyme is a recently discovered ribozyme that is unique in the world of naturally occurring ribozymes in two respects. First, it is a ribozyme that is also a riboswitch. Second, the regulatory effector of the ribozyme, glucosamine-6-phosphate (GlcN6P), is actually a functional group that binds to the ribozyme active site and participates in the acid-base catalysis of RNA self-cleavage. The glmS ribozyme is derived from a self-cleaving RNA sequence found in the 50 -untranslatedregion (50 - UTR) of the glmS message; it cleaves itself, inactivating the message, when the cofactor GlcN6P binds. GlcN6P production is thus regulated in many Gram-positive bacteria via this ribozyme-mediated negative-feedback mechanism.

The hammerhead ribozyme
Because it is small and has a simple cleavage mechanism, the hammerhead ribozyme is perhaps the best experimentally characterized RNA enzyme, and therefore, is a clear candidate for
Structural studies. The hammerhead motif consists of three base-paired stems flanking a central core of 15 conserved nucleotides. The conserved central bases are essential for ribozyme activity. Most of these conserved bases cannot form conventional Watson-Crick base pairs, but instead form more complex structures, which mediate RNA folding and catalysis. Substitution of any of the conserved bases with other naturally occurring bases, or sometimes even artificial alteration of their functional groups, results in diminished catalytic activity. In addition, two sets of base pairs in stem III and one pair in stem II are conserved; changing these to other base pairs either impairs or abolishes catalytic function.

my project on GENE THERAPY

2011-07-27T07:47:00.000-07:00

GENE THERAPY_________________________________________
GENE THERAPY

Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease.
Gene therapy is an experimental technique that uses genes to treat or prevent disease. 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.
It is a technique for correcting defective genes that are responsible for disease development. The most common form of gene therapy involves the insertion of functional genes into an unspecified genomic location in order to replace a mutatedgene, but other forms involve directly correcting the mutation or modifying normal gene that enables a viral infection. Although the technology is still in its infancy, it has been used with some success.

APPROACHES OF GENE THERAPY

• 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.

TYPES OF GENE THERAPY_______________________________
Gene therapy may be classified into the two following types:
Germ line gene therapy
In the case of germ line gene therapy, germ cells, i.e., sperm or eggs are modified by the introduction of functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations. This new approach, theoretically, should be highly effective in counteracting genetic disorders and hereditary diseases. However, many jurisdictions prohibit this for application in human beings, at least for the present, for a variety of technical and ethical reasons.
Somatic gene therapy
In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations.
Somatic cells are non-reproductive. Somatic cell therapy is viewed as a more conservative, safer approach because it affects only the targeted cells in the patient, and is not passed on to future generations. In other words, the therapeutic effect ends with the individual who receives the therapy. This type of therapy presents unique problems of its own. Often the effects of somatic cell therapy are short-lived. Because the cells of most tissues ultimately die and are replaced by new cells, repeated treatments over the course of the individual's life span are required to maintain the therapeutic effect. Transporting the gene to the target cells or tissue is also problematic.
Regardless of these difficulties, however, somatic cell gene therapy is appropriate and acceptable for many disorders, including cystic fibrosis, muscular dystrophy, cancer, and certain infectious diseases. Clinicians can even perform this therapy in utero, potentially correcting or treating a life-threatening disorder that may significantly impair a baby's health or development if not treated before birth.

CATEGORIES OF SOMATIC GENE THERAPY________________________
Somatic gene therapy can be broadly split into two categories:

EX VIVO TECHNIQUE
Ex vivo, which means exterior (where cells are modified outside the body and then transplanted back in again). In some gene therapy clinical trials, cells from the patient’s blood or bone marrow are removed and grown in the laboratory. The cells are exposed to the virus that is carrying the desired gene. The virus enters the cells and inserts the desired gene into the cells’ DNA. The cells grow in the laboratory and are then returned to the patient by injection into a vein. This type of gene therapy is called ex vivo because the cells are treated outside the body.
IN VIVO TECHNIQUE
In vivo, which means interior (where genes are changed in cells still in the body). This form of gene therapy is called in vivo, because the gene is transferred to cells inside the patient’s body.

Uses of gene therapy
Gene therapy is being used in many ways. For example, to:
• Replace missing or defective genes;
• Deliver genes that speed the destruction of cancer cells;
• Supply genes that cause cancer cells to revert back to normal cells;
• Deliver bacterial or viral genes as a form of vaccination;
• Provide genes that promote or impede the growth of new tissue; and;
• Deliver genes that stimulate the healing of damaged tissue.
A large variety of genes are now being tested for use in gene therapy.
Examples : a gene for the treatment of cystic fibrosis (a gene called CFTR that regulates chloride); genes for factors VIII and IX, deficiency of which is responsible for classic hemophilia (hemophilia A) and another form of hemophilia (hemophilia B), respectively; genes called E1A and P53 that cause cancer cells to undergo cell death or revert to normal; AC6 gene which increases the ability of the heart to contract and may help in heart failure; and VEGF, a gene that induces the growth of new blood vessels (angiogenesis) of use in blood vessel disease.
A short synthetic piece of DNA (called an oligonucleotide) is being used by researchers to "pre-treat" veins used as grafts for heart bypass surgery. The piece of DNA seems to switch off certain genes in the grafted veins to prevent their cells from dividing and thereby prevent atherosclerosis.
Delivery of genes into cells
Genes can be carried into cells by viruses. Viral vectors or carriers take advantage of the natural ability of a virus to enter a cell and deliver genetic material to the nucleus of the cell that contains its DNA. In developing virus carriers, the DNA coding for some or all of the normal genes of the virus to be used as a carrier are removed and replaced with a treatment gene. Most of these virus carriers are engineered so that they are able to enter cells, but they cannot reproduce themselves and so are innocuous.
Genes can also be delivered within tiny synthetic "envelopes" of fat molecules. Cell membranes contain a very high concentration of fat molecules. The fat molecule "envelope" can carry the therapeutic gene into the cell by being admitted through the cell membrane as if it were one of its own molecules.
Genes can also gain entrance into cells when an electrical charge is applied to the cell to create tiny openings in the membrane that surrounds a cell. This technique is called electroporation.
Route of administration of gene therapy
The choice of route for gene therapy depends on the tissue to be treated and the mechanism by which the therapeutic gene exerts its effect. Gene therapy for cystic fibrosis, a disease which effects cells within the lung and airway, may be inhaled. Most genes designed to treat cancer are injected directly into the tumor. Proteins such as factor VIII or IX for hemophilia are also being introduced directly into target tissue (the liver).

PROBLEMS OF GENE THERAPY____________________________
Some of the problems of gene therapy include:
• Short-lived nature of gene therapy – Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
• Immune response – Anytime a foreign object is introduced into human tissues, the immune system has evolved to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a possibility. Furthermore, the immune system's enhanced response to invaders that it has seen before makes it difficult for gene therapy to be repeated in patients.
• Problems with viral vectors – Viruses, the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient —toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
• Multigene disorders – Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.
• Chance of inducing a tumor (insertional mutagenesis) - If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.

APPLICATIONS OF GENE THERAPY________________________
A "bionic chip"
A new "bionic chip" has been developed to help gene therapists using electroporation to slip fragments of DNA into cells. Electroporation was originally a hit-or-miss technique because there was no way to determine how much of an electrical jolt it took to open the cell membrane.
The "bionic chip" solves this problem. It contains a single living cell embedded in a tiny silicon circuit. The cell acts as a diode, or electrical gate. When it is hit with just the right charge, the cell membrane opens, allowing the electricity to pass from the top to the bottom of the bionic chip. By recording what voltage caused this phenomenon to occur, it is now posssible to determine precisely how much electricity it takes to pry open different types of cells.

