Hartl, Jones - Genetics. Principlers and analysis - 1998, страница 11

Little else is known about the vegetative [growth] phase of these viruses. Theexperiments reported in this paper show that one of the first steps in the growth of T2 is the release from itsprotein coat of theOur experiments show clearly that a physical separation of the phage T2 into genetic andnongenetic parts is possible.nucleic acid of the virus particle, after which the bulk of the sulfur-containing protein has no furtherfunction.

. . . Anderson has obtained electron micrographs indicating that phage T2 attaches to bacteria by itstail. . . . It ought to be a simple matter to break the empty phage coats off the infected bacteria, leaving the phageDNA inside the cells. . . . When a suspension of cells with 35S- or 32P-labeled phage was spun in a blender at10,000 revolutions per minute, . . . 75 to 80 percent of the phage sulfur can be stripped from the infectedcells. . . .

These facts show that the bulk of the phage sulfur remains at the cell surface during infection. . . . Littleor no 35S is contained in the mature phage progeny. . . . Identical experiments starting with phage labeled with 32Pshow that phosphorus is transferred from parental to progeny phage at yields of about 30 phage per infectedbacterium. .

. . [Incomplete separation of phage heads] explains a mistaken preliminary report of the transfer of35S from parental to progeny phage. . . . The following questions remain unanswered. (1) Does any sulfur-freephage material other than DNA enter the cell? (2) If so, is it transferred to the phage progeny? (3) Is the transferof phosphorus to progeny direct or indirect? . .

. Our experiments show clearly that a physical separation of thephage T2 into genetic and nongenetic parts is possible. The chemical identification of the genetic part must waituntil some of the questions above have been answered. . . . The sulfur-containing protein of resting phageparticles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as aninstrument for the injection of the phage DNA into the cell.

This protein probably has no function in the growthof the intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from theexperiments presented.Source: Journal of General Physiology 36: 39–56activity from 32P-labeled phage was found to be associated with the bacteria; however, when the infecting phagewas labeled with 35S, only about 20 percent of the radioactivity was associated with the bacterial cells. From theseresults, it was apparent that a T2 phage transfers most of its DNA, but not much of its protein, to the cell it infects.The critical finding (Figure 1.4) was that about 50 percent of the transferred 32P-labeled DNA, but less than 1percent of the transferred 35S-labeled protein, was inherited by the progeny phage particles.

Because some proteinwas transferred to infected cells and transmitted to the progeny phage, the Hershey-Chase experiment was notnearly so rigorous as the transformation experiments in implicating DNA as the genetic material. Nevertheless,owing to its consistency with the DNA hypothesis, the experiment was very influential.The transformation experiment and the Hershey-Chase experiment are regarded as classics in the demonstration thatgenes consist of DNA. At the present time, many research laboratories throughout the world carry out theequivalent of the transformation experiment on a daily basis, generallyPage 9using bacteria, yeast, or animal or plant cells grown in culture.

These experiments indicate that DNA is the geneticmaterial in these organisms as well as phage T2.There are no known exceptions to the generalization that DNA is the genetic material in all cellularorganisms.It is worth noting, however, that in a few types of viruses, the genetic material consists of another type of nucleicacid called RNA.1.2—DNA Structure: The Double HelixEven with the knowledge that genes are DNA, many questions still remained.

How does the DNA in a geneduplicate when a cell divides? How does the DNA in a gene control a hereditary trait? What happens to the DNAwhen a mutation (a change in the DNA) takes place in a gene? In the early 1950s, a number of researchers began totry to understand the detailed molecular structure of DNA in hopes that the structure alone would suggest answersto these questions.

The first essentially correct three-dimensional structure of the DNA molecule was proposed in1953 by James Watson and Francis Crick at Cambridge University. The structure was dazzling in its elegance andrevolutionary in suggesting how DNA duplicates itself, controls hereditary traits, and undergoes mutation. Evenwhile the tin sheet and wire model of the DNA molecule was still incomplete, Crick could be heard boasting in hisfavorite pub that "we have discovered the secret of life."In the Watson-Crick structure, DNA consists of two long chains of subunits twisted around one another to form adouble-stranded helix.

