Bacterial genetics lec 5

Bacterial genetics
Genetics: Science dealing with heredity - constancy and change in physiologic functions that form properties of organism Unit- Gene DNA segment carrying in its nucleotide sequence information for specific physiologic or biochemical property. Bacterial Genetic material single circular DNA E.Coli mol. Wt 2 x 109. 5 x 106 base pairs-code for 2000 proteins Average Mol. Wt 50,000 Mycoplasma mol. Wt 5 x 108, Human 3x109 base pairs - encode 100000 proteins

Bacterial genetics
Bacteria - Haploid: single chromosome, single copy of gene. mutation loss of trait Human - Diploid: pair of chromosomes. copy of gene (allele) dominant other recessive Phenotype: collective physiologic or structural properties of cell. Genotype: sequence of DNA within a gene/ organization of gene. Phenotype variation: because of genes capacity to permit growth under conditions of selection, resistance gene grow in presence of Penicillin.

Bacterial genetics
Restriction enzymes cleave DNA at specific sites DNA Restriction fragments, if introduced in plasmids amplified many times. PCR Polymerase chain reaction amplified DNA segment placed near promotors expression of protein technology of genetic engineering Genetic Code: written as sequence of bases of nucleotides. Read as group of 3 nucleotides sequence called Codon . Each codon amino acid Gene: series of codon read in series from starting point to end point at other end. Written in 5/ - 3/ direction. nucleotides sequence corresponds to amino acid sequence in protein written in direction from N to C terminus

Bacterial genetics
Mutations: permanent change in DNA sequence or gene leading to altered phenotype i.e. insertion of different amino acid or loss of encoded protein. Mutant organism identified by difference in appearance, physiology or growth properties compared to normal. Mutation results from 3 types of molecular changes. Base substitution Frame shift Transposons or insertion sequences

Bacterial genetics
Base substitution: insertion of single base in place of another. During replication due to error or chemical modification of base. Mutation can be Missense: insertion of different amino acid Nonsense: generate termination codon stop protein synthesis prematurely. Loss of protein function Frame shift: insertion or deletion of single base pair change of reading frame of entire subsequent sequence insertion of wrong amino acid inactive protein. Generally followed by stop codon. Acridine dyes Point mutation: involve single base pair Transposons or insertion sequences: integrated into DNA large scale changes in genes effecting function

Mutations
Spontaneous: occur as result of normal cellular operations or random interaction with environment. Can occur in all organisms in spite of tendency to repair. Induced: occur due to exposure to mutagens Mutagens: substances or factors which cause mutation
Physical Biological Chemical

mutagens
Physical:
X ray: DNA damage in 3 ways. Breaks covalent bond that holds ribose-PO4 chain, produce free radicals attacking bases, alters electron in base changing H bonding UV light: cross linkage of adjacent pyrimidine bases to form dimers inability to replicate

Biological:
Viruses: Bacteriophages inserted in chromosome

mutagens
Chemical: act by changing physical or chemical structure of DNA.
Nitrous acid- alter existing base which form H bond with wrong base Bromouracil: act as base analogue inserted instead of thymine, binds with guanine G-C pair instead of A-T. Iodoxuridine: act as thymidine base analogue Acridine dyes Alkylating agents Benzpyrene: cause frame shift mutation

Mutations
Conditional lethal mutation Expressed only under certain conditions. Temperature sensitive organism mutation amino acid change replicate in low temp 32o C not at 37oC. Experimental influenza vaccine

Transfer of DNA
Occurs within bacterial cells or between bacterial cells Transfer of DNA within bacterial cells: Transposons Programmed rearrangement Transposon / jumping genes/ mobile sequences: genetic elements of several Kbp of DNA including information for migration from one locus to other, within or between DNA of bacteria, plasmid or bacteriophage. Replicate as part of chromosome where inserted. Detection or genetic exploitation done by selecting information they carry. Transposition: process by which sequences are copied & inserted to new site in genome.

Transfer of DNA
Simple Transposon/ insertion sequences: genes for transposase protein (mediate excision & integration) in centre & inverted repeats (IR) on either sides. Molecular parasites, cause mutation or inactivate genome where inserted. Complex Transposon: 4 domains, IR on either sides, gene for transposase, repressor gene ( regulate transposae synthesis and gene product of 4th domain, gene for enzyme mediating resistance, toxins, other enzymes Programmed rearrangement: movement of gene from one silent site to an active site where it is expressed. Encode variants of antigen. Help escape immune response. N.gonorrhoea, B.recurrentis

Transfer of DNA
Transfer of DNA between cells: 3 methods Conjugation Transduction Transformation Conjugation: mating of 2 cells, DNA transfer from donor to recepient. Process controlled by F plasmid (fertility F factor). It has genes for required proteins eg pilin for sex pilus. F+ attaches to receptor on FReeling in of pilus cells drawn together Cleavage of F factor Transfer of one strand of DNA across conjugal bridge Complementory strand synthesized- doudle stranded

Conjugation

Hfr cells: Fplasmid of some F+ cells integrated into DNA acquire ability of transferring chromosome into another cells. Single DNA strand having a piece of F plasmid in front and remaining behind. Complete DNA transfer in 100 mins breakage of attachment only portion of DNA transferred. Genes near leading piece usually. Variable genes transferred because of variable attachment of plasmid in DNA. DNA recombine with recepient DNA.

High frequency recombination

Transfer of DNA between cells

Transduction
DNA transfer by bacteriophage. During viral replication in bacteria, a piece of DNA may be inserted in viral coat. When it infects other bacteria, DNA integrates into bacterial DNA. The infected bacteria acquires new trait lysogenic conversion. It may convert non pathogen to pathogen. Generalised Transduction: virus carries segment from any part of DNA after fragmentation. frequency of 1in 1000 particles Specialized Transduction: integrated viral DNA is excised and carries with it adjacent part of bacterial DNA. Genes are specific because phages integrate at specific sites

Transduction

Transfer of DNA between cells

Transformation: transfer of DNA from one cell to other by itself. 2 methods Dying bacteria release DNA which is taken up b recipient cells. Lab introduction of DNA extracted from bacteria into another bacteria Transfection: introduction of purified DNA into eukaryotic cell. Used in genetic engineering

Recombination
Process of integration of introduced DNA into host cell. 2 types Homologous: two DNA pieces have extensive homologous regions which pair upand exchange pieces by processes of breakage and reunion Non homologous: little if any homology is necessary