GENE THERAPY IN FUTURE______________________________
The potential of gene therapy is great but, compared to its promise, the results to date are still quite limited. However, the benefits of gene therapy are believed to be on the near horizon. Gene therapy is one of the hottest areas of medical research today.
The remarkable advances in genetics, including the human genome project, have opened new doors for the exploration of gene therapy. New technologies are needed to speed the progress of gene therapy. As these new technologies such as the "bionic chip" arrive, we believe that, without a doubt, gene therapy will play an increasingly important and prominent part in medicine in the decades to come.

rna-structure-prediction

2011-07-23T11:13:00.000-07:00

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Developmental genetics

2011-07-23T10:21:00.001-07:00

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http://www.slideshare.net/xulkifal/developmental-genetics-4294650
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dna and rna Viruses

2011-07-23T10:19:00.000-07:00

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Genes anticipation

2010-07-03T00:29:00.000-07:00

In genetics, anticipation is a phenomenon whereby the symptoms of a genetic disorder become apparent at an earlier age as it is passed on to the next generation. In most cases, an increase of severity of symptoms is also noted. Anticipation is common in trinucleotide repeat disorders such as Huntington's disease and myotonic dystrophy where a dynamic mutation in DNA occurs. All of these diseases have neurological symptoms. Prior to the understanding of the genetic mechanism for anticipation, it was debated whether anticipation was a true biological phenomenon or whether the earlier age of diagnosis was related to heightened awareness of disease symptoms within a family.

Trinucleotide Repeats and Expansion

Trinucleotide repeats are apparent in a number of loci in the human genome. They have been found in introns, exons and 5' or 3' UTR's. They consist of a pattern of three nucleotides (e.g. CGG) which is repeated a number of times. During meiosis, unstable repeats can undergo triplet expansion (see later section); in this case, the germ cells produced have a greater number of repeats than are found in the somatic tissues.
The mechanism behind the expansion of the triplet repeats is not well understood. One hypothesis is that the increasing number of repeats influence the overall shape of the DNA, which can have an effect on its interaction with DNA polymerase and thus the expression of the gene.
Disease mechanisms
For many of the loci, trinucleotide expansion is harmless, but in some areas expansion has detrimental effects that cause symptoms. When the trinucleotide repeat is present within the protein-coding region, the repeat expansion leads to production of a mutant protein with gain of function. This is the case for Huntington's disease, where the trinucleotide repeat encodes a long stretch of glutamine residues. When the repeat is present in an untranslated region, it could affect the expression of the gene in which the repeat is found (ex. fragile X) or many genes through a dominant negative effect (ex. myotonic dystrophy).
In order to have a deleterious effect, the number of repeats must cross a certain threshold. For example, normal individuals have between 5 and 30 CTG repeats within the 3' UTR of DMPK, the gene that is altered in myotonic dystrophy. If the number of repeats becomes greater than 50, the person is only mildly affected - perhaps having only cataracts. However, meiotic instability could result in a dynamic mutation that increases the number of repeats in offspring inheriting the mutant allele. Once the number of copies reaches over 100, the disease will manifest earlier in life (although the individual will still reach adulthood before the symptoms are evident) and the symptoms will be more severe - including electrical myotonia. As the number progresses upwards past 400, the symptoms show themselves during childhood or infancy.

Examples of Diseases showing Anticipation

Some examples of diseases showing anticipation, and some of the corresponding repeat sequences.

Spinocerebellar ataxia

Spinocerebellar ataxia (SCA) is a progressive, degenerative, genetic disease with multiple types, each of which could be considered a disease in its own right. The first ataxia gene was identified in 1993 for a dominantly inherited type. It was called “Spinocerebellar ataxia type 1" (SCA1). Subsequently, as additional dominant genes were found they were called SCA2, SCA3, etc. Usually, the "type" number of "SCA" refers to the order in which the gene was found. At this time, there are at least 29 different gene mutations which have been found (not all listed).
Many SCAs below fall under the category of polyglutamine diseases, which are caused when a disease-associated protein (i.e. ataxin-1, ataxin-3, etc.) contains a glutamine repeat beyond a certain threshold. In most dominant polyglutamine diseases, the glutamine repeat threshold is approximately 35, except for SCA3 which is beyond 50. Polyglutamine diseases are also known as "CAG Triplet Repeat Disorders" because CAG is the codon which codes for the amino acid glutamine. Many prefer to refer to these also as polyQ diseases since "Q" is the one-letter reference for glutamine.

Signs and symptoms

Spinocerebellar ataxia (SCA) is one of a group of genetic disorders characterized by slowly progressive incoordination of gait and often associated with poor coordination of hands, speech, and eye movements. Frequently, atrophy of the cerebellum occurs, and different ataxias are known to affect different regions within the cerebellum.
As with other forms of ataxia, SCA results in unsteady and clumsy motion of the body due to a failure of the fine coordination of muscle movements, along with other symptoms.
The symptoms of an ataxia vary with the specific type and with the individual patient. Generally, a person with ataxia retains full mental capacity but may progressively lose physical control.

Genetics: A conceptual Approach & Problem Solving CD-ROM

CAUSES
The hereditary ataxias are categorized by mode of inheritance and causative gene or chromosomal locus. The hereditary ataxias can be inherited in an autosomal dominant, autosomal recessive, or X-linked manner.

Many types of autosomal dominant cerebellar ataxias are now known for which specific genetic information is available. Synonyms for autosomal dominant cerebellar ataxias (ADCA) used prior to the current understanding of the molecular genetics were Marie's ataxia, inherited olivopontocerebellar atrophy, cerebello-olivary atrophy, or the more generic term "spinocerebellar degeneration." (Spinocerebellar degeneration is a rare inherited neurological disorder of the central nervous system characterized by the slow degeneration of certain areas of the brain. There are three forms of spinocerebellar degeneration: Types 1, 2, 3. Symptoms begin during adulthood.)

There are five typical autosomal recessive disorders in which ataxia is a prominent feature: Friedreich ataxia, ataxia-telangiectasia, ataxia with vitamin E deficiency, ataxia with oculomotor apraxia (AOA), spastic ataxia. Disorder Subdivisions: Friedreich's ataxia, Spinocerebellar ataxia, Ataxia telangiectasia, Vasomotor ataxia, Vestibulocerebellar, Ataxiadynamia, Ataxiophemia, Olivopontocerebellar atrophy, and Charcot-Marie-Tooth disease.

There have been reported cases where a polyglutamine expansion may lengthen when passed down, which often can result in an earlier age-of-onset and a more severe disease phenotype for individuals who inherit the disease allele. This falls under the category of genetic anticipation.

Treatment

There is no known cure for spinocerebellar ataxia, which is a progressive disease (it gets worse with time), although not all types cause equally severe disability.
Treatments are generally limited to softening symptoms, not the disease itself. The condition is considered to be irreversible. A person with this disease will usually end up needing to use a wheelchair, and eventually they may need assistance to perform daily tasks.
The treatment of incoordination or ataxia, then mostly involves the use of adaptive devices to allow the ataxic individual to maintain as much independence as possible. Such devices may include a cane, crutches, walker, or wheelchair for those with impaired gait; devices to assist with writing, feeding, and self care if hand and arm coordination are impaired; and communication devices for those with impaired speech.
Many patients with hereditary or idiopathic forms of ataxia have other symptoms in addition to ataxia. Medications or other therapies might be appropriate for some of these symptoms, which could include tremor, stiffness, depression, spasticity, and sleep disorders, among others.
Both onset of initial symptoms and duration of disease can be subject to variation. If the disease is caused by a polyglutamine trinucleotide repeat CAG expansion, a longer expansion may lead to an earlier onset and a more radical progression of clinical symptoms.

