Molecular Biology Practical No.2 DNA as a carrier PDF

Molecular Biology Practical No.2 DNA as a carrier of the genetic information While the chemists were working out the structure of nuclein, the biologists were attempting to elucidate the transformation of the genetic information through cells. A number of experiments were conducted on both bacteri...

Molecular Biology Practical No.2 DNA as a carrier of the genetic information While the chemists were working out the structure of nuclein, the biologists were attempting to elucidate the transformation of the genetic information through cells. A number of experiments were conducted on both bacteria and viruses and the first clue that the DNA was the carrier of the genetic informations came from the experiments of the English physician Frederick Griffith in 1928,whichwas one of the first experiments suggesting that, bacteria are capable of transferring genetic information. Dr. Griffith began studying Streptococcus pneumoniae (figure1.1) in his laboratory in the hopes of developing a vaccine against it. As so often happens in scientific research, Griffith never found what he was looking for (there is still no vaccine for pneumonia), but instead, he made one of the most important discoveries in the field of biology: a phenomenon he called "transformation." Figure (1.1):Streptococcus pneumoniae in Gram-stained smears. The organisms from a colony growing on agar medium (left) are gram positive and lancet shaped and appears in pairs and short chains. In a Gram stain of cerebrospinal fluid from a patient with pneumococcal meningitis (right), the organisms are mostly diplococci. The capsule (arrow) can be seen around some bacteria, outlined by the pink proteinaceous material of the fluid. Dr. Griffith had isolated two strains of S. pneumoniae, one of which was virulent or pathogenic (meaning it causes sickness or death, in this case, pneumonia), and one which was non- virulent or harmless. The pathogenic strain looked smooth when 1 grown on agar plate due to a protective polysaccharide coat surrounding the bacteria and so he named this strain S, for smooth. The harmless strain of S. pneumoniae lacked the protective coat and produce a rough appearing colony on an agar plate; these forms are referred to as R, for rough (figure 1.2). It was found that, Capsular polysaccharides are the primary basis for the pathogenicity of the organism. They are antigenic and form the basis for classifying pneumococci by serotypes. Ninety serotypes have been identified, based on their reaction with type-specific antisera. Figure (1.2): An illustration of the Rough (R) and smooth (S) Streptococcus pneumoniae cells. Dr. Griffith was interested in the origins of the different strains of S. pneumonia and why some types were virulent, whereas others were not. He observed that injecting small amounts of living type IIIS bacteria into mice caused the mice to develop pneumonia and die; on autopsy, he found large amounts of type IIIS bacteria in the blood of the mice. Whereas, when he injected type IIR bacteria into mice, the mice lived, and no bacteria were recovered from their blood (figure 1.3). Figure (1.3): Transforming experiment: Rough (IIR) and Smooth (IIIS) Streptococcus pneumoniae cells. 2 Next, Dr. Griffith noticed that, if he applied heat to the IIIS strain of bacteria, and then injected them into mice, the mice would no longer get sick and die. He thus hypothesized that excessive heat kills the bacteria. However, Dr. Griffith didn't stop there – he decided to try something else; he mixed living IIR bacteria (which are not pathogenic) with heat-killed IIIS bacteria, and then he injected the mixture into mice. Surprisingly, the mice got pneumonia infections and eventually died after five days (figure 1.4). When Griffith examined the blood collected from the hearts of these mice, he observed live type IIIS bacteria. Figure (1.4): Illustration of F. Griffith's discovery of transformation in S. pneumoniae using mice. Dr. Griffith’s results had several possible interpretations, all of which he considered: The first possibility, it could have been the case that, he had not sufficiently sterilized the type IIIS bacteria and thus a few live bacteria remained in the culture. Any live bacteria injected into the mice would have multiplied and caused pneumonia. Griffith knew that this possibility was unlikely, because he had used only heat-killed type IIIS bacteria in the control experiment, and they never produced pneumonia in the mice. 3  The second possibility was that, the live type IIR bacteria had mutated to the virulent S form. Such a mutation would cause pneumonia in the mice, but it would produce type IIS bacteria, not the type IIIS that Griffith found in the dead mice. The most appropriate explanation for the unusual results was that, these bacteria can somehow be genetically "re-programmed" into a slightly different version of themselves. One strain of bacteria, in this case the IIR strain of S. pneumoniae, can be changed into something else, presumably because of the transfer of genetic material from a donor, in this case the heat- killed IIIS strain to the non- virulent IIR. Scientists around the world began repeating this experiment, but in slightly different ways, trying to discover exactly what was happening. It became clear that, when the IIIS bacteria are killed by heat, they break open and many substances are released. Something in this mixture can be absorbed by living bacteria IIR, leading to a genetic transformation transforming the IIR to live IIIS. But because the mixture contains protein, RNA, DNA, lipids, and carbohydrates, the question remained – which molecule is the "transforming agent?" 4