Microbial Genetics

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Genetics and Genes

Genetics – the study of heredity

The science of genetics explores:

1. Transmission of biological traits from parent

to offspring

2. Expression and variation of those traits

3. Structure and function of genetic material

4. How this material changes

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Levels of genetic study

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Levels of Structure and Function of

the Genome

• Genome – sum total of genetic material of an

organism (chromosomes + mitochondria/chloroplasts

and/or plasmids)

– genome of cells – DNA

– genome of viruses – DNA or RNA

• DNA complexed with protein constitutes the genetic

material as chromosomes.

• Bacterial chromosomes are a single circular loop.

• Eukaryotic chromosomes are multiple and linear.

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Chromosome is subdivided into genes, thefundamental unit of heredity responsible for a given trait.

– site on the chromosome that provides information for a certain cell function

– segment of DNA that contains the necessary code to make a protein or RNA molecule

Three basic categories of genes:

1. Genes that code for proteins - structural genes

2. Genes that code for RNA

3. Genes that control gene expression - regulatory genes

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• All types of genes constitute the genetic

makeup – genotype.

• The expression of the genotype creates

observable traits – phenotype.

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Genomes Vary in Size

• Smallest virus – 4-5 genes

• E. coli – single chromosome containing

4,288 genes; 1 mm; 1,000X longer than cell

• Human cell – 46 chromosomes containing

31,000 genes; 6 feet; 180,000X longer than

cell

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DNA• Two strands twisted into a helix

• Basic unit of DNA structure is a nucleotide

• Each nucleotide consists of 3 parts:

– a 5 carbon sugar - deoxyribose

– a phosphate group

– a nitrogenous base – adenine, guanine, thymine, cytosine

• Nucleotides covalently bond to form a sugar-phosphate linkage – the backbone

– each sugar attaches to two phosphates –

• 5′ carbon and 3′ carbon

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DNA

• Nitrogenous bases covalently bond to the 1′ carbon of each sugar and span the center of the molecule to pair with an appropriate complementary base on the other strand

– adenine binds to thymine with 2 hydrogen bonds

– guanine binds to cytosine with 3 hydrogen bonds

• Antiparallel strands 3′ to 5′ and 5′ to 3′

• Each strand provides a template for the exact copying of a new strand

• Order of bases constitutes the DNA code

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Significance of DNA Structure

1. Maintenance of code during reproduction

- constancy of base pairing guarantees

that the code will be retained.

2. Providing variety - order of bases

responsible for unique qualities of each

organism.

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DNA Replication

• Making an exact duplicate of the DNA involves 30 different enzymes

• Begins at an origin of replication

• Helicase unwinds and unzips the DNA double helix

• An RNA primer is synthesized by primase

• DNA polymerase III adds nucleotides in a 5′ to 3′ direction

– leading strand – synthesized continuously in 5′ to 3′ direction

– lagging strand – synthesized 5′ to 3′ in short segments; overall direction is 3′ to 5′

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• DNA polymerase I removes the RNA

primers and replaces them with DNA.

• When replication forks meet, ligases link

the DNA fragments along the lagging strand

to complete the synthesis.

• Separation of the daughter molecules is

complete.

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As replication proceeds

one strand loops down

and final separation

occurs when enzyme cuts

and releases two

completed molecules.

DNA replication is semiconservative because

each chromosome ends up with one new

strand of DNA and one old strand.

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Applications of the DNA code

• Information stored on the DNA molecule is

conveyed to RNA molecules through the

process of transcription.

• The information contained in the RNA

molecule is then used to produce proteins in

the process of translation.

