DNA sequencing.

The term DNA sequencing refers to sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.
Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology, forensic biology and biological systematics. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes.

RADIO ACTIVELY LABLLED SEQUENCING GEL.

The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of dye-based sequencing methods with automated analysis, DNA sequencing has become easier and orders of magnitude faster.

History:

RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the complete genome of Bacteriophage MS2, identified and published by Walter Fiers and his coworkers at the University of Ghent (Ghent, Belgium), between 1972 and 1976.
 Prior to the development of rapid DNA sequencing methods in the early 1970s by Frederick Sanger at the University of Cambridge, in England and Walter Gilbert and Allan Maxam at Harvard, a number of laborious methods were used. For instance, in 1973, Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis.
The chain-termination method developed by Sanger and coworkers in 1975 soon became the method of choice, owing to its relative ease and reliability.

Maxam–Gilbert sequencing:
In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases.
Although Maxam and Gilbert published their chemical sequencing method two years after the ground-breaking paper of Sanger and Coulson on plus-minus sequencing,
Maxam–Gilbert sequencing rapidly became more popular, since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA. However, with the improvement of the chain-termination method (see below), Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity prohibiting its use in standard molecular biology kits, extensive use of hazardous chemicals, and difficulties with scale-up.
The method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first "cut" site in each molecule. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA fragment, from which the sequence may be inferred.
Also sometimes known as "chemical sequencing", this method originated in the study of DNA-protein interactions (footprinting), nucleic acid structure and epigenetic modifications to DNA, and within these it still has important applications.

Chain-termination methods:

Because the chain-terminator method (or Sanger method after its developer Frederick Sanger) is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice. The key principle of the Sanger method was the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.

The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.
The newly synthesized and labeled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence.

CAPILLARY ELECTROPHORESIS.

DNA fragments are labeled with a radioactive or fluorescent tag on the primer (1), in the new DNA strand with a labeled dNTP, or with a labeled ddNTP. (click to expand)

Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5’ end with a fluorescent dye. Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. The later development by Leroy Hood and coworkers  of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.

Chain-termination methods have greatly simplified DNA sequencing. For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.

Dye-terminator sequencing:

Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths.

Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis (see figure to the left).
This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.

DYE TERMINATOR READ

Challenges:

Common challenges of DNA sequencing include poor quality in the first 15–40 bases of the sequence and deteriorating quality of sequencing traces after 700–900 bases. Base calling software typically gives an estimate of quality to aid in quality trimming.
In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain parts of the cloning vector. In contrast, PCR-based cloning and emerging sequencing technologies based on pyrosequencing often avoid using cloning vectors. Recently, one-step Sanger sequencing (combined amplification and sequencing) methods such as Ampliseq and SeqSharp have been developed that allow rapid sequencing of target genes without cloning or prior amplification.
Current methods can directly sequence only relatively short (300–1000 nucleotides long) DNA fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide.

Automation and sample preparation:

Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day. DNA sequencers carry out capillary electrophoresis for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Sequencing reactions by thermocycling, cleanup and re-suspension in a buffer solution before loading onto the sequencer are performed separately. A number of commercial and non-commercial software packages can trim low-quality DNA traces automatically. These programs score the quality of each peak and remove low-quality base peaks (generally located at the ends of the sequence). The accuracy of such algorithms is below visual examination by a human operator, but sufficient for automated processing of large sequence data sets.

Amplification and clonal selection:

Genomic DNA is fragmented into random pieces and cloned as a bacterial library. DNA from individual bacterial clones is sequenced and the sequence is assembled by using overlapping DNA regions.(click to expand)

