Biochemistry and Molecular Biology - Belyasova N.A. 2002

Molecular Foundations and Mechanisms of Heredity
Organization of the Cellular Genetic Apparatus

The evolution of our understanding of DNA as the molecule that encodes cellular hereditary information unfolded in The history of biochemistry over a span of about two decades through several distinct stages. Such a prolonged debate among researchers regarding one of the fundamental questions of natural science was driven, on the one hand, by conservative views on the Structure of Nucleic Acids as "simply organized molecules." Given the limited knowledge of the time, both DNA and RНК were assumed to be polymers formed by the repetition of tetranucleotides. Meanwhile, protein molecules had begun to be studied earlier than other cellular macromolecules, and by 1928 a certain degree of progress had been achieved in understanding their Organization: it was known that they consist of at least 20 Amino Acids arranged in a random order, which allows for a vast array of polypeptide structures.

The historical path leading to the ESTABLISHMENT OF THE postulate "DNA is the carrier of hereditary information" is a remarkable example of the elegance of human thought, sheds light on many patterns of trait inheritance in living organisms, and is well worth studying.

The first direct proof of the genetic role of DNA came from F. Griffith's pneumococcal transformation experiments in 1928. Griffith worked with Two Types of Diplococcus pneumoniae strains: S-forms, which form smooth, shiny colonies on Agar media (from the English smooth), and R-forms, characterized by a rough colony surface (from the English rough). The S-forms were highly virulent to mice and caused Pneumonia in them. However, S-form pneumococci killed by heating to 65° C did not cause disease or death in the mice. The R-forms were of low virulence and rarely caused illness in mice.

Griffith discovered that if mice were infected with a mixture of living R-forms and heat-killed (to 65° C) S-forms, the animals fell ill, and viable S-form pneumococci could be isolated from their Blood—specifically of the same serotype as the heat-killed S-forms. This observation led Griffith to conclude that a "transformation" of one type of Bacteria (R) into another type (S) occurs within the mouse Organism, and that the transforming factor must be a substance determining hereditary properties contained within the heat-killed Cells. Since Proteins undergo Denaturation at the temperatures used (60–65 °C), Griffith hypothesized that the transforming factor was most likely DNA rather than protein.

Since Griffith's experiments, this method of Genetic information transfer has been known as transformation. It was later discovered that The Nature of the pneumococcal Cell surface is determined by two Gene alleles: the S allele controls The Cell's ability to form a polysaccharide capsule, which gives colonies a smooth surface and protects pneumococci from the mouse's immune system; if the R allele is present in the cell, the capsule is not formed, and the cells are easily recognized and destroyed by the host's immune system.

At the time, Griffith's experimental results and Conclusions were rejected by the scientific community as defying traditional views on these processes. It took the Replication of similar in vitro Procedures, carried out in 1944 by American researchers Avery, MacLeod, and McCarty, to change this. These scientists transformed a growing culture of R-type pneumococci using DNA isolated from S-strain cells. It turned out that some bacteria acquired The ability to synthesize a polysaccharide capsule and, consequently, became pathogenic to mice. Furthermore, purified DNA was the sole factor capable of conferring this property upon R-cells. In addition, these experiments revealed that Proteolytic Enzymes had no effect on transformation, whereas treating the transforming factor with Nucleases prevented the transformation process altogether. Finally, the experiments showed that the S-type bacteria resulting from transformation retained the ability to pass on this acquired trait (capsular Polysaccharide synthesis) to their progeny. The evidence obtained by the American scientists regarding The Role of DNA in the storage and transmission of hereditary information was fundamental and made history, although it was not appreciated immediately for the reasons mentioned above. Moreover, The Study of the basics of heredity in 1944 was only just beginning, and it had not yet been definitively established that bacteria possess genes entirely analogous to those of higher organisms.

The definitive proof of the genetic role of DNA came from experiments conducted by Alfred Hershey and Martha Chase in 1952. They successfully demonstrated that DNA is the carrier of hereditary information in the T2 bacteriophage. The Essence of the experiments was as follows. One culture of Escherichia coli cells was grown in a medium containing radioactive phosphorus isotopes (³²Р), while another was grown in the presence of sulfur isotopes (³⁵S), resulting in the incorporation of these isotopes into the cell contents. Each of the labeled bacterial cultures was then used to produce a T2 lysate. Different lysates of isotope-labeled phages were obtained: one contained T2 particles with ³⁵S incorporated into the protein (capsid), and the other contained T2 particles with ³²Р incorporated into the DNA. Radioactive labels made it possible to trace the paths of the phage protein and DNA during its reproduction.

The lytic cycle begins with the attachment of the phage particle to the cell surface, and after a certain time, the phage DNA is injected into the cell. This was confirmed by the results of centrifuging the Suspensions at designated stages: initially, both phages and bacteria sedimented together (with ³⁵S and ³²Р detected in the pellet). However, after a certain time, most of the sulfur-isotope-labeled protein could be detached from the cells by agitating the suspension, whereas most of the phosphorus-isotope-labeled DNA remained attached to the bacteria and was recovered in the pellet. This strongly suggested that the DNA ends up inside the cells.

Removing empty phage coats ("ghosts") from the culture does not affect subsequent events: the bacteria lyse, and phage progeny are released in exactly the same way as when the "ghosts" remain on the cell surface. It turned out that the removal of "ghosts" is accompanied by the removal of at least 80% of the ³⁵S, while the bulk of the ³²Р remains inside the cells and is subsequently passed on to the progeny (during phage reproduction). Thus, it became abundantly clear that DNA, rather than protein, directs The process of phage reproduction within cells.

The Hershey-Chase experiment served as conclusive proof of the genetic role of DNA and drew attention to the work performed on pneumococci a few years earlier. Several factors contributed to this: by 1952, research into The structure of Nucleic Acids had made great strides, dispelling the notion that these molecules were conservative; the experiment was performed on a bacteriophage whose mechanisms of trait inheritance were well known to be analogous to those of higher organisms; and finally, Mutations were demonstrated in the T2 phage, and recombination of mutant genes was described just as in higher organisms.

Additional proof of the genetic role of DNA was the discovery of infectious properties in a purified DNA preparation from the tobacco mosaic virus.