Genetics of Viruses (With Diagram)

In this article we will discuss about the genetics of viruses.

History of Viruses:

Viruses were first discovered in 1899 when M. W. Beijerinck noticed the existence of micro­organisms invisible in the microscope, that could pass through filters that stopped bacteria. In 1917 Felix d’Herelle gave the name bacteriophage (meaning eater of bacteria) to one such microbe that was parasitic on, and capable of killing a certain rod-shaped bacterium.

Work on phage genetics was initiated in 1930s independently by Max Delbruck, Martin Schlesinger and F. M. Burnet. Delbruck, who was educated as a physicist, is credited with the most important contributions in phage genetics. In the 1940s he, along with Salvatore Luria and A. D. Hershey discovered genetic recombination in phages. Thereafter, phages have been extensively used as tools for the study of gene structure and function.

Viruses are obligate intracellular parasites designated in relation to the host cell they parasitise. Thus we have bacteriophages parasitic on bacteria, fungal viruses on fungus and cyanophages on blue green algae. Structurally viruses are the smallest organisms (exceptions being the viroids) consisting of a nucleic acid (either DNA or RNA) and a protein coat or capsid.

Inside the host cell they exist in the form of replicating nucleic acid molecules devoid of protein coats. The nucleic acid is double-stranded DNA or single-stranded RNA in most viruses. The phage φX174 and some coliphages have single-stranded DNA; the reoviruses and some plant viruses have double stranded RNA as their genetic material.

All the DNA viruses and most RNA viruses have a single molecule of nucleic acid of constant length in circular or linear form. In different viruses the length varies from a few thousand nucleotides to over 250,000 nucleotides.

After penetration of host cells there is synthesis of enzymes for viral nucleic acid replication and for production of virus-specific proteins. Viruses also regulate the growth and differentiation of host cells by taking control of their metabolic machinery. In the following account the most studied bacteriophages and cancer viruses are described.

Phages consist of protein and nucleic acid. The much investigated T-phages infecting E. coli are named as T1, T2, T3, T4…. T7 (T stands for “type”). The T-even phages namely T2, T4 and T6 are closely related as they share some common features such as presence of 5-hydroxy-methyl-cytosine instead of cytosine. The T-phages consist of a hexagonal head of protein containing a double helical DNA molecule.

In the phage T4 the tail consists of two hollow tubes, the outer sheath and the inner hollow core or needle. There is a base plate below the tail with tail fibres and tail pins (Fig. 18.1). The phage φX 174 is without tail and has a single stranded DNA molecule; lambda has a tail but no tail fibres or pins.

Life Cycle of Virulent Bacteriophages:

Phages which lead to destruction or lysis of host cell are called virulent. The T-phages become attached (adsorb) to the host E. coli cell. The polysaccharide cell wall of the bacterium recognises specific phage types for attachment. The process of attachment involves uncoiling of tail fibers along the base plate. The tail sheaths contract and the needle penetrates the bacterial cell wall and DNA is injected.

Once inside the host cell, T4 phage DNA takes control of the host cell machinery. Normal activities of bacterial DNA stop, and synthesis of phage DNA and coat protein components begins. Host cell DNA disintegrates and disappears.

These activities are regulated by three sets of phage genes expressed in a sequence. First, the early gene produces viral messenger RNA. This is translated on host ribosomes to form protein products that inhibit E. coli RNA synthesis.

The enzymes required for replication of phage DNA are also synthesised by the early genes. The second set of gene products release nucleases that digest the E. coli chromosome to provide free nucleotides for phage DNA synthesis. Third, the late genes control synthesis of phage coat proteins and tail components. The DNA is incorporated into the protein coats by a packaging process involving condensation of DNA (Fig. 18.2).

In T-even phages replication of phage DNA produces DNA molecules many times longer than the phage chromosome called concatamers. These are cleaved into smaller segments of required length so that each maturing phage head receives a headful of DNA.

