Prescott's Microbiology,4/e

		Microbiology, 4/e Prescott, Harley, Klein
		Instructor Resources

13 Microbial Genetics: General Principles

CHAPTER OVERVIEW

This chapter presents the basic concepts of molecular genetics: storage and organization of genetic information in the DNA molecule, mutagenesis, and repair. The role of microorganisms in screening procedures for mutagenic agents is also described. Primary emphasis is given to the genetics of bacteria.

CHAPTER OBJECTIVES

After reading this chapter you should be able to:

o discuss the nature of the genetic code

o define a gene and discuss controlling elements, such as promoters and operators

o discuss the four parts (promoter, leader, coding region, trailer) associated with a bacterial gene

o discuss the nature and causes of mutations

o discuss the various genetic repair mechanisms and their limitations

CHAPTER OUTLINE

I. Introduction

A. Clone-a population of cells that are genetically identical

B. Genome-all the genes present in a cell or virus

C. Genotype-the specific set of genes an organism possesses

D. Phenotype-the collection of characteristics of an organism that an investigator can observe

II. DNA as the Genetic Material

A. Griffith (1928) demonstrated the phenomenon of transformation: nonvirulent bacteria could become virulent when live, nonvirulent bacteria were mixed with dead, virulent bacteria

B. Avery, MacLeod, and McCarty (1944) demonstrated that the transforming principle was DNA

C. Hershey and Chase (1952) showed that for the T2 bacteriophage, only the DNA was needed for infectivity; therefore, they proved that DNA was the genetic material

III. The Genetic Code-the manner in which genetic instructions to direct polypeptide production are stored within the genome

A. Colinearity-the DNA base sequence corresponds in linear fashion to the amino acid sequence of the protein whose synthesis it directs

B. Establishment of the genetic code-each codon that specifies a particular amino acid must be three bases long for each of the 20 amino acids to have at least one codon

C. Organization of the code

1. Degeneracy-there are up to six different codons for a single amino acid

2. Sense codons-61 codons that specify amino acids

3. Stop (nonsense) codons-three codons (UGA, UAG, UAA) that do not specify an amino acid, and that are used as termination signals

4. Wobble-describes amino acid specificity leniency: the third position of an mRNA codon is less important than the first or second position in determining the encoded amino acid

a. Codons differing only in the third position frequently specify the same amino acid

b. Wobble eliminates the need for a unique tRNA for each codon because the first two positions are sufficient to establish hydrogen bonding between the mRNA and the aminoacyl-tRNAs

IV. Gene Structure

A. Gene-a linear sequence of nucleotides that is within the genomic nucleic acid molecule, and that has a fixed start point and end point

1. Encodes a polypeptide, a tRNA, or an rRNA

2. Controlling elements (e.g., promoters) that regulate expression of a gene may be considered as part of the gene itself, or they may be considered as separate regulatory sequences

3. With some exceptions, genes are not overlapping

4. The segment that encodes a single polypeptide is also called a cistron

B. Procaryotic vs. eucaryotic genes

1. Procaryote-coding information is normally continuous although some bacterial genes are interrupted

2. Eucaryote-most genes have coding sequences (exons) that are interrupted by noncoding sequences (introns); one exception are the genes that code for histones, which lack introns

C. Genes that code for proteins

1. Sense strand-the one strand which contains coding information

2. Antisense strand-the strand complementary to the sense strand

3. Promoter-a sequence of bases that is usually situated upstream from the coding region which serves as a recognition/binding site for RNA polymerase

a. Recognition site-site of initial association with RNA polymerase (35 bases upstream of transcription initiation site)

b. Binding site (Pribnow box)-sequence that favors DNA unwinding before transcription begins (approximately 10 bases upstream of transcription initiation site)

c. Consensus sequences-idealized base sequences at these and other positions found most often at those positions when comparing the sequences of different bacteria

4. Leader sequence-a transcribed sequence that is not translated but which contains a consensus sequence known as the Shine-Dalgarno sequence which serves as the recognition site for the ribosome

5. Coding region-the sequence that begins immediately downstream of the leader sequence; starts with the sense sequence 3¢TAC5¢, which gives rise to mRNA codon 5¢AUG3¢, the first translated codon (specifies N-formylmethionine)

6. Trailer region-nontranslated region located immediately downstream of the translation terminator sequence and before the transcription terminator

7. Regulatory sites-the operators and CAP binding sites previously discussed that are found associated with some genes

D. Genes that code for tRNA and rRNA

1. Promoter, leader, and trailer regions are also found for these genes; they are removed after transcription

2. More than one tRNA gene may be made from a single transcript; they are separated by a noncoding spacer region which is removed after transcription