Large-scale sequencing aims at sequencing very long DNA pieces, such as whole chromosomes. Common approaches consist of cutting (with restriction enzymes) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented DNA is cloned into a DNA vector, and amplified in Escherichia coli. Short DNA fragments purified from individual bacterial colonies are individually sequenced and assembled electronically into one long, contiguous sequence.
This method does not require any pre-existing information about the sequence of the DNA and is referred to as de novo sequencing. Gaps in the assembled sequence may be filled by primer walking. The different strategies have different tradeoffs in speed and accuracy; shotgun methods are often used for sequencing large genomes, but its assembly is complex and difficult, particularly with sequence repeats often causing gaps in genome assembly.
Most sequencing approaches use an in vitro cloning step to amplify individual DNA molecules, because their molecular detection methods are not sensitive enough for single molecule sequencing. Emulsion PCR isolates individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. Polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing.
Another method for in vitro clonal amplification is bridge PCR, where fragments are amplified upon primers attached to a solid surface, used in the Illumina Genome Analyzer. The single-molecule method developed by Stephen Quake's laboratory (later commercialized by Helicos) is an exception: it uses bright fluorophores and laser excitation to detect pyrosequencing events from individual DNA molecules fixed to a surface, eliminating the need for molecular amplification.

High-throughput sequencing:

The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once. High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.

Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS):

The first of the "next-generation" sequencing technologies, MPSS was developed in 1990s at Lynx Therapeutics, a company founded in 1992 by Sidney Brenner and Sam Eletr. MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences. Because the technology was so complex, MPSS was only performed 'in-house' by Lynx Therapeutics and no machines were sold; when the merger with Solexa later lead to the development of sequencing-by-synthesis, a more simple approach with numerous advantages, MPSS became obsolete. However, the essential properties of the MPSS output were typical of later "next-gen" data types, including hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels. Lynx Therapeutics merged with Solexa in 2004, and this company was later purchased by Illumina.

454 pyrosequencing:

A parallelized version of pyrosequencing was developed by 454 Life Sciences. The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other. 454 Life Sciences has since been acquired by Roche Diagnostics.

Illumina (Solexa) sequencing:

Solexa, now part of Illumina developed a sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed (bridge amplification). One type of nucleotide at a time is then added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides and the dye is chemically removed from the DNA, allowing a next cycle.

SOLiD sequencing:

Applied Biosystems' SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing.

Future methods:

Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced. Mass spectrometry may be used to determine mass differences between DNA fragments produced in chain-termination reactions.
 DNA sequencing methods currently under development include labeling the DNA polymerase, reading the sequence as a DNA strand transits through nanopores, and microscopy-based techniques, such as AFM or electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments (>5,000 bp) by nucleotide labeling with heavier elements (e.g., halogens) for visual detection and recording.
In microfluidic Sanger sequencing the entire thermocycling amplification of DNA fragments as well as their separation by electrophoresis is done on a single glass wafer (approximately 10 cm in diameter) thus reducing the reagent usage as well as cost. In some instances researchers have shown that they can increase the throughput of conventional sequencing through the use of microchips.Research will still need to be done in order to make this use of technology effective.
In October 2006, the X Prize Foundation established an initiative to promote the development of full genome sequencing technologies, called the Archon X Prize, intending to award $10 million to "the first Team that can build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 100,000 bases sequenced, with sequences accurately covering at least 98% of the genome, and at a recurring cost of no more than $10,000 (US) per genome."

vitamin B12

CAULIFLOWER MOSAIC VIRUS(CamV)

Cauliflower mosaic virus

Cauliflower mosaic virus
Virus classification
Group:	Group VII (dsDNA-RT)
Family:	Caulimoviridae
Genus:	Caulimovirus
Species:	Cauliflower mosaic virus

Cauliflower mosaic virus (CaMV) is the type member of the caulimoviruses, one of the six genera in the Caulimoviridae family, pararetroviruses that infect plants (Pringle, 1999). Pararetroviruses replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA

Structure

The CaMV particle is an icosahedron with a diameter of 52 nm built from 420 capsid protein (CP) subunits arranged with a triangulation T = 7, which surrounds a solvent-filled central cavity (Cheng et al., 1992). It contains a circular double-stranded DNA molecule of about 8.0 kB, interrupted by sitespecific discontinuities resulting from its replication by reverse transcription. After entering the host, the single stranded nicks in the viral DNA are repaired, forming a supercoiled molecule that binds to histones. This DNA is transcribed into a full length, terminally redundant 35S RNA and a subgenomic 19S RNA.