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Gene-Protein Connection

1. Each triplet of nucleotides on the RNA specifies a particular amino acid.

2. A protein’s primary structure determines its shape and function.

3. Proteins determine phenotype. Living things are what their proteins make them.

4. DNA is mainly a blueprint that tells the cell which kinds of proteins to make and how to make them.

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RNAs

• Single-stranded molecule made of nucleotides

– 5 carbon sugar is ribose

– 4 nitrogen bases – adenine, uracil, guanine, cytosine

– phosphate

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RNA• 3 types of RNA:

– messenger RNA (mRNA) – carries DNA message

through complementary copy; message is in triplets

called codons

– transfer RNA (tRNA) – made from DNA;

secondary structure creates loops; bottom loop

exposes a triplet of nucleotides called anticodon

which designates specificity and complements

mRNA; carries specific amino acids to ribosomes

– ribosomal RNA (rRNA) – component of ribosomes

where protein synthesis occurs

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Transcription

1. RNA polymerase binds to promoter region upstream

of the gene.

2. RNA polymerase adds nucleotides complementary

to the template strand of a segment of DNA in the 5′

to 3′ direction.

3. Uracil is placed as adenine’s complement.

4. At termination, RNA polymerase recognizes signals

and releases the transcript.

• 100-1,200 bases long

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Translation

• Ribosomes assemble on the 5′ end of a mRNA transcript.

• Ribosome scans the mRNA until it reaches the start codon, usually AUG.

• A tRNA molecule with the complementary anticodon and methionine amino acid enters the P site of the ribosome and binds to the mRNA.

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Translation Elongation

• A second tRNA with the complementary anticodon fills the A site.

• A peptide bond is formed.

• The first tRNA is released and the ribosome slides down to the next codon.

• Another tRNA fills the A site and a peptide bond is formed.

• This process continues until a stop codon is encountered.

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Translation Termination

• Termination codons – UAA, UAG, and

UGA – are codons for which there is no

corresponding tRNA.

• When this codon is reached, the ribosome

falls off and the last tRNA is removed from

the polypeptide.

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The Master Genetic Code

• Represented by the mRNA codons and the

amino acids they specify

• Code is universal

• Code is redundant

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Polyribosomal complex allows for the synthesis of

many protein molecules simultaneously from the

same mRNA molecule.

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Eucaryotic Transcription and

Translation

1. Do not occur simultaneously – transcription occurs in the nucleus and translation occurs in the cytoplasm.

2. Eucaryotic start codon is AUG, but it does not use formyl-methionine.

3. Eucaryotic mRNA encodes a single protein, unlike bacterial mRNA which encodes many.

4. Eucaryotic DNA contains introns – intervening sequences of noncoding DNA- which have to be spliced out of the final mRNA transcript.

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Genetics of Animal Viruses

• Viral genome - one or more pieces of DNA

or RNA; contains only genes needed for

production of new viruses

• Requires access to host cell’s genetics and

metabolic machinery to instruct the host cell

to synthesize new viral particles

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Regulation of Protein Synthesis and

Metabolism

• Genes are regulated to be active only when

their products are required.

• In procaryotes this regulation is coordinated

by operons, a set of genes, all of which are

regulated as a single unit.

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Operons

• 2 types of operons:

– inducible – operon is turned ON by substrate:

catabolic operons- enzymes needed to metabolize

a nutrient are produced when needed

– repressible – genes in a series are turned OFF by

the product synthesized; anabolic operon –

enzymes used to synthesize an amino acid stop

being produced when they are not needed

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Lactose Operon: Inducible Operon

Made of 3 segments:

1. Regulator- gene that codes for repressor

2. Control locus- composed of promoter and

operator

3. Structural locus- made of 3 genes each coding

for an enzyme needed to catabolize lactose –b-galactosidase – hydolyzes lactose

permease - brings lactose across cell membrane

b-galactosidase transacetylase – uncertain function

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Lac Operon

• Normally off

– In the absence of lactose, the repressor binds with the operator locus and blocks transcription of downstream structural genes.

• Lactose turns the operon on.

– Binding of lactose to the repressor protein changes its shape and causes it to fall off the operator. RNA polymerase can bind to the promoter. Structural genes are transcribed.

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Arginine Operon: Repressible

• Normally on and will be turned off when

nutrient is no longer needed

• When excess arginine is present, it binds to

the repressor and changes it. Then the

repressor binds to the operator and blocks

arginine synthesis.