CLONING

2010-07-01T01:20:00.000-07:00

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Cloning
- “ Cloning” in biology is the process of similar producing populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually.
Types Of Cloning
- Reproductive cloning
- Therapeutic cloning
- Recombinant DNA cloning
Reproductive Cloning
- Reproductive cloning is a type of cloning which is performed for the purpose of creating a duplicate copy of another organism.
- In somatic cell nuclear transfer, scientists extract the nucleus of a somatic cell, a cell which can come from anywhere in the body, and insert it into an egg which has had its nucleus removed.
Continued…
- The egg is stimulated, and it begins dividing and growing, developing into an embryo which can be implanted into a gestational surrogate and carried to term.
- It is also possible to manipulate the genetic material used in reproductive cloning using recombinant DNA technology to alter DNA.
Therapeutic Cloning
- Therapeutic cloning is cloning which is performed for the purpose of medical treatment. For example, it could theoretically be used to grow a replacement organ, to generate skin for a burn victim, or to create nerve cells for someone suffering from brain damage or a neurological condition.
Formally, this type of cloning is called somatic cell nuclear transfer. It involves extracting the nucleus of a cell, and putting the nucleus into an egg which has been de-nucleated. Then, the egg is allowed to divide and grow. In therapeutic cloning, the growing egg is used as a source of stem cells, which are undifferentiated cells which can grow into a wide variety of different types of cells.
RECOMBINANT DNA CLONING
- The fragment of DNA to be amplified is first inserted into a cloning vector.
- MOST COMMONLY USED VECTOR:-
- The most popular vectors currently in use consist of either small circular DNA molecules (plasmids) or bacterial viruses (phage).
- The vectors contain genetic information that allows bacterial DNA replication machinery to copy them.
- After insertion of the foreign DNA, the plasmid or phage vector is re-introduced into a bacterial cell.
- The growing bacterial culture replicates the foreign DNA, along with the vector, in hundreds of copies per cell.
CONTINUE…
- This process yields multiple, identical clones of the original recombinant molecule.
USES OF RECOMBINANT DNA CLONING
- Recombinant DNA cloning is used to produce genetic material in large enough quantities for scientific use. There are a variety of uses for this reproduced, or cloned, genetic material.
- Microbial Cells
- Isolation of Proteins
- Identification of Mutations
- Diagnosis of Hereditary Diseases
- Transferring Genes Between Organisms
Ethical Issues Of Cloning
- Cloning has become a contentious ethical and scientific issue in some parts of the world.
- Some people believe that life begins at conception, and they feel that reproductive cloning is unnatural and that it could potentially violate their religious beliefs.
Several nations have passed resolutions to explicitly ban human cloning, out of concern about ethical issues.
Advantages Of Cloning
- Medical treatment is that it would allow doctors to grow replacements for missing and damaged body parts for their patients. This would eliminate organ and tissue shortages
- Using cloned body parts would also eliminate the need for immunosuppressive drugs, and reduce the risk of rejection and other problems which are commonly associated with transplants.
- Cloning research on mice has suggested that new nerve cells can be grown with reproductive cloning techniques and used to repair damaged brains, an application which could be useful for people with dementia, Alzheimer's disease, or strokes.Investigative Reports: The Human Cloning Race [VHS]

VIRAL GENETICS

2010-06-30T23:12:00.001-07:00

Viral Genetics

Introduction to Viruses:

A virus is a biological agent that reproduces inside the cells of living hosts. When infected by a virus, a host cell is forced to produce many thousands of identical copies of the original virus, at an extraordinary rate. Unlike most living things, viruses do not have cells that divide; new viruses are assembled in the infected host cell. Over 2,000 species of viruses have been discovered. Notable human diseases caused by viruses include SARS, influenza and hepatitis C

A virus consists of two or three parts: all viruses have genes made from either DNA or RNA, long molecules that carry the genetic information; all have a protein coat that protects these genes; and some have an envelope of fat that surrounds them when they are not within a cell. Viruses vary in shape from the simple helical and icosahedral to more complex structures. Viruses are about 100 times smaller than bacteria, and it would take 30,000 to 750,000 of them, side by side, to stretch to 1 centimeter

Life-cycle of Viruses:

When a virus infects a cell, the virus forces it to make thousands more viruses. It does this by making the cell copy the virus's DNA or RNA, making viral proteins, which all assemble to form new virus particles.

There are six basic, overlapping stages in the life cycle of viruses in living cells.

Attachment: the binding of the virus to specific molecules on the surface of the cell. This specificity restricts the virus to a very limited type of cell. For example, the human immunodeficiency virus (HIV) infects only human T cells, because its surface protein, gp120, can only react with CD4 and other molecules on the T cell's surface. Plant viruses can only attach to plant cells and cannot infect animals. This mechanism has evolved to favor those viruses that only infect cells in which they are capable of reproducing.
Penetration follows attachment; viruses penetrate the host cell by endocytosis or by fusion with the cell.
Uncoating: happens inside the cell when the viral capsid is removed and destroyed by viral enzymes or host enzymes, thereby exposing the viral nucleic acid.
Replication of virus particles is the stage where a cell uses viral messenger RNA in its protein synthesis systems to produce viral proteins. The RNA or DNA synthesis abilities of the cell produce the virus's DNA or RNA.
Assembly takes place in the cell when the newly created viral proteins and nucleic acid combine to form hundreds of new virus particles.
Release occurs when the new viruses escape or are released from the cell. Most viruses achieve this by making the cells burst, a process called lysis. Other viruses such as HIV are released more gently by a process called budding.

Viral Genetics:

Viral genetics, the study of the genetic mechanisms that operate during the life cycle of viruses, utilizes biophysical, biological, and genetic analyses to study the viral genome and its variation. The virus genome consists of only one type of nucleic acid, which could be a single or double stranded DNA or RNA. Single stranded RNA viruses could contain positive-sense (+RNA), which serves directly as mRNA or negative-sense RNA (-RNA) that must use an RNA polymerase to synthesize a complementary positive strand to serve as mRNA. Viruses are obligate parasites that are completely dependent on the host cell for the replication and transcription of their genomes as well as the translation of the mRNA transcripts into proteins. Viral proteins usually have a structural function, making up a shell around the genome, but may contain some enzymes that are necessary for the virus replication and life cycle in the host cell.

Classification of Viruses:

Viruses are classified in two families depending on whether they have RNA or DNA genomes and whether these genomes are double or single stranded. Further subdivision into types takes into account whether the genome consists of a single RNA molecule or many molecules as in the case of segmented viruses. Four types of bacteriophages are widely used in biochemical and genetic research. These are the T phages, the temperate phages typified by bacteriophage lambda, the small DNA phages like M13, and the RNA phages. Animal viruses are subdivided in many classes and types. Class I viruses contain a single molecule of double stranded DNA and are exemplified by adenovirus, simian virus 40 (SV40), herpes viruses, and human papillomaviruses. Class II viruses are also called parvoviruses and are made of single stranded DNA that is copied in to double stranded DNA before transcription in the host cell. Class III viruses are double stranded RNA viruses that have segmented genomes which means that they contain 10-12 separate double stranded RNA molecules. The negative strands serve as template for mRNA synthesis. Class IV viruses, typified by poliovirus, have single plus strand genomic RNA that serves as the mRNA. Class V viruses contain a single negative strand RNA which serves as the template for the production of mRNA by specific virus enzymes. Class VI viruses are also known as Retroviruses and contain double stranded RNA genome.

RNA Viruses:

An RNA virus is a virus that has RNA (ribonucleic acid) as its genetic material. This nucleic acid is usually single-stranded RNA (ssRNA) but may be double-stranded RNA (dsRNA).

Single stranded RNA viruses:

Single stranded RNA viruses can be further classified according to the sense or polarity of their RNA into negative-sense and positive-sense RNA viruses. Some are positive in that they have a "sense" strand of RNA (coded information about how to build proteins) as their genetic material. Positive-sense viral RNA is identical to viral mRNA and thus can be immediately translated by the host cell. And other RNA viruses are negative in that they have an "antisense" strand (the paired opposite of the coded information). Negative-sense viral RNA is complementary to mRNA. Negative-strand or antisense-strand RNA viruses are as opposed to positive-strand or sense-strand RNA viruses. As such, purified RNA of a positive-sense virus can directly cause infection though it may be less infectious than the whole virus particle. Purified RNA of a negative-sense virus is not infectious by itself as it needs to be transcribed into positive-sense RNA.

Definition of Negative-strand RNA virus:

Also known as an antisense-strand RNA virus, a virus whose genetic information consists of a single strand of RNA that is the negative or antisense strand which does not encode mRNA (messenger RNA) and is complementary to it. Therefore it must be copied into the complementary plus-sense mRNA before proteins can be made. Thus, besides needing to code for an RNA-dependent RNA-polymerase, these viruses also need to package it (polymerase) in the virion so that they can make mRNAs upon infecting the cell. The positive-sense RNA molecule then acts as viral mRNA, which is translated into proteins by the host ribosome. The resultant protein goes on to direct the synthesis of new virions, such as capsid proteins and RNA replicase, which is used to produce new negative-sense RNA molecules. Examples of negative-strand RNA viruses include influenza virus (orthomyxovirus), measles viruses (paramyxoviruses), and rabies virus (rhabdovirus).