In all phages new phage particles are released by cell lysis, aided by the enzyme lysozyme which is synthesised under the direction of phage DNA. E. L. Ellis and Max Delbruck in 1939 showed that about 100 new phage particles are produced from a single virus within 30 minutes.

Temperate Phages and Prophage:

All phages are not virulent. Many phages infect and live inside the host cell without causing lysis or cell destruction. Such a non-virulent virus is called symbiotic or temperate phage. Phage lambda (λ) is one studied in detail.

It has a linear, double helical DNA molecule with a unique feature— the 3′ end of each strand is longer by 12 nucleotides than the 5′ end of the opposite strand. The nucleotide sequences of the single stranded extended regions are complementary to each other.

When phage lambda infects E. coli cell it can either cause host cell lysis (lytic response), or it may become integrated into the E. coli chromosome and enter the state of lysogeny (Fig. 18.3 A). The integrated viral DNA called prophage is replicated along with the host chromosome and is transmitted to the cell progeny.

In case the prophage is excised from the host chromosome, it replicates and produces progeny virus particles which are liberated through cell lysis. That is why the term lysogenic is used for bacteria which carry such a virus. Alternatively, the integrated lambda genome can remain in the host cell as prophage for many generations.

When the linear molecule of lambda DNA enters the cell, it becomes circular by forming H bonds between the base sequences of the extended region at the 3′ end followed by joining by ligase enzyme.

There is a mechanism of phage repression which inhibits the lytic cycle and allows for the lysogenic state. The DNA of phage λ has a gene (the C1 gene) which produces a repressor protein. The λ repressor binds to a specific receptor site in the lambda DNA.

The repressor is involved in the integration of the λ genome into the bacterial chromosome at the specific λ attachment site to form the prophage (Fig. 18.3 B). When the receptor site in λ DNA is not occupied by the repressor, the DNA is replicated and all the genes are expressed to form the viral proteins.

But when the repressor molecule is bound to its receptor site, replication of DNA and expression of all genes (except Cl gene) are inhibited. The Cl gene continues to produce repressor protein when all the other λ genes are not functioning.

Genetic Recombination in Phages:

Recombination in phages may be vegetative or site specific. Vegetative recombination occurs during the lytic response when the host cell contains a large number of phage DNA molecules.

There are several rounds of replication of viral DNA and recombination occurs due to coming together of homologous sequences in the DNA molecules followed by breakage and exchange of sequences. In site specific recombination there is reciprocal exchange by breakage and reunion between a specific site on the phage DNA (att in lambda) and a site on the bacterial chromosome.

As in Mendelian inheritance, the study of genetic recombination in phages requires identification of phenotypes. Structural details of head and tail are phenotypes visible only in the electron microscope. Other characters like plaque size and shape and host range are more convenient for identifying normal and mutant phenotypes.

Plaque formation can be demonstrated by adding a solution of phage particles on the surface of nutrient agar on which sensitive E. coli cells are growing to form a colony or lawn of cells. Even if one virus particle infects a bacterial cell, it will multiply to form a hundred progeny particles which are released by cell lysis in 15-60 mins. The progeny particles attach to new bacteria and multiply.

After several such cycles of attachment, multiplication and release, all the bacteria lying near the original virus particle are killed. This results in clear, circular zones called plaques in a lawn of healthy bacteria (Fig. 18.4).

The morphology of the plaque is used to identify viral strains and mutants. For example the r (rapid lysis) mutant of T-phage produces a large-sized plaque with smooth edge. The normal, wild type phage (r+) produces small plaques with fuzzy outline.

The host range mutants of phage are able to infect certain additional bacterial strains. Cells of E. coli B strain can become infected with phage T2. A mutant strain E. coli B/2 is resistant to infection by phage T2, because the B/2 mutation changes the cell surface thus preventing attachment of T2. But if a mutation occurs in T2, the mutant phage T2/h (h stands for host range) can adsorb to and infect .E. coli B/2 because it possesses altered tail fibers.

