Molecular Basis of Heredity 192 Structure of DNA 194

Nucleotides—Building Blocks of the Genetic Code Chains of Nucleotides—Directionality of DNA DNA Double Helix—Complementarity Historical Perspective: Discovering the Structure of DNA I Replication of DNA 198

Semiconservative DNA Replication Steps in DNA Replication Unwinding the DNA Double Helix—Replication Forks

Methodology: Polymerase Chain Reaction (PCR) Formation of a New Chain of Nucleotides—DNA Polymerase I Mutations 203

Types of Mutations Methodology: Ames Test Factors Affecting Rates of Mutation I Expression of Genetic Information 205 Genes

Historical Perspective: One Gene—One Polypeptide RNA Synthesis Ribonucleic Acid (RNA)—Functions and Types Transcription ; Initiation and Termination of Transcription Synthesis of mRNA in Prokaryotic and Eukaryotic Cells—Split Genes Protein Synthesis—Translation of the Genetic Code mRNA and the Genetic Code tRNA and Polypeptide Formation Forming the Polypeptide Regulation of Gene Expression 214 Operons

Regulating the Metabolism of Lactose—the lac Operon Catabolite Repression

In this chapter we will:

• Discover the underlying mechanisms of heredity and the biochemical events that enable the passage of hereditary information.

• Examine the properties of DNA (deoxyribonucleic acid), the universal master molecule of life that stores genetic information in all cells—bacterial, human, and other.

• See how DNA molecules are replicated so that hereditary information can be passed from one generation to the next.

• Discover how all of the properties of an organism are determined at the molecular level.

• Learn how genetic expression occurs, seeing how information in the DNA is transferred through RNA (ribonucleic acid) molecules to proteins.

• Learn the following key terms and names:

Ames test nonsense codons

anticodon nucleic acid bases

auxotrophs nucleotides

base pairing operon model

catabolite repression phenotype

codon polycistronic

diploid polymerase chain reaction

DNA gyrase (PCR)

DNA polymerase promoter

double helix prototroph

exons regulatory genes

frame-shift mutations replica plating

genes replication fork

genotype ribosomal RNA (rRNA)

haploid RNA polymerase

heterozygous semiconservative

hnRNA (heterogeneous replication

nuclear RNA) split genes

homozygous structural genes

inducible template strand

introns thymine dimer

lethal mutation transcription

mutagens transfer RNA (tRNA)

mutation translation

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Preview to Chapter 7

Chapter Outline

In 1928 a British microbiologist, Frederick Griffith, was trying to develop a vaccine against pneumonia. He was working with two different strains of the causative bacterium Streptococcus pneumoniae (FIG. 7-1). One strain was pathogenic, killing the mice in­jected with it. The other strain was nonpathogenic. The two strains differed in appearance when viewed under the microscope. The nonpathogenic strain ap­peared rough and was not surrounded by a capsule. The pathogenic strain appeared smooth, surrounded by a polysaccharide capsule. When Griffith injected heat-killed cells of this smooth, pathogenic strain of S. pneumoniae into a mouse, the mouse survived be­cause the dead bacteria were unable to establish an

infection in the mouse. However, when he injected a mouse with living cells of the rough nonpathogenic strain, together with dead smooth bacteria, knowing that neither of them could cause disease alone, the mouse died. Unlike the live, rough bacteria he in- ; jected, the bacteria he isolated from the dead mouse appeared smooth and surrounded by a capsule.

This was a most puzzling observation. Griffith rea­soned that genetic material from the heat-killed bac­teria had somehow entered the living nonpathogens [ and transformed them into pathogenic bacteria. He postulated that heat could kill the pathogenic cells without destroying the substance containing their f hereditary information, which included instructions [

FIG. 7-1 The transformation of Streptococcus pneumoniae shows how the properties of a bacterial strain can be altered by a hereditary substance (later identified as DNA). When cells of S. pneumoniae are heat killed they leak DNA, which can be picked up by other cells and incorporated into the genetic information of those cells. In this manner, avirulent (non­pathogenic) strains of S. pneumoniae that lack the gene for capsule production (virulence factor that contributes to their ability to cause fatal disease) can acquire the gene (DNA) that encodes for capsule production. When this occurs, an avirulent noncapsule-producing strain of S. pneumoniae is transformed into a virulent strain that produces a capsule.

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on how to cause infection and disease. Griffith had, in fact, observed the movement of hereditary mater­ial from one cell to another. The chemical that trans­mitted the hereditary information for causing disease leaked from the dead pathogens and was picked up by the living bacteria, transforming them into pathogens when it became part of their hereditary material.

Other scientists then began to investigate the spe­cific chemical substance that caused the transforma­tion of a nonpathogen to a pathogen. They were look­ing for the molecular basis for heredity. Chemical analyses narrowed the possible hereditary molecules to either proteins or nucleic acids. Most scientists hy­pothesized that proteins were the basis of heredity because their essential roles in metabolism were known. The specific chemical nature of the trans­forming material observed by Griffith, however, re­mained a puzzle until 1944 when Oswald Avery and his co-workers were able to demonstrate the chemi­cal nature of the substance that transformed non­pathogenic S. pneumoniae to pathogenic S. pneumo­niae.

