Chapter 9

 

Microbial Genetics

 

 

Introduction to Genetics

 

 

 

Structure of DNA

§  the 4 bases of DNA are adenine (A), guanine (G), thymine (T), and cytosine (C)

§  sugar- deoxyribose

§  phosphate- PO4

§  ends of the strands are identified by the carbon nearest to the end (3’ or 5’)

 

 

 

DNA Replication

§ DNA Gyrase – unwinds double helix so helicase can bind

§ Target of ciprofloxacin and other floroquinolones

§ Helicase – breaks hydrogen bonds and unzips double helix

§ DNA Polymerase III - builds new DNA strands in a 5 to 3 direction

§ reads the original in a 3 to 5 direction

§ can only add new nucleotides to the 3 end of an already existing chain

§ requires an RNA primer

§ Primase – creates a small section of RNA that initiates DNA replication (primer)

§ DNA Polymerase I – removes & replaces RNA primers with DNA

§ Ligase – seals gaps between DNA fragments

 

 

 

DNA Expression

§ Process by which information is taken from DNA (genotype) and used to make proteins (phenotype)

 

 

Transcription

§ it recognizes promoter and termination sequences on the DNA template

 

 

Translation

§ codon – three-nucleotide sequence that represents one amino acid (genetic code)

§ can be UGA, UAA, or UAG

 

 

 

Diversity in Bacteria: Mutations

§ Chemical mutagens - alter base composition

§ Radiation - gamma, ultraviolet, and X-rays damage DNA

§ missense mutation - substitution of different amino acid

§ nonsense mutation - substitution of a stop codon

§ silent mutation - does not change amino acid

§ used to test environmental and dietary chemicals

 

 

DNA Recombination

§ results in a new genetic strain different from the donor and original recipient

§ normally involves the transfer of plasmids

§ the recipient organism accepts the DNA  into its genetic make-up and expresses it

§ allows for transfer of toxins, virulence factors, or antibiotic resistance genes

 

 

 

Methods of Gene Transfer

§ involves a pilus

§ pilus forms a bridge between 2 organisms and a replicated plasmid is transferred

§ limited to organisms of the same genus

§ cell picks up small pieces of DNA from the environment usually from a lysed cell

§ allows a cell to get genetic material from an entirely different species

§ called transfection in eukaryotic cells

§ Involves infection by bacteriophage

§ Bacteriophage carries genetic material from its previous host cell into the new host

§ Generalized - random pieces of host DNA are picked up during infection

§ Specialized- specific pieces of host DNA are incorporated into bacteriophage

§  May contain bacterial toxins

§ segments of DNA that jump around and insert into DNA

§ widespread in prokaryotic and eukaryotic cells

§ can change traits, replace damaged DNA, or cause drug resistance in bacteria

 

 

Chapter 10

 

Genetic Engineering

 

 

Genetic Engineering

        

 

Tools and Techniques: DNA

                                   

 

 

Tools and Techniques: Enzymes

§ cut DNA into small, workable fragments

§ recognize specific sequences and clip DNA at that location

§ Seals the ends together by rejoining the phosphate-sugar bonds

§ Used to splice pieces of DNA into plasmids and chromosomes – DNA cloning

§ Uses an RNA template to create a strand of DNA

§ Results in copy DNA or cDNA

§ Used to study transcribed DNA

§ DNA moves through the gel based on the size of the fragments

§ Larger fragments move more slowly than smaller fragments     

 

 

Applications of Genetic Engineering

§ DNA is denatured at high temperature/pH and similar strands will re-anneal at low temperature/neutral pH

§ Known, labeled DNA sequences (probes) will base-pair with complementary DNA if present in a test sample

§ Used to detect the presence of genes in a given sample

§ Can diagnose an infection from a single cell within a few hours

§ DNA sequencing – provides the identity and order of nucleotides in a genome

§ DNA profiling – uses restriction endonucleases to provide a “DNA fingerprint” from genetic material

§ Microarray Analysis – used to study patterns of gene expression in cells

 

 

§ Gene is excised from the donor using restriction endonucleases

§ Gene is inserted into a cloning vector, such as a plasmid or virus

§ Cloning vector is introduced into a host cell, which makes protein from the DNA

 

 

Applications of DNA Cloning

§ hormones, enzymes, vaccines

§ recombinant organisms produced through the introduction of foreign genes

§ transgenic microbes, plants, and animals have been developed

§ Cloning vectors (usually viruses) are used to permanently repair a genetic defect in humans