Now E. coli 5/2 can again become resistant to phage T2A if the cells undergo a mutation to become E. coli B/2/h. T2h phage can once again acquire ability to infect E. coli B/2/h after another mutation which changes it to T2h form.

The host range mutants (h) can be identified by adding the phage to a mixture of B and B/2 cells. The h mutants will infect both B and B/2 cells and form plaques distinct from those produced by wild type phage.

It is possible to have mixed infection with phages carrying two different mutations so that both types of phages enter the host bacterium. Due to the mutation, neither of the two phages can multiply inside the host cell to form plaques. But if the two mutations are for different functions, one will complement the other and progeny virus particles will form. This is known as complementation test.

Hershey and Rotman (1948, 1949) carried out the first detailed analysis of genetic recombination in phages by single burst experiments. By this method it is possible to analyse the progeny (recombinants and parentals) released from a single infected bacterial cell. In such an experiment bacteria growing in culture are exposed to mixed infection of two parental phage types.

The infected bacteria are diluted to the extent that a single infected bacterium is present in a culture tube. Hershey and Rotman made crosses between wild type (r +, h +) and the r and h mutants of phage T2, and determined the frequencies of parental and recombinant types in the progeny.

The cross consisted of parental phages with genotypes hr + and h + r used to produce mixed infection in E. coli cells. After cell lysis, progeny virus particles were added to a mixture of B and B/2 cells, and the plaques formed were identified to be of four types (Fig. 18.5). It was found that parental combinations appeared with higher frequency than recombinant phenotypes.

The experiments of Hershey and Rotman demonstrated genetic recombination in T2 phages. A striking feature was that in an individual cross (a single burst) the recombinant phenotypes did not appear with equal frequency. But when the average from many experiments was considered, the proportions of the two recombinant classes were roughly equal.

The occurrence of one recombinant type more frequently than the other in the progeny of an individual cross indicates that there is little, if any, reciprocal exchange of genes. The percentage frequency of recombination between genes could be calculated as follows: no. of recombinant plaques/total plaques scored x 100.

If we assume that the frequency of recombination between two genes is related to the distance separating them, then it is possible to map positions of genes on the phage chromosome. The data on recombination frequencies was utilised by Hershey and Rotman to assign r mutations to 3 different loci in relation to h.

The pattern of recombination in phage is significantly different from that in eukaryotes. In a Mendelian cross, reciprocal genetic exchange is restricted to two of the four non-sister chromatids, and occurs only once during meiotic prophase I. The two classes of recombinant progeny occur with equal frequency, and maximum frequency of crossing over between two loci never exceeds 50 per cent.

A distinctive feature of phage recombination is that pairing and exchange may occur repeatedly throughout the period when free chromosomes are present within the host cell; there are many cycles of phage DNA replication. Maximum amount of recombination between two genes is 30 per cent. When all the progeny arising from a doubly infected cell is analysed the reciprocal recombinants are not present in equal proportions.

That phage chromosomes undergo several successive rounds of exchanges can be shown by infecting a host bacterial cell with three genetically different phages such as xyz which is possible only if each of the three phages has contributed one gene. Figure 18.6 shows how separate exchange events can occur involving breakage and fusion in all the three phage types during the latent period of phage in the host cell.

It is due to multiple rounds of exchanges that recombinants recovered from any one infected cell do not occur with equal frequency. The frequency of recombination between two widely spaced genes is rarely more than 30 per cent can be explained as due to multiple exchanges as follows. Consider a cross between xy and x + y + where the two genotypes are present in equal frequency.

There is an equal probability of xy exchanging with xy i.e. ½ x ½ = ¼. The recombinants (x + y and xy + ) can arise if both xy and x + y + have exchanges with each other, that is a probability of ¼ + ¼ = ½. If each such heterozygous exchange were to produce a recombinant, the maximum of 50 per cent re-combinations would be obtained.