RHABDOVIRUSES (RHABDOVIRIDAE)

Example: Rabies virus. The most intensively studied member is vesicular stomatitis virus. RNA is single stranded, is negative (minus) sense, and codes for 5 proteins. The entire life cycle occurs in the cytoplasm, RNA polymerase and RNA modification enzymes are virally-coded and present in the virion itself. There is no early/late division of gene expression.

Attachment, penetration and uncoating:

The virus adsorbs to cell surface. G (Glycoprotein) is the attachment protein which binds to a receptor on the host cell surface. The attached virus is taken up by endocytosis. The membrane of the virus fuses with the endosome membrane (the acid pH of endosome is important because the G protein needs to be exposed to acidic pH before it can facilitate fusion). As a result of fusion of the viral membrane with the endosome membrane, the nucleocapsid is released into cytoplasm.

Transcription

'Transcription' is used in this context to refer to synthesis of mRNAs. Complete uncoating of the nucleocapsid is not necessary for transcription - the virion RNA polymerase can copy virion RNA when it is in the nucleocapsid form. This is an advantage in that genomic RNA is therefore somewhat protected from ribonucleases. There is one monocistronic mRNA for each of the five virally coded proteins. The mRNAs are capped, methylated, and polyadenylated. Since this is a cytoplasmic, negative-sense RNA virus, the enzymes for mRNA synthesis and modification are packaged in the virion.

Translation

Messenger RNAs are translated on host ribosomes and all five viral proteins are made at the same time. There is no distinction between early and late functions.

RNA replication

RNA replication is the process by which new copies of genome-length RNAs are made. RNA replication occurs in the cytoplasm and is carried out by the viral RNA polymerase. The full length plus strand is coated with nucleocapsid protein as it is made (mRNAs are not coated with this protein, which would interfere with the host protein translation machinery). The new positive strand is copied into full length minus strand, which is also coated with nucleocapsid protein as it is made.

New negative strands may now:

à be used as templates for the synthesis of more full length plus strands.

à be used as templates for the synthesis of more mRNAs.

à be packaged into virions. .

Assembly

The newly made RNAs and polymerase complexes are first packed into nucleocapsid containing G protein and then they are modified in envelop by ribosome on rough endoplasmic reticulum.

Definition of Positive-strand RNA virus:

Also known as a sense-strand RNA virus, a virus whose genetic information consists of a single strand of RNA that is the positive (or sense) strand which usually behaves as mRNA (messenger RNA). This mRNA can be translated immediately upon infection of the host cell, producing a single protein which is modified by host and viral proteins to form the various proteins needed for replication... Replication in positive-strand RNA viruses is via a negative-strand intermediate. Examples of positive-strand RNA viruses include poliovirus (picornavirus), Coxsackie virus, and echovirus. Togaviruses, flaviviruses.

PICORNAVIRUSES (PICORNAVIRIDAE)

These are small (28nm), naked icosahedral viruses (pico=very small). The RNA is single-stranded, plus sense, polyadenylated. It functions as mRNA immediately upon infection. The entire life cycle occurs in the cytoplasm and there is no division into early and late gene expression of the genome. Example is poliovirus.

Adsorption and penetration

A viral protein recognizes a receptor on the host cell membrane (this is important in the tropism of virus). It seems that binding to the receptor alters capsid structure in some way, a channel forms across the cell membrane and the RNA is released into cytoplasm. The mRNA is now available for translation.

Synthesis of viral proteins

Poliovirus virion RNA functions as an mRNA but does not have the methylated cap structure typical of eukaryotic mRNAs - it has a "ribosome landing pad" (known as the internal ribosome entry site or IRES) which enables ribosomes to bind without having to recognize a 5' methylated cap structure. Picornaviruses often interfere with host cell methylated cap recognition. Most host cell translation is cap-dependent, so this inhibits a lot of host protein synthesis but not viral protein synthesis - one way in which these viruses can modify the host cell to their advantage. The mRNA is translated into a single polypeptide (polyprotein), which is cleaved. The cleavages occur before translation is complete ( i.e. on the nascent / growing chain) and are carried out by virally coded proteases. Some of these proteases can work even while part of the polyprotein.

Products of cleavage include: An RNA polymerase (replicase), Structural components of the virion, and Proteases.5

RNA replication

We now have newly made viral proteins to support replication.

1. Viral RNA polymerase copies plus-sense genomic RNA into complementary minus-sense RNA using VPg (or precursor containing VPg), Viral RNA polymerase (replicase), and Certain Host proteins. VPg may act as a primer for RNA synthesis, this would explain why it is at the 5' end of all newly synthesized RNA molecules.

2. New minus sense strands serve as template for new plus sense strands. Again, RNA polymerase and VPg are needed. VPg is linked to the 5' ends of the new plus sense strands (again, it probably functions as a primer).

The new plus strand has three alternative fates:

à It may serve as a template for more minus strands.

à It may be packaged into progeny virions.

à It may be translated into polyprotein (In this case VPg is usually removed prior to translation).

Assembly

When sufficient plus-sense progeny RNA and virion proteins have accumulated, assembly begins. Particles assemble with VPg-RNA inside and 3 proteins in the capsid [VP0, 1 and 3]. VP0 is then cleaved to VP2 and VP4 as the virions mature and these mature virions are infectious. Virions are released following cell lysis. Excess capsids are formed and inclusion bodies may be seen in the cytoplasm.

Single stranded RNA Segmented Viruses:

The term for RNA viruses that explicitly excludes retroviruses is ribovirus. Their RNAs are called segmented because each segment is coding for a particular gene & trait.

Segmented Negative Strand Viruses:

Examples: Orthomyxoviruses , Bunyaviruses (include Hantavirus genus), Arenaviruses

ORTHOMYXOVIRUSES (ORTHOMYXOVIRIDAE)

It is also called Influenza virus. There are three groups of influenza virus: A, B and C. Influenza A virus is most intensively studied and influenza A and B are the most important in human disease. Influenza viruses are pleomorphic virions (that is, they vary in shape). They have negative-sense, single-stranded RNA and an RNA genome that is SEGMENTED. There are eight RNA segments in influenza A. The nucleocapsid is helical. Virions contain RNA polymerase packaged within the virus particle. These viruses are enveloped and have two membrane glycoproteins:

HA - hemaglutinin - This is the attachment and fusion protein
NA - neuraminidase - This is important in release. It removes sialic acid from proteins of the virus and the host cell.

Adsorption and penetration:

The virus adsorbs to receptors on the cell surface and is internalized by endocytosis. At acidic pH of an endosome, HA undergoes a conformational change and fusion occurs. Nucleocapsids are released to cytoplasm and are transported into the nucleus. The mRNA synthesis and replication of viral RNA occurs in the nucleus. This is very unusual for an RNA virus.

Double Stranded RNA Virus:

The double-stranded (ds)RNA viruses represent a diverse group of viruses that vary widely in host range (humans, animals, plants, fungi, and bacteria), genome segment number (one to twelve), and virion organization (T-number, capsid layers, or turrets). Members of this fascinating group include the rotaviruses, renowned globally as the commonest cause of gastroenteritis in young children, and bluetongue virus, an economically important pathogen of cattle and sheep.

Viruses with dsRNA genomes are currently grouped into six families: Reoviridae, Birnaviridae, Totiviridae, Partitiviridae, Hypoviridae, and Cystoviridae. Of these six families, the Reoviridae is the largest and most diverse in terms of host range.

The double stranded RNA viruses replicate in the cytoplasm, and their replication is monocistronic(mRNA that can encode only one polypeptide per RNA molecule). Example is the Reovirus.

REOVIRUS FAMILY (REOVIRIDAE)

The Reovirus family includes:

the members of the reovirus genus
the members of the rotavirus genus
the members of the orbivirus genus
Colorado tick fever virus

Reoviruses have icosahedral symmetry and a multiple layered capsid (inner and outer capsid). The RNA is double stranded. There are 10-12 segments (depending on the genus of the Reovirus family). There are some significant differences in the life cycle of members of the reovirus family and of the rotavirus family. Due to their clinical importance in humans, focus is on rotaviruses.

