Class 12 Molecular Basis of Inheritance-I NEET AIIMS
Molecular Basis of Inheritance-I
Introduction
l Even though the discovery of nuclein by F. Meischer (1869) and the proposition of inheritance by G.J. Mendel (1866) were almost of the same time, that DNA acts as genetic material took about 60 years to be discovered (F. Griffith, 1928) and about 75 years to be proven (Avery et al. 1944). Previous discoveries by G.J. Mendel, T.Boveri, W. Sutton, T.H. Morgan and others proved that genes were present in the chromosomes present inside the nucleus. But the question of what molecule (either protein, DNA or RNA; the constituents of the chromosome) was the genetic material, had not been answered.
l Genes should be capable of three major functions:
Ä They should be replicate and be inherited by the progeny faithfully.
Ä They should be susceptible to an occasional change by way of mutation and such a change should be stably inherited.
Ä They should be able to carry all the information necessary to program the functions of a cell.
l In the first instance, it appeared that proteins would be the ideal substance to be the genetic material because proteins are made up of twenty amino acids. The diversity that can be generated on the basis of the number of residues of each amino acid in a given protein and the sequence of amino acids would be enormous. But in 1949, A. Mirsky and H. Ris reported that all cells of organisms have a constant amount of DNA but different amounts of proteins,
Definition
l The chemical substance which stores biological information in a coded form transfers it to the next generation and expresses it in the offspring is called genetic material.
Evidence in Favour of DNA as Genetic Material
l Different evidence in favour of genetic material are :
Bacterial Transformation
l It is the change in the genetic constitution of a bacterium by picking genes of its dead relatives present outside the medium and integrating the same into its nucleoid. In 1928, Frederick Griffith, a British Medical Officer, studied two strains of Streptococcus pneumoniae, also called Pneumococcus pneumoniae ( Diplococcus pneumoniae) :
Ä Capsulated or S III type (virulent) – Smooth colony
Ä Non-capsulated or RII type (avirulent) – Rough colony
l Four sets of experiments were performed. He injected live, rough bacteria into mice. The mice did not die. The mice injected with live, smooth bacteria died, but the mice injected with heat-killing, smooth bacteria survived. Griffith then injected mice with a mixture of live, rough bacteria and heat-killing, smooth bacteria. Although neither of these alone was harmful, their mixture caused pneumonia and killed the mice. Moreover, he found live, smooth bacteria in the dead mice.
l Griffith concluded that the heat-killing S III bacteria were somehow responsible for converting live avirulent RII cells virulent SIII ones. Calling the phenomenon transformation principle might be some part of a polysaccharide capsule or some compound required for capsule synthesis, although the capsule alone did not cause pneumonia. To use Griffith’s term, the transforming principle from the dead SIII cells served as “pabulum” for the RII cells. However, the biochemical nature of genetic material was not defined from his experiments.
l In 1944, O.T. Avery, C. Macleod and M. Mc Carty separated the extract of heat-killed S. III type into protein, DNA and carbohydrate fractions. Each fraction was separately added to a culture medium containing live R II types. Only the cultural medium that received the DNA fraction of S III type produced the live S cells. This proved that DNA was the transforming agent. When DNA fraction was treated with deoxyribonuclease enzyme, R II type is not converted into S III type. This confirmed that DNA is genetic material. DNA is not destroyed if the cells are killed by heat.
l They also discovered that protein-digesting enzymes (proteases) and RNA –digesting enzymes (R Nases) did not affect transformation, so the transforming substance was not a protein or RNA.
l The work of Rollin Hotchkiss also confirmed that the critical factor in the transformation was DNA and not protein. The transformation has now been shown to occur in haemophilus influenza, Bacillus subtilis, Shigella para dysenteriae, and Escherichia coli.
Bacteriophage Multiplication (Transduction or Bacterial infection)
l The second major piece of evidence supporting DNA as the genetic material was provided during the study of the bacterium, E.coli and one of its infecting viruses, bacteriophage, often referred to simply as a phage. In 1952, Alfred Hershey and Martha J. Chase by his Wareing -blender experiment confirmed that DNA from the bacteriophage enters the host cell and carries the necessary information for the formation of new phages.
l They devised an experiment with two different preparations of phage. In one preparation, the protein part was made radioactive (35S) and in the other, DNA was made radioactive (32P). DNA contains phosphorus (P), but not sulphur, effectively labels DNA, and because proteins contain sulphur (S), not phosphorus, labels protein. This was the key point in the experiment. These two phage preparations were made to infect a culture of E. coli.
l Soon after infection, the E. coli cells were gently agitated in a blender to loosen the adhering phage particles and the culture was centrifuged. The researchers found that when phage containing radioactive DNA was used to infect E. coli, the bacteria showed radioactivity. But, if phage containing radioactive protein was used as an infecting agent, the bacteria contained very little or no radioactivity. This experiment indicated that during infection with the virus, it was the DNA that entered into the bacteria.
l Moreover, when such bacteria were allowed to grow further, they lysed and new viral particles were released. This finding suggests that it is the viral DNA and not protein that contains information for the production of more viral particles and, therefore, DNA should be the genetic material.
l A.D. Hershey, M. Delbruck and S.E. Luria shared the Nobel Prize in medicine for 1969, for their contribution to replication and recombination of viruses.
Transfection Experiment
l In 1960, George Guthrie and Robert Sinsheimer purified DNA from bacteriophage, a small phage that contains a single-stranded circular DNA molecule of some 5386 nucleotides. When added to E. Coli protoplasts, also called spheroplast (wall-less cells obtained by treating cells with enzyme lysozyme), the purified DNA resulted in the production of complete bacteriophage. This process of infection by only the viral nucleic acid, called transfection proved conclusively that DNA alone contains all the necessary information for the production of mature phages. Thus, the evidence supporting the conclusion that DNA serves as genetic material was further strengthened.
RNA AS GENETIC MATERIAL IN PLANT VIRUSES
l In some viruses RNA instead of DNA serves as the genetic material, (for example, Tobacco Mosaic Virus, QB bacteriophage, etc). RNA as genetic material in plant virus (e.g., TMV) was shown by H. Fraenkel-Conrat (1957) in California. When the RNA part was made to infect tobacco plants, it caused tobacco mosaic disease, whereas the protein part was made to infect tobacco, no infection was caused. This confirms RNA as genetic material.
Heinz Fraenkel-Conrat and B Singer separated and isolated RNA and coated proteins from TMV and HRV (Holmes ribgrass virus) and reconstituted hybrid or chimeric viruses by using RNA of one virus and protein of the other, and vice-versa. When this hybrid virus was spread on tobacco leaves, the lesions that developed corresponded to the type of RNA in the reconstituted virus. Again, it was concluded that RNA serves as the genetic material in these viruses.
l Norman R. Pace and Sol Spiegelman (1965, ’66) isolated RNA from QB and allowed it to replicate in vitro by using an enzyme, RNA replicase. (isolated from host E.coli cells following normal infection). When RNA replicated in vitro was added to E. coli protoplasts, infection and viral multiplication occurred. Thus RNA serves as the genetic material in this phage.
l Following infection of the host cell, RNA of retroviruses serves as a template for the synthesis of the complementary DNA molecule by the process of reverse transcription under the direction of an RNA-dependent DNA polymerase enzyme called reverse transcriptase. This DNA is incorporated into the genome of the host cell, and when the host DNA is transcribed, copies of the original RNA chromosomes are produced. Retroviruses include HIV as well as RNA tumour viruses.