However, when x and y are far apart they also tend to assort independently to produce xy, x + y +, x + y and xy + in the ratio 1:1:1:1. Thus, only half of all the heterozygous matings produce recombinants, i.e., half of 50 per cent = 25 per cent. In this way for genes that are widely spaced recombination frequency is always less than 50 per cent.

Recombination frequency in phages is influenced by factors like number of DNA molecules pregent in an infected cell, replication rates of different genotypes, and by proportions of the parental genotypes. By performing three-point crosses in phages, Streisinger and Berne (1960) demonstrated that T-phages had one linkage group of genes. This was later confirmed along with the circular nature of the chromosome.

Negative Interference in Phage Crosses:

In eukaryotes a crossover in one region suppresses chiasma formation in an adjacent region. Double crossovers therefore, are fewer than expected and the phenomenon is known as positive interference. It is measured in terms of the ratio of double crossovers observed to those expected i.e., coincidence.

When coincidence is less than 1.0, positive interference has occurred. In phage negative interference occurs so that double crossovers are observed more often than expected and coincidence is greater than 1.0. This is partly due to the successive rounds of exchanges between phage DNA molecules in the host cell.

Negative interference is of two types. One called high negative interference is localised within those regions of the chromosomes which have a high concentration of enzymes involved in recombination.

This type of interference is observed in eukaryotes also within genes separated by very small gap distances. The low negative interference is found only in viruses, it occurs between genes widely separated from each other, and is probably due to multiple rounds of exchanges in DNA molecules.

Heterozygosity in Phages:

Phages are haploid organisms with each gene represented once in the single DNA molecule. The experiments of Hershey and Chase in 1951 indicated that the phage DNA could be partially ‘diploid’ for some genes. When E. coli is doubly infected with r+ and r phages and the infected bacteria are plated on agar before bursting, some of the plaques formed are “mottled” in appearance (Fig. 18.7).

Such plaques contain a mixture of r+ and r phages. That a single phage has given rise to both r and r+ progeny can be explained by assuming the formation of a heteroduplex, a hybrid DNA molecule with one strand containing r gene, the other r+ gene.

Such a heterozygous condition leads to the occurrence of different base sequences at a specific site (r locus) in the two DNA strands. Such mismatched sites in DNA are acted upon by certain correction systems which restore homozygosity by removing bases in one strand and substituting bases complementary to those in the other strand.

Mapping Genes in Phage Lambda:

Various methods of gene mapping have been applied to the lambda chromosome. In one method, the sites of mutations are physically located by breaking up the chromosome into fragments of specific lengths. Isolated DNA helices from lambda are stirred in the blender at a certain speed which breaks the helix into two half lengths. Increasing the speed of the blender produces quarter fragments, and so on.

Fragments of different lengths are separated from each other by density gradient centrifugation in caesium chloride (CsCl). The various fragments of lambda have different AT: GC ratios.

Therefore fragments of higher GC content which are denser, separate from the lighter AT rich fragments. Moreover, one half of the lambda genome is much richer in GC than the other half. The purified halves are used in DNA transformation experiments to find out which genes are located in each fragment.

A physical method of mapping the phage genome is as follows. The DNA of lambda is denatured by heating to 100°C, the high temperature breaking all the H-bonds to yield single strands (denaturation).

If the separated strands are gradually cooled, the H-bonds reform to yield double helical molecules of DNA (renaturation). If DNA from two different viral genomes is denatured, then allowed to re-anneal, heteroduplex molecules are formed of which each strand comes from a different viral genome.

The re-natured DNA is observed in the electron microscope. Wherever a deletion or a mutated region is present in a strand, the corresponding segment of the other strand will not be able to pair with it, but will extend outwards in the form of a loop.

This is a convenient method of detecting physical locations of deletions, mutations and mismatched bases. The data can be compared with the genetic map prepared from recombination frequencies.