ROTAVIRUSES

(rota = wheel (from appearance of virions in the electron-microscope))

Adsorption, penetration and uncoating:

It is still not clear what exactly what happens in-vivo. There appears to be a need for a protease to remove some of the outer layer of the capsid and to generate an "intermediate sub-viral particle" (ISVP) before the virus can enter the cytoplasm. In vivo, the ISVPs are probably generated by protease digestion in the GI tract. A viral attachment protein is then exposed on the ISVP, probably at the vertices, and binds to host cell receptors. The activated ISVP enters the cytoplasm directly or via endocytosis. In the cytoplasm, the virion RNA is copied by the viral RNA polymerase while still in a nucleocapsid that has fewer proteins associated with it than are associated with the ISVP or the virion.

Transcription and translation:

Double stranded RNA does not function as an mRNA and so the initial step is to make mRNA (transcription). The mRNAs are made by virally-coded RNA polymerase packaged in the virion. The RNA is capped and methylated by virion packaged enzymes. It is then extruded from the vertices of the capsid. The mRNAs are translated and the resulting viral proteins assemble to form an immature capsid. The mRNAs are packaged into the immature capsid and are then copied within the capsid to form double stranded RNAs (It is not known how the virus ensures that each particle gets one copy of the 11 different mRNAs) .More mRNAs are now made by the newly formed immature capsids.

Assembly

More proteins are made and eventually the immature capsids bud into the lumen of the endoplasmic reticulum. In doing so, they acquire a transient envelope which is lost as they mature. This is a very odd feature of the rotaviruses. Then the release probably occurs via cell lysis.

Double-stranded RNA Segmented viruses:

Double-stranded RNA viruses (Group III) contain from one to a dozen different RNA molecules, each of which codes for one or more viral proteins. They include: orthoreoviruses, rotavirus, phytoreoviruses, and bluetongue virus

Structure of Orthoreoviruses ( reoviruses ):

Reoviruses are non-enveloped and characterized by concentric capsid shells that encapsidate a segmented dsRNA genome. In particular, reovirus has eight structural proteins and ten segments of dsRNA. A series of uncoating steps and conformational changes accompany cell entry and replication. The icosahedral structures of intact virions, infectious subviral particles that bind to cell surface receptors and core particles, which mediate RNA transcription, ths is how reovirus completes its lifecycle.

Structure and Functions of the Orthoreovirus σ3 Protein:

Protein s3 serves a number of distinct roles in the orthoreovirus life cycle. It forms the outermost layer of the reovirus particle, it imparts significant environmental stability to virions. Virion σ3 also plays a critical role as a determinant of cell entry, as it must be degraded from particles during their activation for membrane penetration. Early in infection σ3 is thought to serve a regulatory function through its capacity to bind dsRNA and interfere with dsRNA-activated innate immune pathways.

Retroviruses:

Retroviruses comprise a large and diverse family of enveloped RNA viruses defined by common taxonomic denominators that include structure, composition, and replicative properties. The virions are 80–100 nm in diameter, and their outer lipid envelope incorporates and displays the viral glycoproteins

The shape and location of the internal protein core are characteristic for various genera of the family. The virion RNA is 7–12 kb in size, and it is linear, single-stranded, non-segmented, and of positive polarity. The hallmark of the family is its replicative strategy which includes as essential steps reverse transcription of the virion RNA into linear double-stranded DNA and the subsequent integration of this DNA into the genome of the cell. Retroviruses contain RNA as the hereditary material in place of the more common DNA. In addition to RNA, retrovirus particles also contain an enzyme called reverse transcriptase (or RTase) that can both copy minus strand DNA from genomic RNA, catalyze the synthesis of a complementary plus DNA strand. The resulting double stranded DNA is integrated in the host chromosome and is transcribed by the host's own machinery. The resulting transcripts are either used to synthesize proteins or produce new viral particles. These new viruses are released by budding, usually without killing the host cell. Both HIV and HTLV viruses belong to this class of viruses.

Retroviruses are broadly divided into two categories—simple and complex—distinguishable by the organization of their genomes. The retrovirus family is split up into 7 genera: the Alpha retroviruses, the Beta retroviruses, Gamma retroviruses, Delta retroviruses, Epsilon viruses (all of which used to be classified as one genus, the oncoviruses), the Lentiviruses (which includes HIV) and the Spumaviruses.

All retroviruses contain three major coding domains with information for virion proteins:

gag, which directs the synthesis of internal virion proteins that form the matrix, the capsid, and the nucleoprotein structures;
pol, which contains the information for the reverse transcriptase and integrase enzymes; and
env, from which are derived the surface and transmembrane components of the viral envelope protein.

An additional, smaller, coding domain present in all retroviruses is pro, which encodes the virion protease. Simple retroviruses usually carry only this elementary information, whereas complex retroviruses code for additional regulatory non-virion proteins derived from multiply spliced messages.

Virus Replication

A detailed summary of the steps of virus replications is as follows:

1) Receptor binding and membrane fusion.

2) Internalization and un-coating of virion.

3) Reverse transcription of RNA to form double-stranded DNA

4) Entry of viral DNA, into host nucleus.

5) Integration of viral DNA into cellular DNA to form the provirus.

6) Transcription of provirus to form viral RNA.

7) Splicing of viral RNAs and export from host nucleus.

8) Translation of viral RNAs to form viral proteins.

9) Assembly of virion and packaging of viral RNA genome.

10) Budding and release of new virions.

11) Proteolytic processing of proteins and virion maturation.

Human Retroviruses:

Human T-Cell lymphotropic Virus Types I and II:

HTLV- I and II is included in the retroviridae family because of its nucleotide sequence and genome structure. Morphologically they are named the primate T-cell leukemia/lymphoma viruses. They are biologically distinct from the human immunodeficiency viruses in the lentivirus genus. Clinical manifestations of HTLV-I are linked with the development of adult T-cell leukemia/lymphoma and a progressive neurological disease called HTLV-I associated myelopathy. HTLV-II is also linked with leukemia and neurologic disease cases as well.

Human Immunodeficiency Viruses(HIV)

Human immunodeficiency viruses are part of the lentivirus genus. It includes the disease subtypes HIV-1 and HIV-2, the third and fourth human retroviruses discovered. HIV enters the host cell through the CD4 molecule and chemokine receptor as a dual receptor system. The biology of HIV-1 has been highly researched due to the pressing concerns of an HIV global pandemic and push for vaccine and treatment development.

DNA Viruses:

Dna virus is a virus that has DNA (deoxyribonucleic acid) as its genetic material. They are usually Large, Icosahedral, enveloped in Lipoproteins, Do not have polymerase enzymes, and cause Latent infection. Examples include Poxviruses, Herpesviruses, hepadnaviruses, Hepatitis b.

HERPESVIRUSES :

Larger virions than adenoviruses(180 - 200nm). Larger genome (three to five times) than adenoviruses. Linear, double-stranded DNA, Enveloped, icosahedral virus (this means that lipid solvents readily inactivate these viruses)

Adsorption and penetration

Many herpesviruses, including herpes simplex virus, can fuse directly with the plasma membrane (results in partial uncoating). Such fusion with the plasma membrane has implications for both the virus and the host cell. Among these are:

i) Since the fusion protein is active at physiological pH, if it is inserted into the host cell membrane during the virus growth cycle, the infected cell can potentially fuse with other cells and form syncytia.

ii) The viral membrane leaves a "footprint" in the cell plasma membrane and this is a possible clue that the cell is infected. Capsids are transported towards the nucleus and the DNA passes into the nucleus (probably via nuclear pores).

Early phase

Early transcription (the mRNAs made during this phase are the alpha and beta mRNAs). Herpes viruses use host RNA polymerase. However, a virion tegument protein (VP16) enters the nucleus upon infection and is important as part of the transcription factor complex recognized by the host RNA polymerase. The virus uses host mRNA modification enzymes.

