Hershey and Chase: the role of DNA on the T2 phage life cycle

In 1952, Alfred D. Hershey and Martha Chase performed several experiments with T2 bacteriophage, a virus that infects bacteria. Their results convinced even the sceptics that DNA, and not protein, was the genetic material.

Electron micrographs indicate that T2 bacteriophage infects Escherichia coli by injecting its DNA into the bacterium while leaving its protein coat on the outside. The phage takes over the genetic machinery of the host cell to make new phages. Eventually, the bacterial cell bursts (a process called lysis), releasing new phages to infect other bacteria (figure 1).

Hershey and Chase wanted to test the hypothesis that only the viral DNA entered the bacterium. They made use of the fact that DNA contains phosphorus but not sulphur, whereas protein contains sulphur but not phosphorus.

§ With some T2 phages, they labelled the viral DNA with a radioactive isotope of phosphorus (³²P). With other T2 phages, they labelled the viral protein coat with a radioactive isotope of sulphur (³⁵S).

§ They added the viruses to a culture of E. coli and gave them enough time to infect their host cells (but not enough time to reproduce).

§ The viral coats were then separated from the infected bacteria by shaking the mixture vigorously in a blender.

§ When E. coli was infected with a T2 phage containing ³⁵S (labelled Protein), little radioactivity occurred within the bacterial cells.

§ With a T2 phage containing ³²P (labelled DNA), the bacterial cells were radioactive. Moreover, when the bacterial cells burst open, the new viruses that emerged were radioactively labelled with ³²P. When the protein was labelled, new viruses were only slightly radioactive.

Quick check:

1. How can the harmless rough strain of pneumococcus be transformed into the pathogenic smooth strain?

2. How can the DNA in the disease-causing smooth strain of bacteria bt extracted from RNA and proteins?

3. Describe the distribution of protein and DNA in T2 bacteriophage.

4. Explain how they can each be labelled.

5. Explain the significance of Griffith’s work on Pneumococcus.

6. Describe how Avery and other workers analysed the transforming factor.

7. Describe Hershey and Chase’s experiment.

8. Express the main idea of each paragraph in a single sentence in English.

9. Suggest a suitable title for each paragraph of the text.

10. Divide the text into an introduction, principal part and conclusion.

■ Text 8. The One Gene One Polypeptide Hypothesis

Phenylketonuria (PKU) occurs in about one in 10 000 live births among white Europeans. If untreated, a patient may have an IQ (intelligence quotient) of less than 20 (the average IQ is 100).

The disorder is treated by reducing the intake of phenylalanine in the diet to an absolute minimum. A child with PKU must avoid products that are rich in phenylalanine such as drinks and confectionery that are sweetened with aspartame. (Aspartame contains a mixture of two amino acids: aspartic acid and phenylalanine.)

High blood levels of phenylalanine are not damaging in adulthood (presumably because brain growth is complete), so except while pregnant or breast feeding, adults with PKU can eat a normal diet.

In the 1940s and early 1950s, researchers established that genes are made of DNA. At the same time, other researchers wanted, to know how genes determine inherited characteristics. Clues came from research carried out in the early 1900s by Sir Archibald Garrod. He observed that two human inherited diseases - alkaptonuria and phenylketonuria (PKU) - were each caused by absence of a specific enzyme. (He called these diseases 'inborn errors of metabolism'.)

Alkaptonuria People suffering from alkaptonuria lack an enzyme called homogenistic acid oxidase. This enzyme breaks down the amino acids tyrosine and phenylalanine. When the enzyme is absent, an intermediate product known as homogenistic acid accumulates. This causes a dark brown discoloration of the skin and eyes, and progressive damage to the joints, especially the spine.

Phenylketonuria

Normally, phenylalanine is converted into another amino acid by a transferase(an enzyme which helps transfer a chemical group from one organic molecule to another). This enzyme is absent from people with PKU. This means that phenylalanine accumulates in the blood. High concentrations of phenylalanine damage the nervous system, leading to severe mental retardation. Nowadays, routine postnatal screening detects the condition early enough that the diet can be modified to prevent brain damage (see Fact of life).

Garrod's observations indicated that genes probably exert their effects through enzymes, but the evidence was only circumstantial. Scientists wanted more direct proof that genes brought about their effects by determining which enzymes were made in cells. This proof came with the work of George Beadle and Edward Tatum on Neurospora crassa.

Beadle and Tatum: the one gene - one enzyme hypothesis

Neurospora crassa is a common pink mould (a fungus) which is a particularly damaging pest in bakeries because it can turn bread mouldy. It reproduces by spores and grows in the bread as a mycelium (a mass of threads). It has several features which make it suitable for genetic research. One of the most important is its ability to produce haploid spores asexually. These spores are identical, and have only one set of chromosomes. They therefore have only one allele for each characteristic (spread 19.3). This means that a recessive mutation is not masked by a dominant allele; it is always expressed in the haploid organism.

Neurospora can grow on a culture medium called minimal medium.This contains sugar, a source of nitrogen, mineral ions, and the vitamin biotin. The fungus can synthesise all the other carbohydrates, fats, proteins, and nucleic acids it needs using enzymes produced by its cells.

§ Beadle and Tatum grew Neurospora on minimal medium and exposed the culture to a dosage of X-rays that caused the formation of mutations.

§ Occasionally a mutant spore was produced that was unable to grow on minimal medium. However, it would grow and reproduce if provided with all 20 amino acids.

§ After isolating a mutant Neurospora, Beadle and Tatum attempted to grow it on 20 different minimal media, each of which was supplemented with a different single amino acid.

§ They discovered that the mutant that could not grow on the minimal medium needed only one particular amino acid in order to grow and reproduce normally.

§ They concluded that the mutant lacked the enzyme required to

synthesise that particular amino acid.

Further experiments indicated that other mutants lacked different enzymes, each of which was dictated by a particular gene. In each case, Beadle and Tatum found that the inability to synthesise a specific enzyme was inherited in a normal Mendelian manner. They concluded that each gene in an organism coded for the production of one enzyme. This became known as the one gene-one enzyme hypothesis.The hypothesis was soon extended to a onegene-one protein hypothesiswhen it was shown that proteins other than enzymes could also be determined by specific genes.

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