The genetic method of constructing a gene map of lambda consists in performing two-point and three-point crosses. The technique based on linkage studies in higher organisms, was applied by Kaiser (1955) for mapping genes in lambda.

The genome of lambda has been worked out to be a linear DNA molecule about 3.8 x 10⁶ Daltons, corresponding to about 465 kilo-base (one kilo-base equals a length of 1000 bases). There are about 50 genes arranged in functional blocks (Fig. 18.8). The proteins involved in assembly of the phage particles are coded by the left half of the DNA molecule. The head protein genes (A-F) are all located to the left of the tail protein genes (Z-J).

The DNA segment in the b2 region between the J gene and the attachment site (att) does not perform any function necessary to phage growth. The point at which the phage integrates into the host chromosome (lysogeny) is denoted by att. The region from the attachment site onwards to Y controls genes which control the various phage mediated recombination events, that is, site-specific recombination (integration) and general recombination.

The region from Y to ell is the control region that regulates genetic expression of the various operons in the genome. The OP region controls replication of phage DNA, and Q region controls the functioning of the late genes of lambda.

The genes S and R at the right end of the genome code for proteins needed to lyse the cell. The sequence of genes on lambda is similar to that in other lambdoid phages. In fact many regions in their genomes are homologous when heteroduplex molecules are observed.

The RNA Phages:

Some bacteriophages contain a single RNA molecule as the genetic material. They are classified into groups: phages f2, R17, MS2 and a few more are placed together in one group, Qβ belongs to another group. RNA phages have an exceedingly small amount of genetic material, there being 3,600 nucleotides in f2 and over 4,000 in Qβ.

The single-stranded RNA is extensively folded into hairpin loops, giving rise to a characteristic flower arrangement (Fig. 18.9). Besides the hairpin loops, the secondary structure of RNA phages is also due to some base pairing between complementary base sequences located apart from each other.

The entire genome of Qβ has only 3 genes controlling the synthesis of the following proteins:

“Maturation” or A protein, coat protein, and one subunit (ii) of the enzyme Qβ replicase (Fig. 18.10). A fourth protein is also synthesised, starting from the coat protein gene by a read through of the codons beyond the termination site.

Infection involves attachment of specific RNA phages to the sex pilus and injection of RNA into the host cell. The infecting RNA (+strand) serves as a template for synthesis of the complementary minus (-) strand by the enzyme Qβ-replicase. A base sequence near the 3′ end of the RNA molecule, called Qβ replicase recognition gene regulates the start of replication by Qβ replicase.

The – strand is synthesised in the 5′ to 3′ direction and serves as the template for the synthesis of the + strand. The – strand requires the replicase and a protein factor for its synthesis, the plus strand only the replicase. The minus strand is used repeatedly for synthesis of + strand, so that the number of + strands far exceeds that of the – strands.

The enzyme Qβ replicase consists of four subunits (Fig. 18.11). Only one polypeptide chain (subunit II) is coded for by the viral genome. The remaining three subunits represent host cell proteins.

Tumour Viruses and Cancer:

Besides mutations, some RNA and DNA viruses are also considered to be causal agents of cancer. They possess the ability to transform normal cells into cancerous ones. A cancerous cell is one which has uncontrolled growth and division and some altered surface characteristics.

Among DNA tumour viruses are SV40 (a simian virus), mouse polyoma, adenoviruses which produce symptoms like common cold and tumours, and herpes group of viruses which produce mononucleosis, Burkitt’s lymphoma and some other cancers.

The RNA viruses include Rous Sarcoma virus (RSV) which causes solid tumours in chickens, and some viruses causing leukemias and sarcomas. The ability of cancer cells to multiply indefinitely is transmitted from parent to progeny cells. Tumour cells do not show virus particles in the EM. That is because in most cases part or all of the genetic material of a virus becomes inserted into the host chromosome (called provirus).