Genome

The promoter of the 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV genome. It is well known for its use in plant transformation. It causes high levels of gene expression in dicot plants. However, it is less effective in monocots, especially in cereals. The differences in behavior are probably due to differences in quality and/or quantity of regulatory factors. The promoter was named CaMV 35S promoter ("35S promoter") because the coefficient of sedimentation of the viral transcript, whose expression is naturally driven by this promoter, is 35S. It is one of the most widely used, general-purpose constitutive promoters. It was discovered at the beginning of the 1980s, by Chua and collaborators at the Rockefeller University.
The 35S RNA is particularly complex, containing a highly structured 600 nucleotide long leader sequence with six to eight short open reading frames This leader is followed by seven tightly arranged longer ORFs that encode all the viral proteins (reviewed by Hohn and Fütterer, 1997). The mechanism of expression of these proteins is very special. The ORF VI protein (encoded by the 19S RNA) controls translation reinitiation of major open reading frames on the polycistronic 35S RNA, which is normally only happens on bacterial mRNA's. TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 (Park et al., 2001).

Genomic map of CaMV

• ORF I - Movement protein
• ORF II - Insect transmission factor
• ORF III
• ORF IV - Capsid protein
• ORF V - Protease, reverse transcriptase and RNaseH
• ORF VI - Translational activator / Inclusion body protein
• ORF VII - Unknown (dispensable)

Replication

CaMV replicates by reverse transcription. Initially all the gaps present in the genome are sealed and the covalently closed DNA then associates with host histones to form a super coiled mini chromosome. Transcription of this mini chromosome produces 35s RNA which translates protein as well as forming dsDNA by the process of reverse transcription. New viral particles are produced which are targeted to inclusion bodies & are released outside.
The Cauliflower Mosaic Virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. It is inserted into transgenic plants in a form which is different to that found when it is present in its natural brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible.
CaMV contains about 8 kb double-strand DNA genome and produces spherical particles. CaMV infections are systemic, and even its DNA is infectious when inoculated on abraded plant surfaces. The CaMV genome has 8 tightly packed genes, of which only two small genes, genes II and VII, are nonessential; as a result, only these two genes can be replaced/deleted without a loss of infectivity. In addition, modified CaMV genomes exceeding the natural genome size (8024 bp) by even a few hundred bp are not packaged into virions. These two factors seriously limit the size of DNA insert clonable in CaMV. Bacterial dhfr (dihydrofolate reductase) gene was inserted in the CaMV genome in place of gene II, and was successfully expressed in plants. EncyclopediaCauliflower mosaic virus (CaMV) is the type member of the caulimoviruses, one of the six genera in the Caulimoviridae family, pararetroviruses that infect plants Pararetroviruses replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA .Structure Cauliflower mosaic virus (CaMV) is the type member of the caulimoviruses, one of the six genera in the Caulimoviridae family, pararetroviruses that infect plants. Pararetroviruses replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA
Structure
The CaMV particle is an icosahedron with a diameter of 52 nm built from 420 capsid protein (CP) subunits arranged with a triangulation T = 7, which surrounds a solvent-filled central cavity (Cheng et al., 1992). It contains a circular double-stranded DNA molecule of about 8.0 kB, interrupted by sitespecific discontinuities resulting from its replication by reverse transcription. After entering the host, the single stranded nicks in the viral DNA are repaired, forming a supercoiled molecule that binds to histones. This DNA is transcribed into a full length, terminally redundant 35S RNA and a subgenomic 19S RNA.Genome
The promoter of the 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV genome. It is well known for its use in plant transformation. It causes high levels of gene expression in dicot plants. However, it is less effective in monocots, especially in cereals. The differences in behavior are probably due to differences in quality and/or quantity of regulatory factors. The promoter was named CaMV 35S promoter ("35S promoter") because the coefficient of sedimentation of the viral transcript, whose expression is naturally driven by this promoter, is 35S. It is one of the most widely used, general-purpose constitutive promoters. It was discovered at the beginning of the 1980s, by Chua and collaborators at the Rockefeller University.
The 35S RNA is particularly complex, containing a highly structured 600 nucleotide long leader sequence with six to eight short open reading frames (ORFs)
 This leader is followed by seven tightly arranged longer ORFs that encode all the viral proteins . The mechanism of expression of these proteins is very special. The ORF VI protein (encoded by the 19S RNA) controls translation reinitiation of major open reading frames on the polycistronic 35S RNA, which is normally only happens on bacterial mRNA's. TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 .
 ORF I Movement protein ORF II Insect transmission factor ORF III ORF IV Capsid protein ORF V Protease, reverse transcriptase and RNaseH ORF VI Translational activator / Inclusion body protein ORF VII Unknown (dispensable)Replication
CaMV replicates by reverse transcription. Initially all the gaps present in the genome gets sealed. The covalently closed DNA associates with host histones to form a super coiled mini chromosome. Transcription of the former produces 35s RNA which translates protein as well as forms dsDNA by the process of reverse transcription. New viral particles are produced which gets targeted to inclusion body & is released outside. The Cauliflower Mosaic Virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant.
It is inserted into transgenic plants in a form which is different to its naturally occurring state arising in its natural brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible. Cauliflower Mosiac Virus (CaMV) -
CaMV contains about 8 kb double-strand DNA genome and produces Spherical particles. CaMV infections are systemic, and even its DNA is infectious when inoculated on abraded plant surfaces.