Avery hypothesized that a nucleic acid, deoxyri­bonucleic acid (DNA), rather than protein was the hereditary molecule. He designed experiments to prove this. In Avery's experiments the transforming principle of S. pneumoniae, which had been shown to be predominantly DNA with a trace of protein, was treated sequentially with an enzyme that destroys protein and an enzyme that destroys DNA (FIG. 7-2). Avery observed that the protein-destroying enzyme did not affect the ability of the material to transform nonpathogenic S. pneumoniae into pathogenic S. pneu­moniae, whereas treatment with the DNA-destroying enzyme eliminated such transformation. Based on these observations, Avery concluded that the trans­forming principle must be DNA.

Despite this quite convincing demonstration, the scientific community was not ready to accept that DNA was the universal hereditary molecule. Most scientists remained convinced that proteins would eventually be shown to be the basis of heredity for organisms other than bacteria. Another set of experi­ments conducted with bacteriophage (viruses that replicate within bacterial cells), however, added con­vincing evidence that nucleic acids, not proteins, are the source of hereditary information. These experi­ments, conducted in 1952 by Alfred Hershey and Martha Chase, examined the replication of bacterio­phage T2. Although bacteriophage are not living cells, they were known to contain DNA and protein, making them good simple models to examine whether it is protein or DNA that carries hereditary information.

Hershey and Chase used two different radioactive labels to track the movement of protein and DNA separately (FIG. 7-3). Most proteins contain sulfur but none contain phosphorus. Thus the radioactive isotope ³⁵S can be used to label the bacteriophage protein. DNA contains phosphorus but no sulfur, so they used the radioactive isotope ³²P to label the bac­teriophage DNA. Thus Hershey and Chase cleverly devised a method for following both the DNA and protein components of bacteriophage T2. When they added bacteriophage that had been labelled with ^3SS to a culture of growing cells of the bacterium Escherichia coli, they observed that the ³⁵S label re­mained outside of the bacterial cells. Thus protein did not enter the bacterial cells. In contrast, when they similarly added ³²P-labelled bacteriophages, the ³²P label entered the interior of the bacterial cells. This indicated that DNA was the material that entered the cells and therefore must be the substance that carried the hereditary information. The progeny bacterio­phages produced from the replication of the original

FIG. 7-2 To prove that the hereditary substance was DNA, enzymes that degrade proteins were added to cell extracts. These enzymes did not eliminate transformation, showing that the substance was not a protein. In contrast, the addition of a DNA-destroying enzyme eliminated transformation.

bacteriophage contained ³²P and not ³⁵S, indicating further that the hereditary material passed from one generation to the next, was, in fact, DNA. Although subsequent experiments have shown that another nucleic acid (ribonucleic acid [RNA]) sometimes is the hereditary substance for viruses, it was now

STRUCTURE OF DNA

Nucleotides—Building Blocks of the Genetic Code

To understand how DNA stores and transmits hered­itary information, it is necessary to examine the chemical structure of this molecule. DNA is a large, high-molecular-weight molecule, called a macromole­cule. It is composed of many subunits called nu­cleotides (FIG. 7-4). Each nucleotide subunit of DNA has three parts: deoxyribose (a 5-carbon sugar), phosphate, and one of four nitrogenous bases (some­times referred to as nucleic acid bases). The four dif­ferent nitrogenous bases that occur in DNA are ade­nine (A), thymine (T), guanine (G), and cytosine (C).

These four nucleotides are like an "alphabet" that makes up the genetic code. They establish the first important property of DNA as the chemical basis for heredity—the ability to encode the genetic informa­tion. This is achieved by linking the nucleotides in a specific order—much as the letters of the alphabet are joined to form words.

The hereditary information is coded by the order in which the four different nucleotides occur within the DNA macromolecule.

Chains of Nucleotides—Directionality of DNA

Individual nucleotides are linked to form a long chain consisting of several million nucleotides. The bonds holding the nucleotides together are covalent and hence strong. This is important for the long-term stability of the hereditary macromolecule. Within this chain, nucleotides are locked together in order, thereby establishing the sequences that encode the genetic information. Once encoded in the chain of DNA, the information remains intact unless acted on by a destructive force, such as certain chemicals or ra­diation.

The chemical bonds holding the chains of nu­cleotides together are called 3'-5' phosphodiester bonds (FIG. 7-5). They are so-named because phos-

firmly established that DNA is the hereditary mole­cule for many viruses and all living cells.

DNA is the substance that transmits the hereditary information of many viruses and all cellular organ­isms.

CHAPTER 7 MICROBIAL GENETICS: REPLICATION AND EXPRESSION OF GENETIC INFORMATION

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