Download Untitled

1
2
3
۴
۵
A second interpretation 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. Many mutations would be required for type II bacteria to mutate to type III bacteria, and the chance of all the mutations occurring simultaneously was impossibly low.
Griffith finally concluded that the type IIR bacteria had somehow been transformed, acquiring the genetic virulence of the dead type IIIS bacteria. This transformation had produced a permanent, genetic change in the bacteria. Although Griffith didn’t understand the nature of transformation, he theorized that some substance in the polysaccharide coat of the dead bacteria might be responsible. He called this substance the transforming principle..
After 10 years of research, Avery, Colin MacLeod, and
Maclyn McCarty succeeded in isolating and purifying the
transforming substance. They showed that it had a chemical
composition closely matching that of DNA and quite different
from that of proteins. Enzymes such as trypsin and
chymotrypsin, known to break down proteins, had no effect
on the transforming substance. Ribonuclease, an enzyme
that destroys RNA, also had no effect. Enzymes capable of
۶
destroying DNA, however, eliminated the biological activity
of the transforming substance (Figure 10.3). Avery,
MacLeod, and McCarty showed that purified transforming
substance precipitated at about the same rate as purified
DNA and that it absorbed ultraviolet light at the same wavelengths
as DNA. These results, published in 1944,
6
At the time of the Hershey–Chase study (their paper was published in 1952), biologists did not understand exactly how phages reproduce. What they did know was that the T2 phage is approximately 50% protein and 50% nucleic acid, that a phage infects a cell by first attaching to the cell wall, and that progeny phages are ultimately produced within the cell. Because the progeny carry the same traits as the infecting phage, genetic material from the infecting phage must be transmitted to the progeny, but how this genetic transmission takes place was unknown.
Hershey and Chase grew one batch of E. coli in a medium containing 32P and infected the bacteria with T2 phage so that all the new phages would have DNA labeled with 32P (Figure 10.5). They grew a second batch of E. coli in a medium containing 35S and infected these bacteria with T2 phage
so that all these new phages would have protein labeled with 35S. Hershey and Chase then infected separate batches of unlabeled E. coli with the 35S‐ and 32P‐labeled phages. After allowing time for the phages to infect the cells, they placed the E. coli cells in a blender and sheared off the then‐empty
protein coats (ghosts) from the cell walls. They separated out the protein coats and cultured the infected bacterial cells. When phages labeled with 35S infected the bacteria, most of the radioactivity was detected in the protein ghosts and little was detected in the cells. Furthermore, when new phages
٧
emerged from the cell, they contained almost no 35S (see Figure 10.5). This result indicated that, although the protein component of a phage is necessary for infection, it does not enter the cell and is not transmitted to progeny phages.
In contrast, when Hershey and Chase infected bacteria with 32P‐labeled phages and removed the protein ghosts, the bacteria were still radioactive. Most significantly, after the cells lysed and new progeny phages emerged, many of these phages emitted radioactivity from 32P, demonstrating that DNA from the infecting phages had been passed on to the progeny (see Figure 10.5). These results confirmed that DNA, not protein, is the genetic material of phages.
7
At the time of the Hershey–Chase study (their paper was published in 1952), biologists did not understand exactly how phages reproduce. What they did know was that the T2 phage is approximately 50% protein and 50% nucleic acid, that a phage infects a cell by first attaching to the cell wall, and that progeny phages are ultimately produced within the cell. Because the progeny carry the same traits as the infecting phage, genetic material from the infecting phage must be transmitted to the progeny, but how this genetic transmission takes place was unknown.
Hershey and Chase grew one batch of E. coli in a medium containing 32P and infected the bacteria with T2 phage so that all the new phages would have DNA labeled with 32P (Figure 10.5). They grew a second batch of E. coli in a medium containing 35S and infected these bacteria with T2 phage
so that all these new phages would have protein labeled with 35S. Hershey and Chase then infected separate batches of unlabeled E. coli with the 35S‐ and 32P‐labeled phages. After allowing time for the phages to infect the cells, they placed the E. coli cells in a blender and sheared off the then‐empty
protein coats (ghosts) from the cell walls. They separated out the protein coats and cultured the infected bacterial cells. When phages labeled with 35S infected the bacteria, most of the radioactivity was detected in the protein ghosts and little was detected in the cells. Furthermore, when new phages
٨
emerged from the cell, they contained almost no 35S (see Figure 10.5). This result indicated that, although the protein component of a phage is necessary for infection, it does not enter the cell and is not transmitted to progeny phages.
In contrast, when Hershey and Chase infected bacteria with 32P‐labeled phages and removed the protein ghosts, the bacteria were still radioactive. Most significantly, after the cells lysed and new progeny phages emerged, many of these phages emitted radioactivity from 32P, demonstrating that DNA from the infecting phages had been passed on to the progeny (see Figure 10.5). These results confirmed that DNA, not protein, is the genetic material of phages.
8
٩
Watson and Crick investigated the structure of DNA, using all available information about
the chemistry of DNA to construct molecular models. By applying the laws of structural
chemistry, they were able to limit the number of possible structures that DNA could assume.
They tested various structures by building models made of wire and metal plates. With their
models, they were able to see whether a structure was compatible with chemical principles
and with the X-ray images.
١٠
DNA and its building blocks
DNA = nucleotides which are linked covalently into a polynucleotide chain with a sugarphate backbone from which the bases extend.
The arrowheads at the ends of the DNA strands indicate the polarities of the two strands,
which run antiparallel to each other in the DNA molecule. In the diagram at the bottom left
of the figure, the DNA molecule is shown straightened out; in reality, it is twisted into a
double helix, as shown on the right. For details see Figure 4-5
11
١٢
١٣
Complementary base pairs in the DNA double helix.
The shapes and chemical structure of the bases allow hydrogen bonds to form efficiently
only between A and T and between G and C. where atoms that are able to form hydrogen
bonds can be brought close together without distorting the double helix. As indicated, two
hydrogen bonds form between A and T, while three form between G and C.The bases can
pair in this way only if the two polynucleotide chains that contain them are antiparallel to
each other.
14
Phosphodiester bond
15
DNA is an alpha helix (righthanded, or clockwise). 10 bp per a rotation; so each base pair is twisted 36 degrees relative to the adjacent bases. The base pairs are 0.34 nm apart; so each rotation of the molecule encompasses 3.4 nm. The diameter of the helix is 2 nm. The spiraling of the nucleotide strands creates major and minor grooves in the helix, features that are important for the binding of some proteins (regulate the expression regulators).
١۶
A radically different secondary structure, called Z‐DNA (Figure 10.15c), forms a left‐handed helix. In this form, the sugar–phosphate backbone zigzags back and forth, giving rise to its name. A Z‐DNA structure can result if the molecule contains particular base sequences, such as stretches of alternating C and G nucleotides. Recently, researchers have found that Z‐DNA‐specific antibodies bind to regions of the DNA that are being transcribed into RNA, suggesting that Z‐DNA may play some role in gene expression.
١٧
Bacterial DNA is frequently methylated to distinguish it from foreign, unmethylated DNA
that may be introduced by viruses; bacteria use proteins called restriction enzymes to cut up
any unmethylated viral DNA.
In eukaryotic Methylation can also affect the three-dimensional structure of the DNA
molecule. A and C are commonly methylated in bacteria.
18