Chapter 9

 

Microbial Genetics

 

 

Introduction to Genetics

§ For a species to survive, its genetic material must be accurately duplicated and transferred to daughter cells

§ Genetic material is located on chromosomes

§ Genotype - refers to an organisms genetic makeup

§ Phenotype - expression of the genotype to create certain traits

 

 

 

Structure of DNA

§ Discovered by James Watson and Francis Crick (1953)

§ DNA stores genetic information (genes) for the production of proteins

§ Nucleotides - composed of a nitrogenous base, sugar, and phosphate

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

§  sugar- deoxyribose

§  phosphate- PO4

§ Bases attach to 1’ carbon

§ Hydroxyl (-OH) group on the 3’ carbon

§ Phosphate group on 5’ carbon

§ Nucleotides arranged in chains (strands)

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

§ DNA is a complimentary, antiparallel, double helix

§ Bases are held together by Hydrogen bonds

§ Base Pairing Rules:

§ A with T

§ G with C

 

 

 

DNA Replication

§ Semi-Conservative, Bidirectional Replication

§ Results in 2 molecules; each with 1 original strand (template) and 1 new strand for future generations

§ Uses multiple enzymes:

§ DNA helicase – breaks H+ bonds and unwinds double helix

§ DNA gyrase - temporarily breaks DNA strands to release tension

§  Target of ciprofloxacin and other floroquinolones

§ 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

§ DNA ligase – seals gaps between DNA fragments

 

 

 

DNA Expression

§ Transcription-Translation

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

 

 

Transcription

§ mRNA is created from a DNA template

§ mRNA is single-stranded

§ The sugar in mRNA is ribose

§ Thymine (T) is replaced with Uracil (U)

§ RNA polymerase - reads the template DNA strand and creates a complimentary mRNA strand

§ it recognizes promoter and termination sequences on the DNA template

 

 

Translation

§ A polypeptide strand (protein) is created from a mRNA template

§ Occurs in the ribosome

§ Ribosome scans the mRNA strand until it reaches the start codon (AUG)

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

§ tRNA brings in the appropriate amino acid for the AUG codon

§ The next codon is read and another amino acid comes in forming a peptide bond with the previous one

§ Continues until ribosome reaches a stop codon on the mRNA

§ can be UGA, UAA, or UAG

§ The new protein is released to the cell for use or secretion

§ There are 64 different codons that translate to the 20 different amino acids

 

 

 

Diversity in Bacteria

§ Bacteria can adapt to a changing environment by altering their genetic makeup

§ Mutation - change in DNA base sequence

§ Spontaneous mutation – random change due to errors in replication

§ Induced mutation - results from exposure to mutagens

§ Chemical mutagens - alter base composition

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

§ Point mutation - only one base pair changed

§ missense mutation - substitution of different amino acid

§ nonsense mutation - substitution of a stop codon

§ silent mutation - does not change amino acid

§ Frameshift mutation - one or more bases are inserted or removed from DNA

§ Mutations can result in natural selection against antimicrobial medication

§ Ames Test – uses bacteria to screen chemicals for their ability to cause mutations

§ used to test environmental and dietary chemicals

 

 

DNA Recombination

§ The transfer of DNA from one organism to another

§ 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

§ Conjugation

§ involves a pilus

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

§ limited to organisms of the same genus

§ Transformation

§ 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

§ Transduction

§ involves infection by bacteriophage

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

§ generalized - transfer of random pieces of DNA

§ specialized- transfer of specific pieces of DNA

§ Transposons

§ 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

§ Deliberately altering an organism’s genetic information

          

 

Tools and Techniques: DNA

§ DNA strands denature at boiling temperatures

§ Strands renature as they cool

                                          

 

 

Tools and Techniques: Enzymes

§ Restriction endonucleases               

§ cut DNA into small, workable fragments

§ recognize specific sequences and clip DNA at that location

§ Ligase

§ Rejoins the phosphate-sugar bonds of the strand

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

§ Reverse transcriptase

§ Uses an RNA template to create a strand of DNA

§ Results in copy DNA or cDNA

§ Used to study transcribed DNA

§ Gel electrophoresis - samples placed in wells in a soft agar gel are subjected to electrical current

§ 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 sequencing - used to identify DNA mutations or new genes         

§ provides identity and order of DNA nucleotides  

§ Nucleic acid hybridization - used to identify complementary nucleotide sequences in unknown samples

§ DNA is denatured at high temperature/pH

§ 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

§ Polymerase Chain Reaction (PCR) - can create millions of copies of a specific segment of DNA

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

§ Use primers of known sequence and DNA polymerases to amplify a segment of DNA over and over

§ DNA cloning - involves removal of a gene from one organism and expressing it in another organism

§ 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

§ Protein production for commercial or medical use

§ hormones, enzymes, vaccines

§ Genetically Modified Organisms (GMOs)

§ recombinant organisms produced through the introduction of foreign genes

§ transgenic microbes, plants, and animals have been developed

§ Gene Therapy

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