Biochemistry and Molecular Biology - Belyasova N.A. 2002

Molecular Foundations and Mechanisms of Heredity
Organization of the Cellular Genetic Apparatus
Establishment of the "DNA as the Carrier of Genetic Information" Postulate

The Evolution of the concept of DNA as the molecule responsible for encoding cellular hereditary information spanned approximately twenty years in The history of biochemistry, unfolding across several stages. This 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." Due to insufficient understanding at the time, both DNA and RНК were believed to be polymers formed by the monotonous repetition of tetranucleotides. In contrast, protein molecules began to be studied earlier than other cellular macromolecules. By 1928, significant progress had been made in understanding their Organization: it was known that they consist of at least 20 Amino Acids arranged in random order, which accounts for the vast diversity of polypeptide structures.

The historical ESTABLISHMENT OF THE postulate "DNA is the carrier of hereditary information" is a remarkable testament to the ingenuity of human thought, shedding 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 experiments on pneumococcal transformation in 1928. Griffith worked with two distinct strains of Diplococcus pneumoniae: 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 in mice, causing Pneumonia. However, when heated to 65 °C, these S-form pneumococci lost their ability to cause disease and death in mice. The R-forms were of low virulence and rarely induced illness.

Griffith discovered that if mice were infected with a mixture of live 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 matching the serotype of the heat-killed S-Cells. This observation led Griffith to conclude that a "transformation" of one bacterial type (R) into another (S) occurred within the mouse Organism. The transforming factor had to be a substance containing hereditary properties derived from the heat-killed cells. Since Proteins undergo Denaturation at the temperatures used (60–65 °C), Griffith hypothesized that the transforming factor was likely DNA rather than protein.

Ever 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 their smooth appearance and protects the pneumococci from the mouse's immune system; if the R allele is present, 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 went far beyond traditional concepts of these processes and were not accepted by the scientific community. It took the Replication of similar experiments in vitro in 1944 by American researchers Avery, MacLeod, and McCarthy 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. These experiments also revealed that transformation was unaffected by Proteolytic Enzymes; conversely, treating the transforming factor with Nucleases prevented the transformation process. Finally, the results demonstrated that the resulting S-type bacteria possessed the ability to acquire and transmit this new trait (capsular Polysaccharide synthesis) to their progeny. While the evidence obtained by the American scientists regarding The Role of DNA in storing and transmitting hereditary information was fundamental and made history, it was not immediately appreciated for the reasons mentioned above. In addition, The Study of the fundamentals of heredity was only in its infancy in 1944, and it had not yet been definitively established that bacteria possess genes entirely analogous to those of higher organisms.

Definitive proof of the genetic role of DNA came from experiments conducted by Alfred Hershey and Margaret Chase in 1952. They successfully proved that DNA is the carrier of hereditary information in the T2 bacteriophage. The Essence of their 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 cellular contents. Each labeled bacterial culture was then used to produce a T2 lysate. This yielded different batches of isotopically labeled phages: one contained T2 particles with ³⁵S incorporated into the protein coat (capsid), and the other contained T2 particles with ³²Р within their DNA. These radioactive labels made it possible to trace the paths of the phage protein and DNA during reproduction.

The lytic cycle begins with the attachment of the phage particle to the cell surface, after which the phage DNA is injected into the cell. This was confirmed by centrifuging the Suspensions at specific stages: initially, both phages and bacteria sedimented together (with ³⁵S and ³²Р detected in the pellet). However, after a certain time, shaking the suspension caused the bulk of the sulfur-labeled protein to detach from the cells, whereas the majority of the phosphorus-labeled DNA remained attached and was recovered in the pellet. This provided strong evidence that the DNA had entered the cells.

Removing the empty phage protein shells ("ghosts") from the culture did not affect subsequent events: the bacteria lysed and released progeny phages just as they did when the "ghosts" remained on the cell surface. It turned out that removing the "ghosts" resulted in the removal of at least 80% of the ³⁵S, while the bulk of the ³²Р remained inside the cells and was subsequently passed on to the progeny during phage reproduction. Thus, it became unmistakably clear that DNA, rather than protein, directs the reproduction of the phage inside cells.

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

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