foundations in microbiology - warner pacific universityclasspages.warnerpacific.edu/sramos/bio370...
TRANSCRIPT
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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.
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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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