DNA is the Genetic Material

Welcome to Transformative Advances in Molecular Biology, a YouTube series prepared by graduate students in a journal colloquium at the University of Florida. Dr. Mark Settles and I decided that instead of the traditional discussion of contemporary science literature, that it'd be fun and informative to look backwards to review the seminal discoveries that seeded contemporary molecular biology. This series offers student presentations of milestone papers in this area.

This is the story of DNA as the hereditary material, as told by Derek and Tingting.

Firstly, let's look at the background. There are 3 very important milestones during the process of DNA research. The first one is Griffith's discovery of transformation of pneumococcal types in 1928. The second one is in 1944, Avery and his colleagues demonstrated that The substance including transformation of pneumococcal types is DNA. Until 1952, the process of bacterial infection by phage is found and we knew that DNA is the real hereditary material. This slide shows us the famous Griffith's experiment. Living R strain and heat killed S strain pneumococci are not virulent for mice, while living S strain is lethal. In 1928, Griffith reported that mice unexpectedly died after being injected with a mixture of living R strain and heat-killed S strain bacteria. Griffith was also able to recover infectious type 2 S pneumococci From the hearts of the dead mice, indicating that the changes were heritable. From these results, Griffith hypothesized that a substance could be transferred from non-living S cells of one serotype to living R cells of another serotype and transform R-streng bacteria into a lethal form, which is named transforming principle later.

Avery, MacLeod, and McCarty undertook an experiment in 1944 based on the findings of Griffith. Starting with large aliquots of virulent smooth bacterial cultures, they successfully isolated a highly purified sample with strong capability to transform rough cultures into smooth cultures. Using an elementary chemical analysis shown in Table 1, they determined that several independent preparations of transforming principle had a chemical composition close to that known for sodium deoxyribonucleate, or DNA. To verify this assertion, they subjected preparations of the transforming principle to crude enzyme preparations from several animal and bacterial sources. Only those sources known to contain depolymerase for deoxyribonucleate, known today as DNase, fully inactivated the transforming principle. Based on these findings, it was speculated that the transforming principle was likely to be DNA, or a factor very closely associated with the DNA molecule in the preparation. Subsequent work by Hershey and Chase was undertaken in later years to demonstrate unequivocally that DNA was the transforming agent. Hershey and Chase leveraged the bacteriophage system to demonstrate their findings. Table 1 shows that whole phage labeled with either radioactive phosphorus, which only labels DNA, or radioactive sulfur, which only labels protein, can be separated by osmotic shock. However, when phage is adsorbed to sensitive bacteria, both protein and DNA can be recovered from the precipitate. This finding suggests that during the course of infection, DNA remains protected either by the bacterial membranes, the protein coat of the phage, or both.

Table 2 shows us the effect of phage adsorption on sensitivity to DNS. There are 4 treatments here. They are phage adsorbed to live bacteria, bacteria heated before or after infection, and heated unadsorbed phage. Phage was labeled with P32 and S35, co-cultured with bacteria in adsorption media for 5 minutes, then washed and centrifuged. The low percentage of p32 in non-sedimentable acetate shows that phage adsorbed to live bacteria is protected from DNase. After heating unadsorbed phage at different temperatures, we know only high, very high temperature, which is more than 90 degrees, caused the release of phage DNA. However, phage adsorbed to bacteria which are killed by 80°C heating become sensitive to DNase. Therefore, they supposed that adsorption to bacteria might help the release of phage DNA since the protein shells of phage don't break at 80°C.

Similar to the outcome of disrupting bacterial membranes by heat shock, Freezing and thawing infected cells also released the phage DNA to be sensitive to degradation by DNase. Much of the phage DNA remains associated with the bacteria, but disruption of the bacterial membrane exposes the DNA to DNase. This suggests that phage DNA becomes part of an organized structure involving the bacterial membranes during the course of the infection process.

This table shows us that the sensitization Of phage DNA to DNase is due to liberation of the DNA from the protein coat. According to the table, 80% of phage was inactivated, and the sulfur was in the sediment with bacterial cells. Most of the DNA was either surviving phage or liable to DNase. One disadvantage of this experiment is that it was not known if the liberated DNA represents all or only some of the contents of the inactivated phage.

The key experiment conducted by Hershey and Chase was to utilize a Waring blender to show that phage protein can be separated from living bacterial cells in infected cultures, whereas phage DNA remains associated with the bacterial cell. After running an infected culture in their blender for 2 minutes, nearly 80% of the phage protein could be recovered independent of the bacterial cells, as shown in the figure. However, only about 20% of the DNA could be recovered. Nearly all of the infected bacteria remained viable during the experiment. This suggests that DNA is harbored within a living bacterial cell, and that most of the protein plays no part in phage replication.

Table 8 shows us the sulfur transfer from parental phage to progeny is less than 1%. From the table, we know that striping reduces the S-35 in all fractions. After separation, the radioactive tracer then was found in the low-speed sediment, protein shells. In high-speed sediment, which contains most of phage progeny, sulfur is much less. Mechanical separation of protein coats from infected bacteria eliminates nearly all traces of S-35. from fractions containing phage progeny.

So to summarize, the Hershey-Chase experiment demonstrated that DNA is the transforming principle and the hereditary material by showing that phage DNA moves into the bacterial cell during the course of infection, and that the ability of phage to replicate is dependent on the transfer of DNA into the bacterial cell. Protein plays little role in the process of phage replication. But is important for phage DNA to enter into the bacterial cell.

Finally, let's have a look at the overall conclusion. Firstly, the transforming principle first described by Griffith in 1928 is DNA.

Second, DNA is wholly responsible for transmission of information from parents to progeny.

Thirdly, DNA is capable of carrying information about non-nucleic acid structures of the cell.

Fourth, protein plays little to no role in heritability.

Finally, DNA is real hereditary material.

Thank you for watching, and please check out the other exciting topics in the series.