CaMV genome has 8 tightly packed genes, of which only two small genes, genes II and VII, are nonessential; as a result, only these two genes can be replaced/deleted without a loss of infectivity. In addition, CaMV genomes exceeding the natural genome size (8024 bp) by even a few hundred bp are not packaged into virions. These two features seriously limit the size of DNA insert clonable in CaMV. Bacterial dhfr (dihydrofolate reductase) gene was inserted in the CaMV genome in place of gene II, and was successfully expressed in plants.

The CaMV 35S Promoter

The CaMV 35S promoter is being used in almost all GM crops currently grown or tested, especially GM maize. It is the promoter of choice for plant genetic engineering, as it is a strong and constitutive promoter. Failure to recognise or to ignore its capacity to be universally active in almost any organism is irresponsible and careless and shows a serious lack of scientific rigour and commitment to safety.
Any safety assessment can be expected to be flawed that does not resort to actual laboratory test of the capacity of bacteria and fungi to utilise the particular genes and their promoters.

Risks Associated with the Use of the Cauliflower Mosaic Virus Promoter in Transgenic Crops

The Cauliflower Mosaic Virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. It is potentially dangerous.

            It is inserted into transgenic plants in a form which is different to its naturally ocurring state arising in its natural brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible.

The Use of Cauliflower Mosaic Virus

The majority of crop plant constructions for herbicide or disease resistance employ a Promoter from cauliflower mosaic virus (CaMV). Regardless of the gene transferred, all transfers require a promoter, which is like a motor driving production of the genes' message. Without a promoter, the gene is inactive, but replicated, CaMV is used because it is a powerful motor which drives replication of the retrovirus and is active in both angiosperms and gymnosperms. The CaMV pararetrovirus replication cycle involves production vegetative virus containing RNA which is reverse transcribed to make DNA similar to HIV, Human Leukemia Virus and Human hepatitis B.

The CaMV promoter is preferred above other potential promoters because it is a more powerful promoter than others and is not greatly influenced by environmental conditions or tissue types. CaMV has two Promoters 19S and 35S, of these two the 35S promoter is most frequently used in biotechnology because it is most powerful. The 35S promoter is a DNA (or RNA) sequence about 400 base pairs in length. The use of the CaMV promoter in plants is analogous to the use of retrovirus LTR promoters in retrovirus vectors used in human gene therapy. The majority of human gene therapy trials employ LTR promoters to provide motors to activate genes.

