Chapte8 Microbial genetics 8.1 DNA as genetic material 8.2 Mutation 8.3 DNA repair

Microbial molecular biology and genetics A clone: a population of cells that are derived asexually from a parental cell and are genetically identical. Genome: all the genes present in a cell or virus. Central dogma: DNA  RNA  protein transcriptiontranslation

8.1 DNA as genetic material

The Hershey-Chase experiments

RNA as genetic material TMV and HR P196 图 8-2

8.2 Mutations and Mutants Mutations: initially characterized as altered phenotypes or phenotypic expression, a mutation is a stable, heritable change in the nucleotide sequence of DNA. Phenotype: the observable characteristics of an organism –designated by a capital letter followed by two small letters, with either a plus and minus superscript to indicate the presence or absence of that property: His -, Glu + et al. Genotype: The precise genetic make-up of an organism –designated by three lower case letters followed by a capital letter (all in italics) indicating the particular gene involved: hisC, mutations in the hisC gene would be designated as hisC1, hisC2 et al.

Types of mutations Conditional mutations: those that are expressed only under certain environmental conditions. eg. E.coli: a conditional lethal mutant, low temperature grow; high temperature die. Biochemical mutations: those causing a change in the biochemistry of the cell. Mutations make a microorganism unable to grow on a medium lacking an adequate supply of the pathway’s end product. auxotrophs: the mutant cannot grow on minimal medium (MM) and requires nutrinent supplements. prototrophs: microbial strains that can grow on MM. Resistant mutant: a particular type of biochemical mutant that acquires resistance to some pathogen, chemical, or antibiotic.

Mutations occur in one of two ways: 1. Spontaneous mutations: arise occasionally in all cells and develop in the absence of any added agent. 2. Induced mutations: the result of exposure of the organism to some physical or chemical agent called a mutagen.

Spontaneous mutations without exposure to external agents 1. Errors in DNA replication 2. The action of transposons 3. Frameshifts by the deletion or addition of bases 4. The insertion sequences of DNA segments into genes

Transition mutations: purine for putine or pyrimidine for pyrimidine substitutions. relatively common Transversion mutations: a purine is substituted for a pyrimidine, or a pyrimidine for a purine. rarer

Apurinic site: purine nucleotides lose their base. Apyrimidinic site: cytosine can be deaminated to uracil, which is removed. Reactive forms of oxygen: O·, peroxides eg. C-G  8-oxo-7,8-dihydrodeoxyguanine-A

Induced mutations: mutagens 1. Base analogs: eg. 5-BU, an analogs of T. keto and enol: keto-A; enol-G 2. Specific mispairing: a mutagen changes a base’s structure and therefore alters its base pairing characteristics. eg. methyl-nitrosoguanidine: an alkylating agent that adds methyl groups to guanine, causing it to mispair with thymine. GC-AT transition. methyl-C-T 3. Intercalating agent: distort DNA to induce single nucleotide pair insertions and deletions. eg. proflavin and acridine orange 4. Damaged DNA can no longer act as a template. uv radiation; carcinogens: aflatoxins B1; benzopyrene derivatives.

Mutagens Chemical mutagens: Base analogs Chemicals reacting with DNA Alkylating agents Physical mutagens: UV Ionizing rdiation

Physical mutagen: Radiation Noninonizing (electromagnetic): –UV: induction of pyrimidine dimers Ionizing radiation –X-ray, cosmic rays, gamma rays; causing water and other substances to ionize; such as the generation of hydroxyl radical which reacts with DNA

Molecular basis of Mutation Point Mutation(one base pair mutant) –Silent mutation –Nonsense mutation –Missense mutation(the effect may range from complete loss of activity to no change at all)

Frameshift mutations Occur in protein-encoding gene that including the reading frame If occurs in promoter region, it is not frameshift

Back Mutations or Reversions Point Mutations are reversible Revertant –wild type phenotype that was lost in the mutant is restored. Same site revertants True revertants Second site mutation –Suppressor mutation: restore the wild type phenotype by mutation somewhere else.

Rates of Mutation Spontaneous mutation: 10 -6, Transposition: Nonsense mutation: Missense mutation:

Hypermutation Some starving bacteria might rapidly generate multiple mutations through activation of special mutator genes. This would produce many mutant bacterial cells. In such a random process, the rate of production of favorable mutants would increase, with many of these mutants surviving to be counted. There would appear to be directed or adaptive mutation because many of the unfavorable mutants would die.

Isolation of Mutants Pigmented and nonpigmented mutants Antibiotic-resistant mutants within the inhibition zone Genetic marker, such as the expression of  - galactosidase that produces the blue color

Replica Plating Technique: Isolation of Mutants Velveteen ( 锦绒布） Auxotroph ( 营养缺陷型 ) Prototroph ( 原养型 )

Replica Plating Technique: Isolation of Mutants Penicillin-Selection Method –Penicillin kills only growing cells. If penicillin is added to a population growing in a medium lacking the growth factor required by the designed mutant, the parent cells will be killed, whereas the non-growing mutant cells will be unaffected.

Mutants that are detectable Non-motile Non-capsulated Rough colony Nutritional Sugar fermentation Drug resistant Virus resistant Temperature sensitive Pigmentless Cold sensitive

Mutagenesis and Carcinogenesis The Ames Test Bacteria can be used as screening agents for the potential mutagenicity of chemicals –As it has been found that many mutagenic chemicals are also carcinogenic, capable of causing cancer in animals and humans The fact that a compound is mutagenic in a bacterial system serves as a warning of possible danger Dr. Bruce Ames at the UC Berkeley has developed the Ames Test technique for the screening of carcinogenic compounds.

The Ames Test Histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli have been the major tools for the Ames test. Induction of a phage lambda lysogen has also been used as an assay of DNA damage

Other Methods of Creating Mutations Biological mutagens –Transposon mutagenesis: bacteriophage Mu Site-directed mutagenesis –Technique has been developed to make mutation in a specific DNA site.

8.3 DNA repair 1. Excision repair: first excise, then DNA polymerase I, at last DNA ligase join 2. Removal of lesions (photoreactivation) splitting thymine dimers apart into separate thymines with the help of visible light. 3. Postreplication repair mismatch repair system: excise  replace  seal 4. Recombination repair: recA protein 5. SOS repair

SOS regulatory system Mutations may arise from error prone DNA repair

Questions Experimental evidence of DNA as genetic material? Types of mutations What is Mutagens? Molecular basis of Mutation Rates of Mutation How to detect and isolate mutant? The Ames Test What are DNA repair?