Initially, alpha-mRNAs are transcribed. These are the immediate early mRNAs and are exported to the cytoplasm and translated into alpha-proteins. The α-proteins translated in the cytoplasm are transported into nucleus where they enable the beta-promoters to be used by the host RNA polymerase.

Beta-mRNAs are transcribed by the host RNA polymerase again. (Beta-genes are still "early" since they are transcribed prior to DNA synthesis. Sometimes alpha-genes are called "immediate early" and beta-genes are called "early"). Beta proteins are involved in gene expression regulation. They decrease alpha-gene expression and are needed for gamma gene expression. They are also involved in various aspects of DNA synthesis; for example, herpes beta -genes code for a variety of proteins including DNA polymerase, DNA binding proteins, thymidine kinase, ribonucleotide reductase etc.

Since these beta proteins are virally-coded and not host-coded enzymes, they are potentially weak links in the virus life cycle and thus promising targets for viral chemotherapy

Late phase

DNA replication

Herpesviruses code for several proteins, in addition to the DNA polymerase, that are needed for DNA replication. The precise mechanism of DNA replication is not known. DNA replication is accompanied by a lot of recombination. The replicated DNA is present as long concatameric molecules (tandem repeats of the genome linked head-to-tail). These are cleaved to genome-size lengths when DNA is packaged into the virion.

Late transcription:

By definition, late transcription occurs after DNA replication. Gamma mRNAs are made and are translated in the cytoplasm. Gamma proteins are predominantly structural. There is decreased expression of beta genes in the late stage. This is probably due to down-regulation of transcription of beta genes, by gamma proteins. In herpes viruses there is no apparent organization of the genome into blocks for either early or late transcription.

Assembly

Assembly occurs in the nucleus. A capsid is formed and the DNA enters the capsid. The capsids acquire an envelope by budding through areas of the inner nuclear membrane which have viral membrane proteins inserted into them. These areas have tegument proteins associated with the inner face of the inner nuclear membrane. The virus envelope then fuses with the outer nuclear membrane and the de-enveloped nucleocapisid is delivered into the cytoplasm, where it acquires a more mature tegument. It then becomes re-enveloped by budding into Golgi-derived vesicles and is then released.

The late protein required for transcription of immediate early mRNAs in the next round of infection is packaged in the virion.

Evolution of Viruses:

Viral genomes undergo genetic change by mutation, either spontaneous or induced mutation, and by recombination. Recombination may be either intramolecular or, among viruses with divided genomes, by reassortment.

Mutations:

These arise naturally during viral replication: e.g. due to errors by the genome-replicating polymerase or as a result of the incorporation of tautomeric forms of the bases. Mutation in RNA viruses may be extremely rapid because RNA is a less thermodynamically stable molecule than is DNA, many of the mutations which give rise to the diversity seen with the HIV virus is not a result of the fact that the virus uses an RNA genome, but of the nature of the polymerase enzyme that the virus uses. There is no proof-reading mechanism for RNA polymerases, as there is for DNA polymerases. This situation is compounded in the retroviruses, for there is no proof-reading mechanism for the reverse transcriptase either. Most of these mutations result in non-viable phenotypes. Whether the genetic changes lead to emergence of an altered phenotype depends on natural selection, which may occur within the infected cell, during spread of virus in the body, or the transmission of the virus from one host to the next. Mutations can manually b introduced by Agents acting directly on bases, e.g. nitrous acid, Agents acting indirectly, e.g. base analogs which mis-pair more frequently than normal bases or even by Agents such as UV light or X-rays

Exchange of Genetic material

This involves breaking of covalent bonds within the nucleic acid, exchange of genetic information, and reforming of covalent bonds. This kind of break/join recombination is common in DNA viruses or those RNA viruses which have a DNA phase (retroviruses). The host cell has recombination systems for DNA. If a virus has a segmented genome and if two variants of that virus infect a single cell, progeny virions can result with some segments from one parent, some from the other. This is an efficient process - but is limited to viruses with segmented genomes - so far the only human viruses characterized with segmented genomes are RNA viruses

For the practicing physician, virus evolution may appear to be an academic matter, because evolutionary changes usually occur over a time scale that is long compared with human life. However, sometimes genetic changes in viruses may occur rapidly as a result of evolutionary pressure. For instance, the highly virulent myxoma virus introduced into Australia to control the wild rabbit population evolved in a few years to a much more attenuated strain, enabling infected rabbits to survive for weeks instead of days, thereby increasing chances for transmission. Among influenza viruses, antigenic variation evolves toward decreased affinity for preexisting neutralizing antibodies during the course of an outbreak. Because survival of a virus depends largely on its ability to circulate among its natural hosts, natural selection tends to favor those viruses that are better transmitted (usually less virulent), have a lower susceptibility to antibody, and have a greater ability to persist. Also, the ability of the virus to produce reactions that promote excretion, such as coughing and sneezing in respiratory infections and diarrhea in many enteric infections, is likely to be retained.

The mutations and evolution in viruses is supported by the human population explosion. Evolution allows influenza to remain potentially the most important of all human viral diseases. Genetic reassortment and exchange of influenza viruses between humans and animals, producing antigenic shift, periodically introduce new viruses to the human population; mutation and selection, producing antigenic drift, accounts for year-to-year variations in influenza A subtypes.

References:

Developmental genetics

2010-06-30T22:55:00.000-07:00

Developmental Genetics

Zulkifal Yousaf

DEVELOPMENTAL GENETICS:-

Every multicellular organism begins life as a unicellular, fertilized egg. This single-celled zygote undergoes repeated cell divisions, eventually producing millions or trillions of cells that constitute a complete adult organism. Initially, each cell in the embryo is totipotent—it has the potential to develop into any cell type. Many cells in plants and fungi remain totipotent, but animal cells usually become committed to developing into specific types of cells after just a few early embryonic divisions. This commitment often comes well before a cell begins to exhibit any characteristics of a particular cell type; once the cell becomes committed, it cannot reverse its fate and develop into a different cell type. A cell becomes committed by a process called determination, the mechanism of which is still unknown. For many years, the work of developmental biologists was limited to describing the changes that take place in the course of development, because techniques for probing the intracellular processes behind these changes were unavailable. But, in recent years, powerful genetic and molecular techniques have had a tremendous influence on the study of development. In a few model systems such as Drosophila, the molecular mechanisms underlying developmental change are now beginning to be understood.

The Genetics of Pattern Formation in Drosophila:-

One of the best-studied systems for the genetic control of pattern formation is the early embryonic development of Drosophila melanogaster. Geneticists have isolated a large number of mutations in fruit flies that influence all aspects of their development, and these mutations have been subjected to molecular analysis, providing much information about how genes control early development

Stages in the development and the genes that control that stages:-

Stages in the early development o in the early development of

fruit flies and Developmental Stage Genes

Establishment of main Egg polarity genes

body axes

Determination of number and Segmentation genes

polarity of body segments

Establishment of identity Homeotic genthe genes that control

of each segmentage and the genes that control

each stage

The development of the fruit fly :-

An adult fruit fly possesses three basic body parts: head, thorax, and abdomen.The thorax consists of three segments: the first thoracic segment carries a pair of legs; the second thoracic segment carries a pair of legs and a pair of wings; and the third thoracic segment carries a pair of legs and the halteres (rudiments of the second pair of wings found in most other insects). The abdomen contains nine segments. When a Drosophila egg has been fertilized, its diploid nucleus immediately divides nine times without division of the cytoplasm, creating a single, multinucleate cell. These nuclei are scattered throughout the cytoplasm but later migrate toward the periphery of the embryo and divide several more times. Next, the cell membrane grows inward and around each nucleus, creating a layer of approximately 6000 cells at the outer surface of the embryo . Four nuclei at one end of the embryo develop into pole cells, which eventually give rise to germ cells. The early embryo then undergoes further development in three distinct stages:

(1) the anterior–posterior axis and the dorsal–ventral axis of the embryo are established

(2) the number and orientation of the body segments are determined

(3) the identity of each individual segment is established

Different sets of genes control each of these three stages

Egg-polarity genes :-

The egg-polarity genes play a crucial role in establishing the two main axes of development in fruit flies. You can think of these axes as the longitude and latitude of development: any location in the Drosophila embryo can be defined in relation to these two axes. There are two sets of egg-polarity genes: one set determines the anterior–posterior axis and the other determines the dorsal–ventral axis. These genes work by setting up concentration gradients of morphogens within the developing embryo. A morphogen is a protein whose concentration gradient affects the developmental fate of the surrounding region. The egg-polarity genes are transcribed into mRNAs during egg formation in the maternal parent, and these mRNAs become incorporated into the cytoplasm of the egg. After fertilization, the mRNAs are translated into proteins that play an important role in determining the anterior–posterior and dorsal–ventral axes of the embryo.