V. Mutations and their Chemical Basis

A. Mutation-a stable, heritable change in the genomic nucleotide sequence

1. Conditional mutations-are expressed only under certain environmental conditions

2. Morphological mutations-result in changes in colony or cell morphology

3. Biochemical mutations-result in changes in the metabolic capabilities of a cell

a. Auxotrophs-cannot grow on minimal media; require supplements

b. Prototrophs-can grow on minimal media

4. Resistance mutations-result in acquired resistance to some pathogen, chemical, or antibiotic

5. Apparently directed- or adaptive-mutations may be the result of hypermutation followed by selection of favorable mutations

6. Hypermutation involves activation of special mutator genes

B. Spontaneous mutations-arise occasionally in all cells in the absence of any added agent

1. Causes of spontaneous mutations

a. Errors in DNA replication

b. Damage to DNA from background gamma radiation or heat

c. Insertion of transposons

2. Types of mutations

a. Transition-substitution of one purine for another, or of one pyrimidine for another

b. Transversion-substitution of a purine for a pyrimidine or vice versa

c. Frameshift-deletion of DNA segment resulting in an altered codon reading frame

C. Induced mutations are caused by mutagens that damage DNA or alter its chemistry

1. Base analogs are incorporated into DNA during replication and exhibit base-pairing properties different from the bases they replace

2. Specific mispairing occurs when a mutagen changes a base's structure and thereby alters its pairing characteristics

3. Intercalating agents, which become inserted between the stacked bases of the helix, distort the DNA and thus induce single nucleotide pair insertions or deletions that can lead to frameshifts

4. Many mutagens can severely damage DNA so that it cannot act as a replication template; this would be lethal without the repair mechanisms to restore the DNA; however, the repair mechanisms are error prone, which also leads to mutations

D. The expression of mutations

1. Forward mutation-a conversion from the most prevalent gene form (wild type) to a mutant form

2. Back mutation-a conversion of the mutant nucleotide sequence back to the wild type arrangement

3. Suppressor mutation-a reestablishment of the wild type phenotype by a second mutation in the same or a different gene that overcomes the effect of the first mutation

4. Point mutations-affect only one base pair and are more common than large deletions or insertions

a. Silent mutations are alterations of the base sequence that do not alter the amino acid sequence of the protein because of code degeneracy

b. Missense mutations are alterations of the base sequence that result in the incorporation of a different amino acid in the protein; at the level of protein function, the effect may range from complete loss of activity, to no change in activity at all

c. Nonsense mutations are alterations that produce a translation termination codon, which results in premature termination of the protein during synthesis; location of the mutation within the protein will determine the extent of change in function

d. Frameshift mutations are insertions or deletions of one or two base pairs that thereby alter the reading frame

5. Regulatory mutations are changes in the promoter or operator, they affect the expression of the downstream genes

VI. Detection and Isolation of Mutants

A. Detection

1. Visual observation of changes in colony characteristics

2. Auxotrophic mutants (i.e., those which have lost the ability to synthesize a particular end product and which therefore require its presence in the growth medium) can be detected by replica plating on media with and without the growth factor; mutants are those growing with the factor but not without it

B. Selection of mutations is achieved by finding the environmental condition in which the mutant will grow but the wild type will not (useful for auxotrophic revertants resulting from back mutations, and for resistance mutants)

C. Carcinogenicity testing

1. Most cancer-causing agents (carcinogens) are also mutagens

2. Tests for mutagenicity are used as a screen for carcinogenic potential

3. The Ames test is a widely used mutational reversion test for histidine-requiring auxotrophs of special strains of Salmonella typhimurium

VII. DNA Repair-needed to correct errors in DNA sequences

A. Excision repair

1. Corrects damage that causes distortions of DNA

2. The damaged area is excised, producing a single-stranded gap

3. The gap is filled in by DNA polymerase I and DNA ligase

B. Removal of lesions (such as photoreactivation to remove thymine dimers) reverses damage without removing and replacing bases; although this process is error free, it is limited to the repair of certain kinds of damage

C. Postreplication repair

1. In this process, newly synthesized DNA is proofread for mismatched base pairs

2. Mismatched base pairs are removed and replaced by the action of DNA polymerase I and DNA ligase

D. Recombination repair restores DNA that has damage in both strands by recombination with an undamaged molecule if available (this frequently occurs in rapidly dividing cells where there is another copy of the chromosome not yet parceled out to a daughter cell)

E. SOS repair is used to repair excessive damage that halts replication, but this error-prone process results in many mutations

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