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Antibiotics That Affect Transcription

and Translation

• Rifamycin – binds to RNA polymerase

• Actinomycin D - binds to DNA and halts mRNA chain elongation

• Erythromycin and spectinomycin – interfere with attachment of mRNA to ribosomes

• Chloramphenicol, linomycin and tetracycline-bind to ribosome and block elongation

• Streptomycin – inhibits peptide initiation and elongation

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Mutations: Changes in the Genetic Code

• A change in phenotype due to a change in genotype (nitrogen base sequence of DNA) is called a mutation.

• A natural, nonmutated characteristic is known as a wild type (wild strain).

• An organism that has a mutation is a mutant strain, showing variance in morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals, etc.

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Causes of Mutations

• Spontaneous mutations– random change

in the DNA due to errors in replication that

occur without known cause

• Induced mutations – result from exposure

to known mutagens, physical (primarily

radiation) or chemical agents that interact

with DNA in a disruptive manner

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Categories of Mutations

• Point mutation – addition, deletion or

substitution of a few bases

• Missense mutation – causes change in a

single amino acid

• Nonsense mutation – changes a normal

codon into a stop codon

• Silent mutation – alters a base but does not

change the amino acid

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Categories of Mutations

• Back-mutation – when a mutated gene

reverses to its original base composition

• Frameshift mutation – when the reading

frame of the mRNA is altered by the

addition or deletion of nucleotides in a

newly synthesized DNA

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Repair of Mutations

• Since mutations can be potentially fatal, the cell has several enzymatic repair mechanisms in place to find and repair damaged DNA.

– DNA polymerase – proofreads nucleotides during DNA replication

– Mismatch repair – locates and repairs mismatched nitrogen bases that were not repaired by DNA polymerase

– Light repair – for UV light damage

– Excision repair – locates and repairs incorrect sequence by removing a segment of the DNA and then adding the correct nucleotides

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The Ames Test

• Any compound known to be mutagenic is

considered to be carcinogenic.

• Agricultural, industrial, and medicinal

compounds are screened using the Ames test.

• Indicator organism is a mutant strain of

Salmonella typhimurium that has lost the ability

to synthesize histidine.

• This mutation is highly susceptible to back-

mutation.

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Positive and Negative Effects Of

Mutations

• Mutations leading to nonfunctional proteins are

harmful, possibly fatal.

• Organisms with mutations that are beneficial in

their environment can readily adapt, survive, and

reproduce – these mutations are the basis of

change in populations.

• Any change that confers an advantage during

selection pressure will be retained by the

population.

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DNA Recombination Events

Genetic recombination – occurs when an

organism acquires and expresses genes

that originated in another organism

3 means for genetic recombination in bacteria:

1. Conjugation

2. Transformation

3. Transduction

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Conjugation

• Conjugation – transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via a direct connection

– Gram-negative cell donor has a fertility plasmid (F plasmid, F′ factor) that allows the synthesis of a conjugation (sex) pilus

– recipient cell is a related species or genus without a fertility plasmid

– donor transfers fertility plasmid to recipient through pilus

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Conjugation

• High-frequency recombination – donor’s

fertility plasmid has been integrated into the

bacterial chromosome

• When conjugation occurs, a portion of the

chromosome and a portion of the fertility

plasmid are transferred to the recipient.

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Transformation

• Transformation – chromosome fragments

from a lysed cell are accepted by a recipient

cell; the genetic code of the DNA fragment is

acquired by the recipient

• Donor and recipient cells can be unrelated

• Useful tool in recombinant DNA technology

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Insert figure 9.23transformation

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Transduction

• Transduction – bacteriophage serves as a carrier of DNA from a donor cell to a recipient cell

• Two types:

– generalized transduction – random fragments of disintegrating host DNA are picked up by the phage during assembly; any gene can be transmitted this way

– specialized transduction – a highly specific part of the host genome is regularly incorporated into the virus

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Transposons

• Special DNA segments that have the capability of moving from one location in the genome to another – “jumping genes”

• Cause rearrangement of the genetic material

• Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome

• May be beneficial or harmful

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