Because the mRNAs of the polarity genes are produced by the female parent and influence the phenotype of their offspring, the traits encoded by them are examples of genetic maternal effects. Egg-polarity genes function by producing proteins that become asymmetrically distributed in the cytoplasm, giving the egg polarity, or direction. This asymmetrical distribution may take place in a couple of ways. The mRNA may be localized to particular regions of the egg cell, leading to an abundance of the protein in those regions when the mRNA is translated. Alternatively, the mRNA may be randomly distributed, but the protein that it encodes may become asymmetrically distributed, either by a transport system that delivers it to particular regions of the cell or by its removal from particular regions by selective degradation.

Determination of the dorsal–ventral axis:-

The dorsal– ventral axis defines the back (dorsum) and belly (ventrum) of a fly. At least 12 different genes determine this axis, one of the most important being a gene called dorsal. The dorsal gene is transcribed and translated in the maternal ovary, and the resulting mRNA and protein are transferred to the egg during oogenesis. In a newly laid egg, mRNA and protein encoded by the dorsal gene are uniformly distributed throughout the cytoplasm but, after the nuclei migrate to the periphery of the embryo Dorsal protein becomes redistributed. Along one side of the embryo, Dorsal protein remains in the cytoplasm; this side will become the dorsal surface. Along the other side, Dorsal protein is taken up into the nuclei; this side will become the ventral surface. At this point, there is a smooth gradient of increasing nuclear Dorsal concentration from the dorsal to the ventral side .

The nuclear uptake of Dorsal protein is thought to be governed by a protein called Cactus, which binds to Dorsal protein and traps it in the cytoplasm. The presence of yet another protein, called Toll, can alter Dorsal, allowing it to dissociate from Cactus and move into the nucleus. Together, Cactus and Toll regulate the nuclear distribution of Dorsal protein, which in turn determines the dorsal–ventral axis of the embryo.

Inside the nucleus, Dorsal protein acts as a transcription factor, binding to regulatory sites on the DNA and activating or repressing the expression of other genes. High nuclear concentration of Dorsal protein (as on the ventral side of the embryo) activates a gene called twist, which causes mesoderm to develop. Low concentrations of Dorsal protein (as in cells on the dorsal side of the embryo), activates a gene called decapentaplegic, which specifies dorsal structures. In this way, the ventral and dorsal sides of the embryo are determined.

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5–8 Dorsal protein in the nuclei helps to determine the dorsal–ventral axis of the Drosophila embryo. (a) Relative concentrations of Dorsal protein in the cytoplasm and nuclei of cells in

the early Drosophila embryo. (b) Micrograph of a cross section of the embryo showing the Dorsal protein, darkly stained, in the nuclei along the ventral surface

Determination of the anterior–posterior axis :-

Establishing the anterior–posterior axis of the embryo is a crucial step in early development.We will consider several genes in this pathway. One important gene is bicoid, which is first transcribed in the ovary of an adult female during oogenesis. Bicoid mRNA becomes incorporated into the cytoplasm of the egg and, as it is passes into the egg, bicoid mRNA becomes anchored to the anterior end of the egg by part of its 3_ end. This anchoring causes bicoid mRNA to become concentrated at the anterior end (number of other genes that are active in the ovary are required for proper localization of bicoid mRNA in the egg.) When the egg has been laid, bicoid mRNA is translated into Bicoid protein. Because most of the mRNA is at the anterior end of the egg, Bicoid protein is synthesized there and forms a concentration gradient along the anterior–posterior axis of the embryo, with a high concentration at the anterior end and a low concentration at posterior end. This gradient is maintained by the continuous synthesis of Bicoid protein and its short half-life. The high concentration of Bicoid protein at the anterior end induces the development of anterior structures such as the head of the fruit fly. Bicoid—like Dorsal—is a morphogen. It stimulates the development of anterior structures by binding to regulatory sequences in the DNA and influencing the expression of other genes. One of the most important of the genes stimulated by Bicoid protein is

hunchback, which is required for the development of the head and thoracic structures of the fruit fly.

The development of the anterior–posterior axis is also greatly influenced by a gene called nanos, an egg-polarity gene that acts at the posterior end of the axis. The nanos gene is transcribed in the adult female, and the resulting mRNA becomes localized at the posterior end of the egg . After fertilization, nanos mRNA is translated into Nanos protein, which diffuses slowly toward the anterior end. The Nanos protein gradient is opposite that of Bicoid protein: Nanos is most concentrated at the posterior end of the embryo and is least concentrated at the anterior end. Nanos protein inhibits the formation of anterior structures by repressing the translation of hunchback mRNA. The synthesis of the Hunchback protein is therefore stimulated at the anterior end of the embryo by Bicoid protein and is repressed at the posterior end by Nanos protein. This combined stimulation and repression results in a Hunchback protein concentration gradient along the anterior–posterior axis that, in turn, affects the expression of other genes and helps determine the anterior and posterior structures.

of the dorsal–ventral axis in

fruit flies a nd their actiTn tt anterior–posterior axis in a Drosophila embryo is

determined by concentrations of Bicoid and Nanos proteins

Segmentation genes:-

The fruit fly has segmented body plan. When the basic dorsal–ventral and anterior–posterior axes of the fruit-fly embryo have been established, segmentation genes control the differentiation of the embryo into individual segments. These genes affect the number and organization of the segments, and mutations in them usually disrupt whole sets of segments. The approximately 25 segmentation genes in Drosophila are transcribed after fertilization; so they don’t exhibit a genetic maternal effect, and their expression is regulated by the Bicoid and Nanos protein gradients. The segmentation genes fall into three groups . Gap genes define large sections of the embryo; mutations in these genes eliminate whole groups of adjacent segments. Mutations in the Krüppel gene, for example, cause the absence of several adjacent segments. Pair-rule genes define regional sections of the embryo and affect alternate segments. Mutations in the even-skipped gene cause the deletion of even-numbered segments, whereas mutations in the fushi tarazu gene cause the absence of odd-numbered segments. Segment-polarity genes affect the organization of segments. Mutations in these genes cause part of each segment to be deleted and replaced by a mirror image of part or all of an adjacent segment. For example, mutations in the gooseberry gene cause the posterior half of each segment to be replaced by the anterior half of an adjacent segment. The gap genes, pair-rule genes, and segment-polarity genes act sequentially, affecting progressively smaller regions of the embryo. First, the egg-polarity genes activate or repress the gap genes, which divide the embryo into broad regions. The gap genes, in turn, regulate the pair-rule genes, which affect the development of pairs of segments. Finally, the pairrule genes influence the segment-polarity genes, which guide the development of individual segments.

Homeotic genes:-

After the segmentation genes have established the number and orientation of the segments, homeotic genes become active and determine the identity of individual segments. Eyes normally arise only on the head segment, whereas legs develop only on the thoracic segments. The products of homeotic genes activate other genes that encode these segment-specific characteristics. Mutations in the homeotic genes cause body parts to appear in the wrong segments. Homeotic mutations were first identified in 1894, when William Bateson noticed that floral parts of plants occasionally appeared in the wrong place: he found, for example, flowers in which stamens grew in the normal place of petals. In the late 1940s, Edward Lewis began to study homeotic mutations in Drosophila, which caused bizarre rearrangements of body parts.

Homeotic genes create addresses for the cells of particular segments, telling the cells where they are within the regions defined by the segmentation genes. When a homeotic gene is mutated, the address is wrong and cells in the segment develop as though they were somewhere else in the embryo. Homeotic genes are expressed after fertilization and are activated by specific concentrations of the proteins produced by the gap, pair-rule, and segment-polarity genes. The homeotic genes encode regulatory proteins that

bind to DNA; each gene contains a subset of nucleotides, called a homeobox, that are similar in all homeotic genes.