Antisense genes are genes constructed to have a complementary sequence to a target gene, thus producing a product that combines with a gene message to inactivate it. Antisense is analogous to an antibody which combines with an antigen like a key fitting a lock. Antisense is being used to treat human cancer and HIV infection. Antisense is used to prevent spoilage in tomatos, either by targeting an enzyme degrading cell walls (polygalacturonase), or production of ethylene a hormone promoting ripening (P. Oeller et al. Genetic Engineering 49, 1989; R. Fray and D. Grierson, Trends Genetics 9, 438, 1993). Most frequently antisense targets production of a chemical metabolite producing ethylene. The antisense gene also influenced polyamines spermine and spermidine production through S-adenosylmethionine. The implication is that the plant antisense gene product should be tested in animals to ensure that critical functions including gene replication, sperm activity and gene imprinting are not disrupted.

The perceived hazards of CaMV in crop plants include the consequences of recombination and pseudo recombination. Recombination is the exchanges of parts of genes or blocks of genes between chromosomes. Pseudorecombination is a situation in which gene components of one virus are exchanged with the protein coats of another. Frequently viruses may incorporate cellular genes by recombination or pseudorecombination, it has been noted that such recombinants have selective advantages

It has been shown that the CaMV genes incorporated into the plant (canola) chromosome recombine with infecting virus to produce more virulent new virus diseases. The designers of the experiment questioned the safety of transgenic plants containing viral genes. Recombination between CaMV viruses involves the promoter and may take place either between DNA and DNA or RNA and RNA and frequently creates more severe Infections than either parent. Recently related experiments suggest altered plants may breed deadlier diseases . DNA copies of RNA Viruses are frequently propagated using the CaMV 35S promoter to drive RNA virus production . In conclusion CaMV promoters recombine with the infecting viruses to produce virulent new diseases. CaMV viruses and promoter may incorporate genes from the host creating virulent new diseases.

CaMV can recombine with insect viruses and propagated in insect cells . Thus it is likely that as large numbers of humans consume CaMV modified tomatos recombination between CaMV and hepatitis B viruses will take place creating a supervirus propagated in plants, insects and humans.

Plant biotechnology has grown out of recombinant DNA research that began in the early 1970's. The special nature of recombination has been debated since that time. In recent years, government regulators on the American and European continents, under pressure from well-funded lobby representing the biotechnology industry, have chosen to ignore the special nature of recombination. They have chosen instead to base regulations on existing frameworks for toxic chemicals and pathogenic organisms. Ignoring the special nature of recombination is likely to have costly, if not terminal, environmental consequences. A worst-case example includes the complete cloning of Human Immunodeficiency Virus (HIV) on an E. coli plasmid. When the plasmid is used to transform animal cells, intact HIV viruses are released from the cells. A careless (but legal) release of HIV bacteria to the environment would allow the plasmid to transfer to Salmonella as well as E. coli. Thus, numerous mammals and birds could contain HIV bacteria which could transform the animals, which would in turn produce HIV particles unable to target the animals T-cell receptors but easily transmitted to humans. When all the animals are HIV carriers, human survival would be marginal. The special concerns of recombination in plant biotechnology include the viruses and bacteria used in crop plant construction and gene flow between related crop plants and weeds in the field.

Currently most experts agree that virus diseases such as influenza gain strength for epidemics by alternating between animal hosts (pigs and ducks) and man. Epidemics begin when rare combinations appear in large closely associated populations such as in asia. CaMV can propagate in plant and insect hosts following recombination. It may not be outlandish to predict that CaMV may recombine with related Hepatitis B or for that matter HIV to create a most powerful disease. The salient feature being large number of people or animals consuming large numbers of virus genes incorporated into crop plants making up a major part of human and animal diet.

The use of CaMV promoter is seldom an issue in reviews of safety of gene tinkered crops. Few people have raised the important issue and more often than not their concerns are ignored by government officials "protecting" public safety. This omission may be a fatal one because it has potentially the most damaging impact, and the one perceived at the beginning of gene splicing.