The double helix is right-handed, which means that as one looks along the barrel, each chainfollows a clockwise path as it progresses. You can see the right-handed coiling in Figure 1.5A if you imagineyourself looking up into the structure from the bottom: The "backbone" of each individual strand coils in aclockwise direction.

The subunits of each strand are nucleotides, each of which contains any one of four chemicalconstituents called bases. The four bases in DNA are• Adenine (A)• Thymine (T)• Guanine (G)• Cytosine (C)The chemical structures of the nucleotides and bases are included in Chapter 5. A key point for present purposes isthat the bases in the double helix are paired as shown in Figure 1.5B.At any position on the paired strands of a DNA molecule, if one strand has an A, the partner strand has a T;and if one strand has a G, the partner strand has a C.The pairing between A—T and G—C is said to be complementary: The complementFigure 1.5Molecular structure of a DNA double helix.

(A) A "space-filling" model,in which each atom is depicted as a sphere. (B) A diagram highlightingthe helical strands around the outside of the molecule and the A—T andG—C base pairs inside.Page 10of A is T, and the complement of G is C. The complementary pairing in the duplex molecule means that each basealong one strand of the DNA is matched with a base in the opposite position on the other strand. Furthermore,Nothing restricts the sequence of bases in a single strand, so any sequence could be present along onestrand.This principle explains how only four bases in DNA can code for the huge amount of information needed to makean organism.

It is the sequence of bases along the DNA that encodes the genetic information, and the sequence iscompletely unrestricted.The complementary pairing is also called Watson-Crick pairing. In the three-dimensional structure (Figure 1.5A),the base pairs are represented by the spheres filling the interior of the double helix. The base pairs lie almost flat,stacked on top of one another perpendicular to the long axis of the double helix, like pennies in a roll. Whendiscussing a DNA molecule, biologists frequently refer to the individual strands as single-stranded DNA and tothe double helix as double-stranded DNA or duplex DNA.Each DNA strand has a polarity, or directionality, like a chain of circus elephants linked trunk to tail.

In thisanalogy, each elephant corresponds to one nucleotide along the DNA strand. The polarity is determined by thedirection in which the nucleotides are pointing. The "trunk" end of the strand is called the 5' end of the strand, andthe "tail" end is called the 3' end. In double-stranded DNA, the paired strands are oriented in opposite directions,the 5' end of one strand aligned with the 3' end of the other. The molecular basis of the polarity, and the reason forthe opposite orientation of the strands in duplex DNA, is explained in Chapter 5.Beyond the most optimistic hopes, knowledge of the structure of DNA immediately gave clues to its function:1. The sequence of bases in DNA could be copied by using each of the separate "partner" strands as a pattern forthe creation of a new partner strand with a complementary sequence of bases.2.

The DNA could contain genetic information in coded form in the sequence of bases, analogous to letters printedon a strip of paper.3. Changes in genetic information (mutations) could result from errors in copying in which the base sequence ofthe DNA became altered.In the remainder of this chapter, some of the implications of these clues are discussed.1.3—An Overview of DNA ReplicationIn their first paper on the structure of DNA, Watson and Crick remarked that "it has not escaped our notice that thespecific base pairing we have postulated immediately suggests a copying mechanism for the genetic material." Thecopying mechanism they had in mind is illustrated in Figure 1.6; the process is now called replication. Inreplication, the strands of the original (parent) duplex separate, and each individual strand serves as a pattern, ortemplate, for the synthesis of a new strand (replica).

The replica strands are synthesized by the addition ofsuccessive nucleotides in such a way that each base in the replica is complementary (in the Watson-Crick pairingsense) to the base across the way in the template strand. Although the model in Figure 1.6 is simple in principle, itis a complex process with chemical and geometrical problems that require a large number of enzymes and otherproteins to resolve. The details are discussed in Chapter 5. For purposes of this overview, the important point isthat the replication of a duplex molecule results in two duplex daughter molecules, each with a sequence ofnucleotides identical to the parental strand.In Figure 1.6A, the backbones in the parental DNA strands and those in the newly synthesized strand are shown incontrasting colors.