The DNA Tumour Viruses:

SV40 is a spherical virus about 450 Å in diameter that infects cells of the monkey. When SV40 enters a monkey cell, it may be acted upon by the host cell’s defense mechanisms and may disappear. Sometimes it multiplies to produce about a million progeny viruses present in the nucleus of the host cell.

The cell nucleus no longer functions normally and the cell dies (lytic response). But when SV40 enters a cell other than that of monkey (mouse, hamster or human cell in culture), cell lysis does not occur. Instead, the cell becomes transformed into a cancer cell.

SV40 is one of the smallest DNA viruses (mol. wt. about 28 million). The spherical particle has a protein shell made up of 72 identical capsomeres (Fig. 18.12). Each capsomere is made up of 5 or 6 smaller protein molecules. There are three classes of polypeptide chains (VP-1, VP-2, VP-3) making up the shell protein.

Within the shell is a single circular double helical DNA molecule. Four of the host cell histones H2A, H2B, H3 and H4 are bound to the SV40 DNA (minichromosome). Histone H1 is absent. Recent evidence indicates that the SV40 genome contains as few as 3 genes.

The life cycle of SV40 is controlled by early and late genes. In the early stage there is synthesis of SV40 DNA and mRNA that codes for a protein called the T-antigen. The late genes code for the structural proteins VP1, VP2 and VP3.

Studies with DNA-RNA hybridisation have shown the presence of SV40 DNA sequences integrated into DNA of transformed host cells. Each transformed cell contains about 1 to 20 SV40 genomes. When SV40 DNA is integrated into a human cell, it becomes inserted into chromosome No. 7.

RNA Tumour Viruses and Oncogenes:

The best known RNA tumour virus is Rous sarcoma virus (RSV) and was first discovered by Peyton Rous in 1910 in chickens. It is spherical with a membrane-like lipoprotein coat surrounding an RNA core. The coat contains two different viral specific glycoproteins which project as knobs from the membrane surface.

The life cycle of RSV is similar to that of other RNA tumour viruses. After adsorption to the surface of the host cell, the intact virus particle comes into the cytoplasm.

The membrane is shed and the naked RNA is transcribed into a complementary DNA strand by the enzyme reverse transcriptase coded for by the viral genome. The discovery of reverse transcriptase enzyme by Temin, Mitzutani and Baltimore (rewarded with a Nobel Prize) has been crucial in understanding the genetics of RNA viruses, many of which belong to a family of viruses called retroviruses.

The single-stranded DNA becomes converted into a circular double-stranded DNA molecule called the provirus. The provirus integrates into the host chromosomal DNA by a recombination process. The pro-viral DNA makes RNA transcripts from which virus-specific proteins are synthesised. The RNA cores become enclosed in the newly synthesised capsomere proteins and move to the cell surface.

There they become enveloped by portions of the cell membrane which contain viral- specific membrane glycoproteins. The multiplication of RNA tumour virus does not interfere with normal cellular functions. Although thousands of progeny viruses are released from the cell surface, it does not lead to cell death as in the case of DNA tumour viruses.

However, if the viral genome is carrying a special segment or gene known as oncogene (cancer gene), the activity of this gene can transform a normal cell into a cancerous one. Most tumour viruses can produce transformation also in cells growing in artificial culture media.

How exactly the RSV causes cancer is explained by the study of a very important group of mutants—the deletion mutants which are not able to transform cells into cancerous ones. They have a deletion of the small segment of the genome called oncogene.

The deleted segment is the only feature distinguishing transforming and non-transforming viral strains. It is believed (this view has been strengthened by recent experiments described later) that the segment contains cancer-specific genes.

Experiments conducted by Huebner and Todaro led them to suggest that oncogene-like DNA sequences are not present exclusively in viruses, they are not even peculiar to cancer cells, but appear to be present in the genomes of all cells. In normal cells oncogenes are considered to be repressed, never transcribed and harmless.