Tobacco mosaic virus

Tobacco mosaic virus (TMV) is a positive-sense single stranded RNA virus that infects plants, especially tobacco and other members of the family Solanaceae. The infection causes characteristic patterns (mottling and discoloration) on the leaves (hence the name). TMV was the first virus to be discovered. Although it was known from the late 19th century that an infectious disease was damaging tobacco crops, it was not until 1930 that the infectious agent was determined to be a virus.

History:

In 1883, Adolf Mayer first described the disease that could be transferred between plants, similar to bacterial infections. Dimitri Ivanovski gave the first concrete evidence for the existence of a non-bacterial infectious agent, showing that infected sap remained infectious even after filtering through Chamberland filter candles, in 1892. However, he remained convinced despite repeated failures to produce evidence, that bacteria were the infectious agents. In 1898, Martinus Beijerinck showed that a filtered, bacteria-free culture medium still contained the infectious agent. Wendell Meredith Stanley crystallized the virus in 1935 and showed that it remains active even after crystallization. For his work, he was awarded 1/4 of the Nobel Prize in Chemistry in 1946,even though it was later shown some of his conclusions (in particular, that the crystals were pure protein, and assembled by autocatalysis) were incorrect. The first electron microscopical images of TMV were made in 1939 by Gustav Kausche, Edgar Pfankuch and Helmut Ruska - the brother of Nobel Prize winner Ernst Ruska. In 1955, Heinz Fraenkel-Conrat and Robley Williams showed that purified TMV RNA and its capsid (coat) protein assemble by themselves to functional viruses, indicating that this is the most stable structure (the one with the lowest free energy). The crystallographer Rosalind Franklin worked for Stanley for about a month at Berkeley, and later designed and built a model of TMV for the 1958 World's Fair at Brussels. In 1958, she speculated that the virus was hollow, not solid, and hypothesized that the RNA of TMV is single-stranded. This conjecture was proven to be correct after her death and is now known to be the + strand.
Tobacco mosaic virus has a rod-like appearance. Its capsid is made from 2130 molecules of coat protein (see image to the left) and one molecule of genomic single strand RNA 6400 bases long. The coat protein self-assembles into the rod like helical structure (16.3 proteins per helix turn) around the RNA which forms a hairpin loop structure (see the electron micrograph above). The protein monomer consists of 158 amino acids which are assembled into four main alpha-helices, which are joined by a prominent loop proximal to the axis of the virion. Virions are ~300 nm in length and ~18 nm in diameter. Negatively stained electron microphotographs show a distinct inner channel of ~4 nm. The RNA is located at a radius of ~6 nm and is protected from the action of cellular enzymes by the coat protein. There are three RNA nucleotides per protein monomer. X-ray fiber diffraction structure of the intact virus was studied based on an electron density map at 3.6 Å resolution.
Physicochemical properties:
TMV is a thermostable virus. On a dried leaf, it can withstand up to 120 degrees Fahrenheit (50 °C) for 30 minutes.
TMV has an index of refraction of about 1.57.

Disease cycle:

TMV does not have a distinct over-wintering structure. Rather, it will over-winter in infected tobacco stalks and leaves in the soil, on the surface of contaminated seed (TMV can even survive in contaminated tobacco products for many years). With the direct contact with host plants through its vectors (normally insects such as aphids and leaf hoppers), TMV will go through the infection process and then the replication process.

Infection:

After its multiplication, it enters the neighboring cells through plasmodesmata. For its smooth entry, TMV produces a 30 kDa movement protein called P30 which enlarge the plasmodesmata. TMV most likely moves from cell-to-cell as a complex of the RNA, P30, and replicase proteins.
It can also spread through phloem for longer distance movement within the plant. Moreover, TMV can be transmitted from one plant to another by direct contact. Although TMV does not have defined transmission vectors, the virus can be easily transmitted from the infected hosts to the healthy plants, by human handling.