The homeobox consists of 180 nucleotides and encodes 60 amino acids that serve as a DNA-binding domain; this domain is related to the helix-turn-helix motif. Homeoboxes are also present in segmentation genes and other genes that play a role in spatial development. There are two major clusters of homeotic genes in Drosophila. One cluster, the Antennapedia complex, affects the development of the adult fly’s head and anterior thoracic segments. The other cluster consists of the bithorax complex and includes genes that influence the adult fly’s posterior thoracic and abdominal segments. Together, the bithorax and Antennapedia genes are termed the homeotic complex (HOM-C). In Drosophila, the bithorax complex contains three genes, and the Antennapedia complex has five; they are all located on the same chromosome. In addition to these eight genes, HOM-C contains many sequences that regulate the homeotic genes.

Remarkably, the order of the genes in the HOM-C is the same as the order in which the genes are expressed along the anterior–posterior axis of the body. The genes that are expressed in the more anterior segments are found at the one end of the complex, whereas those expressed in the more posterior end of the embryo are found at the other end of complex. The reason for this correlation is unknown.

Homeobox Genes in Other Organisms:-

After homeotic genes in Drosophila had been isolated and cloned, molecular geneticists set out to determine if similar genes exist in other animals; probes complementary to the homeobox of Drosophila genes were used to search for homologous genes that might play a role in the development of other animals. The search was hugely successful: homeobox-containing (Hox) genes have been found in all

animals studied so far, including nematodes, beetles, urchins, frogs, birds, and mammals. They have even been discovered in fungi and plants, indicating that Hox genes arose early in the evolution of eukaryotes.

In vertebrates, there are four clusters of Hox genes, each of which contains from 9 to 11 genes. Interestingly, the Hox genes of other organisms exhibit the same relation between order on the chromosome and order of their expression along the anterior–posterior axis of the embryo as that of Drosophila. Mammalian Hox genes, like those in Drosophila, encode transcription factors that help determine the identity of body regions along an anterior– posterior axis.

The Control of Development:-

Development is a complex process consisting of numerous events that must take place in a highly specific sequence. The results of studies in fruit flies and other organisms reveal that this process is regulated by a large number of genes. In Drosophila, the dorsal–ventral axis and the anterior–posterior axis are established by maternal genes these genes encode mRNAs and proteins that are localized to specific regions within the egg and cause specific genes to be expressed in different regions of the embryo. The proteins of these genes then stimulate other genes, which in turn stimulate yet other genes in a cascade of control. As might be expected, most of the gene products in the cascade are regulatory proteins, which bind to DNA and activate other genes. In the course of development, successively smaller regions of the embryo are determined .

In Drosophila, first, the major axes and regions of the embryo are established by egg polarity genes. Next, patterns within each region are determined by the action of segmentation genes: the gap genes define large sections; the pair-rule genes define regional sections of the embryo and affect alternate segments; and the segment-polarity genes affect individual segments. Finally, the homeotic genes provide each segment with a unique identity. Initial gradients in proteins and mRNA stimulate localized gene expression, which produces more finely located gradients that stimulate even more localized gene expression. Developmental regulation thus becomes more and more narrowly defined. The processes by which limbs, organs, and tissues form (called morphogenesis) are less well understood, although this pattern of generalized-to-localized gene expression is encountered frequently.

Programmed Cell Death in Development:-

Cell death is an integral part of multicellular life. Cells in many tissues have a limited life span, and they die and are replaced continually by new cells. Cell death shapes many body parts during development: it is responsible for the disappearance of a tadpole’s tail during metamorphosis and causes the removal of tissue between the digits to produce the human hand. Cell death is also used to eliminate dangerous cells that have escaped normal controls.

Cell death in animals is often initiated by the cell itself in a kind of cellular suicide termed apoptosis. In this process, a cell’s DNA is degraded, its nucleus and cytoplasm shrink, and the cell undergoes phagocytosis by other cells without any leakage of its contents . Cells that are injured, on the other hand, die in a relatively uncontrolled manner called necrosis. In this process, a cell swells and bursts, spilling its contents over neighboring cells and eliciting an inflammatory response. Apoptosis is essential to embryogenesis; most multicellular animals cannot complete development if the process is inhibited. Surprisingly, most cells are programmed to undergo apoptosis and will survive only if the internal death program is continually held in check. The process of apoptosis is highly regulated and depends on numerous signals inside and outside the cell. Geneticists have identified a number of genes having roles in various stages of the regulation of apoptosis. Some of these genes encode enzymes called caspases, which cleave other proteins at specific sites (after aspartic acid). Each caspase is synthesized as a large, inactive precursor that is activated by cleavage, ofterv by another caspase.When one caspase is activated, it cleaves other procaspases that trigger even more caspase activity. The resulting cascade of caspase activity eventually cleaves proteins essential to cell function, such as those supporting the nuclear membrane and cytoskeleton. Caspases also cleave a protein that normally keeps an enzyme that degrades DNA (DNAse) in an inactive form. Cleavage of this protein activates DNAse and leads to the breakdown of cellular DNA, which eventually leads to cell death. Procaspases and other proteins required for cell death are continuously produced by healthy cells, so the potential for cell suicide is always present. A number of different signals can trigger apoptosis; for instance, infection by a virus can activate immune cells to secrete substances onto an infected cell, causing that cell to undergo apoptosis. This process is believed to be a defense mechanism designed to prevent the reproduction and spread of viruses. Similarly, DNA damage can induce apoptosis and thus prevent the replication of mutated sequences. Damage to mitochondria and the accumulation of a misfolded protein in the endoplasmic reticulum also stimulate programmed cell death.

Apoptosis in animal development is still poorly understood but is believed to be controlled through cell–cell signaling. The cell death that causes the disappearance of a tadpole’s tail, for example, is triggered by thyroxin, a hormone produced by the thyroid gland that increases in concentration during metamorphosis. The elimination of cells between developing fingers in humans is thought to result from localized signals from nearby cells. The symptoms of many diseases and disorders are caused by apoptosis or, in some cases, its absence. In neurodegenerative diseases such as Parkinson disease and Alzheimer disease, symptoms are caused by a loss of neurons through apoptosis. In heart attacks and stroke, some cells die through necrosis, but many others undergo apoptosis. Cancer is often stimulated by mutations in genes that regulate apoptosis, leading to a failure of apoptosis that would normally eliminate cancer cells.

Evo-Devo: The Study of Evolution and Development:-

“Ontogeny recapitulates phylogeny” is a familiar phrase that was coined in the 1860s by German zoologist Ernst Haeckel to describe his belief—now considered wrong—that organisms repeat their evolutionary history during development. According to Haeckel’s theory, a human embryo passes through fish, amphibian, reptilian, and mammalian stages before developing human traits.

Although ontogeny does not recapitulate phylogeny, many evolutionary biologists today are turning to the study of development for a better understanding of the processes and patterns of evolution. Sometimes called “evo-devo,” the study of evolution through the analysis of development is revealing that the same genes often shape developmental pathways in distantly related organisms. In humans for example, the same gene controls the development of eyes, despite the fact that insect and mammalian eyes are thought to have evolved independently. Similarly, biologists once thought that segmentation in vertebrates

and invertebrates was only superficially similar, but we now know that, in both Drosophila and amphioxus (a marine organism closely related to vertebrates). A gene

called distalless, which creates the legs of a fruit fly, has also been found to also play a role in the development of crustacean branched appendages. This same gene also stimulates body outgrowths of many other organisms, from polycheate worms to starfish. Similar genes may be part of a developmental pathway

common to two different species but have quite different effects. For example, a Hox gene called AbdB helps define the posterior end of a Drosophila embryo; a similar group of genes in birds divides the wing into three segments.

The theme emerging from these studies is that a small, common set of genes may underlie many basic developmental processes in many different organisms. Evo-devo is proving that development can reveal much about the process of evolutions.

REFERENCES:-

Genetics a conceptual approach by Benjamin Pierce.Genetics, A Conceptual Approach 3rd edition