In transformed cells they are stimulated to become active and convert cells into cancerous growth. Some viruses have a single oncogene others have a few of them. It is also likely that RNA tumour viruses become oncogenic (that is they develop the ability to induce tumours) after acquiring the oncogene from the host cell.

It must be noted however, that some tumour viruses are oncogenic only in animals which they do not infect in nature, while other tumour viruses are oncogenic in their natural host.

It is fairly well established now that in oncogenesis the viral genome or portions of it become integrated into the host cell DNA. If the tumour virus does not carry an oncogene, in that case integration of the viral genome produces mutations in the host DNA. When mutations occur at certain sites they bring about cancerous changes in the cell.

The oncogene of RSV is called src (for sarcoma) and was first described by Martin in temperature sensitive conditional mutants of the virus. Weissmann and his colleagues at Zurich conducted a detailed study of src gene in deletion mutants of RSV mentioned earlier.

They could identify the src gene as one segment of RNA near one end of the genome. By applying recombinant DNA techniques RSV was shown to carry a single oncogene src coding for a single protein product and capable of transforming normal cells.

The protein coded by src has been designated pp60v-src and has a molecular weight of about 60,000 Daltons. This protein is a kinase enzyme and acts by phosphorylating the amino acid tyrosine. In other words, the enzyme attaches phosphate ions to tyrosine in protein chains.

Enzymes encoded by oncogenes have also been analysed in polyoma and a few other viruses, and phosphorylation of tyrosine seems to be a common function of oncogene-encoded enzymes. In the case of pp60v-src transformed cells have about 10 times as much phosphorylated tyrosine as compared to a normal cell.

It has also been seen that pp60v-src phosphorylates tyrosine in a membrane protein called vinculin which in transformed cells decreases the ability of cells to adhere to solid surfaces. Decrease in cell adhesion is a common property of cancer cells.

If oncogenes are present within the genetic material of vertebrate animals and humans, it should be possible to locate them. Bishop et al., (1982) prepared radioactive DNA probes complementary to the src gene in RSV and used them for molecular hybridisation with DNA of higher animals and humans. A sequence related to src was indeed found in humans and fishes and designated c-src (c for cellular).

Interestingly c-src has been found to be transcribed and translated in normal cells. Its protein product is similar to pp60v-src in structure and function, that is, it phosphorylates tyrosine. So far 16 retrovirus oncogenes have been found to be present in normal vertebrate cells as cancer genes.

It may be concluded that cancer genes are normal components of the genetic material of higher animals including humans. Very recent evidence for existence of cancer genes has come from the independent works of Weinberg and of Wigler who could transform normal human cells (NIH3T3 cells) in culture by means of DNA fragments cloned from human bladder carcinoma cell lines.

Viroids:

Viroids are the smallest organisms causing infectious disease. All they have is a naked short strand of RNA. The protein coat present in viruses is lacking in viroids. So far, viroids have been identified only in diseases of higher plants, although their association with animal diseases is also suspected.

The best studied viroid is the one causing potato spindle tuber disease (PSTV). The infectious agent is a single stranded linear or circular RNA molecule about 50 nm long having molecular weight of 130,000; Diener (1960) called this agent viroid. The single stranded molecule is folded due to intrachain base pairing. The complete nucleotide sequence of PSTV was worked out by Gross et al., (1978).

The viroid has 359 nucleotides comprised of 73 adenines, 77 uracils, 10 guanines and 108 cytosines. A unique structural feature of PSTV is that it is a closed circular RNA molecule which seems to give the impression of a long double stranded molecule due to intrachain base pairing. The unpaired regions project outwards as loops. Viroids can replicate in the host cell, but do not seem to be translated.

In a few other plant diseases, viroids have been established as the causal organism. Among these are Cadang-cadang, a fatal disease that nearly wiped out coconut-trees in Philippines. The cucumber pale-fruit disease, the stunt disease of hops, and two diseases of chrysanthemums, namely, chlorotic mottle and stunt are all caused by viroids.