Replication:

Following entry into its host via mechanical inoculation, TMV uncoats itself to release its viral RNA strand. Then the viral genome will further replicate to produce multiple mRNAs. The resulting mRNAs encode several proteins, including the coat protein and an RNA-dependent RNA polymerase (RdRp), as well as the movement protein. Thus TMV can replicate its own genome. After the coat protein and RNA genome of TMV have been synthesized, they spontaneously assemble into complete TMV virions in a highly organized process. The protomers come together to form disks composed of two layers of protomers arranged in a helical spiral. The helical capsid grows by the addition of protomers to the end of the rod. As the rod lengthens, the RNA passes through a channel in its center and forms a loop at the growing end. In this way the RNA can easily fit as a spiral into the interior of the helical capsid^.

Host and symptoms:

Like other plant pathogenic viruses, TMV has a very wide host range and has different effects depending on the host being infected. The tobacco mosaic virus has been known to cause a production loss for flue cured tobacco of up to two percent in North Carolina.It is known to infect members of nine plant families, and at least 125 individual species, including tobacco, tomato, pepper (all members of the useful Solanaceae), cucumbers, and a number of ornamental flowers. There are many different strains. The first symptom of this virus disease is a light green coloration between the veins of young leaves. This is followed quickly by the development of a "mosaic" or mottled pattern of light and dark green areas in the leaves. Rugosity may also be seen where the infected plant leaves display small localized random wrinkles. These symptoms develop quickly and are more pronounced on younger leaves. Mosaic does not result in plant death, but if infection occurs early in the season, plants are stunted. Lower leaves are subjected to "mosaic burn" especially during periods of hot and dry weather. In these cases, large dead areas develop in the leaves. This constitutes one of the most destructive phases of tobacco mosaic virus infection. Infected leaves may be crinkled, puckered, or elongated. However, if TMV infects crops like grape and apple, it is almost symptomless.

Transference to humans:

Consumption of tobacco products infected with the tobacco mosaic virus has not been found to have any effect on humans.

 Environment:

TMV is known as the one of the most stable viruses. It has a very wide survival range. As long as the surrounding temperature remains below approximately 40 degrees Celsius, TMV can sustain its stable form. All it needs is a host to infect. If necessary, greenhouses and botanical gardens would provide the most favorable condition for TMV to spread out, due to the high population density of possible hosts and the constant temperature throughout the year.
 Treatment/management:
One of the common control methods for TMV is sanitation, which includes removing infected plants, and washing hands in between each plant treatment. Crop rotation should also be employed to avoid infected soil/seed beds for at least 2 years. As for any plant disease, looking for resistant strains against TMV may also be advised. Furthermore, the cross protection method can be administered, where the stronger strain of TMV infection is inhibited by infecting the host plant with mild strain of TMV, similar to the effect of a vaccine.
In the past 10 years, the application of genetic engineering on a host plant genome has been developed to allow the host plant to produce the TMV coat protein within their cells. It was hypothesized that the TMV genome will re-coated rapidly upon entering the host cell, thus it prevents the initiation of TMV replication. Later it was found that the mechanism that protects the host from viral genome insertion is through gene silencing.
In some cases, spraying milk on the host plant is known to inhibit the TMV infection.

Scientific and Environmental Impact:

The large amount of literature about TMV and its choice for many pioneering investigations in structural biology (including X-ray diffraction), virus assembly and disassembly, and so on, are fundamentally due to the large quantities that can be obtained, plus the fact that it does not infect animals. After growing a few infected tobacco plants in a greenhouse and a few simple laboratory procedures, a scientist can easily produce several grams of virus. As a result of this, TMV can be treated almost as an organic chemical, rather than an infective agent.

 Investigational uses:

Due to its cylindrical high aspect ratio, self-assembling nature, and ability to incorporate metal coatings (nickel and cobalt) into its shell, TMV is an ideal candidate to be incorporated into battery electrodes. Addition of TMV to a battery electrode increases the reactive surface area by an order of magnitude, resulting in an increase in the battery's capacity by up to 6 times compared to a planar electrode geometry.

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