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MICROBIAL GENETICS
Chapter 8
STRUCTURE & FUNCTION
• GENETICS = Study of hereditary
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Includes the study of the genes
Gene replication
Gene products (proteins, rRNA, tRNA)
Traits derived from expressed genes
• GENOME = sum of the cell’s genetic material
– It consists of all of the chromosome(s) of a cell
• GENOTYPE = genetic makeup of cell/organism
• PHENOTYPE = traits due to the expression of the genotype
(the expression of the genes)
CHROMOSOMES
• Physical structure that carries the hereditary information
– Genes are made from DNA with A, T, C, G.
– Double-stranded, helical DNA
– Acts as a template to make RNA
• Prokaryotes: one circular chromosome
– May also contain a plasmid
• Eukaryotes: more than one, linear chromosome
GENES
• E. coli - 1 chromosome
– Approximately 4,000 genes
– DNA = 1 mm long (or 1000 m)
– Cell = 1 m long
• Human cell - 23 pairs of chromosomes
– Approximately 100,000 genes
– Each chromosome = 50 mm long
– Chromatin: DNA + proteins
DNA STRUCTURE
• DNA is made up of nucleotides
– Nitrogenous base + pentose + phosphate
– Sugar-phosphate backbone
– Hydrogen bonds from between A : T and C : G
• Two strands are complementary
• TRANSCRIPTION: DNA ----> mRNA
– RNA = A : U and C : G
• TRANSLATION: mRNA ----> protein
DNA REPLICATION: Overview
• Set of enzymes working in a specific sequence
• Process is very accurate
• Each parental strand acts as a template for the new “daughter”
strand  2 daughter strands produced
– SEMI-CONSERVATIVE
• All DNA is synthesized in a directed manner
– ALWAYS from the 5’ towards the 3’ end
• Begins at the ORGIN of REPLICATION
• Bi-directional - all the way around ----> 2 circular DNA
molecules
THE DOGMA: DNA  RNA  PROTEIN
• REPLICATION: DNA ---> DNA
– Occurs before cell divides
• TRANSCRIPTION: DNA ---> RNA
• TRANSLATION: mRNA ---> PROTEIN
• Three types of RNA
– mRNA – messenger
• “Read” by ribosomes to make protein
– tRNA – transfer
• Carries individual amino acids to the ribosomes for making the new
proteins
– rRNA – ribosomal
• Ribosomes contain proteins + rRNA
PROKARYOTIC GENES
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Transcription occurs in the cytoplasm
No INTRONS or EXONS in prokaryotic genes
RNA is not processed
RNA = POLYCISTRONIC
• POLYCISTRONIC = 1 RNA codes for more than one
gene/protein
TRANSCRIPTION
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Synthesis of RNA from DNA
The nucleotides are A, U, C, G
The enzyme is DNA dependent RNA polymerase
Starts at a site called the promoter
The enzyme elongates in a 5’ to 3’ direction
Termination occurs as it reaches a termination codon
Enzyme is released
TRANSLATION: RNA --> PROTEIN
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RNA is “read” in the 5’-->3’ direction
PROKARYOTES: transcription & translation = coupled - both
occur in the cytoplasm, m-RNA is polycystronic
Three phases to translation:
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INITIATION
ELONGATION
TERMINATION
PROKARYOTES: gene expression is regulated primarily at the
level of transcription
You need ribosomes, t-RNA, m-RNA, amino acids, and
translation factors.
GENETIC CODE
• 4 nucleotides (A, U, C, G)
– Used in combinations of 3 gives 64 sets of triplets
– 1 set of 3 nt = CODON
• 1 START: AUG
• 3 STOPS: UAA, UAG, UGA
• 64 –4 = 60 codons left to code for the 20 different amino acids
that are used in proteins
• THEREFORE ………
– Some of the codons are REDUNDANT that is several codons code for
the same amino acid, the genetic code is DEGENERATE.
MUTATIONS
• STABLE, INHERITED CHANGE IN THE NUCLEOTIDE SEQUENCE OF
THE DNA
• CHANGES THE GENOTYPE
– The change occurs in the DNA and this change is passed on to the daughter cells
• MAY ALTER THE PHENOTYPE
– If the change in the DNA causes a change in a codon to code for a different amino
acid in the protein
• SPONTANEOUS:
– No known cause
– Errors from DNA replication
• INDUCED:
– Caused by a MUTAGENIC AGENT (MUTAGEN)
TYPES OF MUTATIONS
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POINT MUTATION or BASE SUBSTITUTION
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MISSENSE MUTATION: change causes a different aa to be used
NONSENSE MUTATION: nt changes results in a STOP CODON
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Insertion or deletion of 1 or a few bases
OFTEN creates a STOP CODON
FRAMESHIFT MUTATION
MUTAGENS
Physical or chemical factors that cause a change in the DNA (a
mutation)
CHEMICALS
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BASE ANALOGS: similar structure, 2-aminopurine and 5 bromouracil
Nitrous acid
Alkylating agents
Intercalating agents
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IONIZING: Gamma and X-rays
NON-IONIZING: UV light (sun tanning)
RADIATION:
GENETIC TRANSFER & RECOMBINATION
Gene transfer = movement of genetic information between
organisms
In eukaryotic organisms this can occur during the fertilization
of an egg
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In prokaryotic organisms – this is not an essential part of the
life cycle
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When it does occur, DNA is transferred from a DONOR to a
RECIPIENT cell
Combining genes (DNA) from two different cells =
RECOMBINATION
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The resulting cells = a RECOMBINATE
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Transformation, Transduction, and Conjugation
Bacteria have 3 ways to transfer DNA
PLASMIDS
Small, extrachromosomal circular DS DNA
Independently replicates
Can exist in single or multiple copies
Usually not essential for normal bacterial growth
Conjugation allows for transfer between 2 cells
PLASMID TYPES
• CONJUGATION FUNCTIONS
– F FACTORS (Genes for SEX PILI & for transfer to another cell)
• RESISTANCE to ANTIBIOTICS
– R FACTORS
• RESISTANCE to HEAVY METALS
• RESISTANCE to BACTERIOPHAGE INFECTION
• BACTERIOCIN PRODUCTION
– Small molecules that kill other bacteria
• TOXIN PRODUCTION
• VIRULENCE DETERMINANTS
– Factors for attachment to other cells
R FACTORS
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Codes for resistance to antibiotics
Two groups of genes
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RTF = RESISTANCE TRANSFER FACTOR
• Genes for plasmid transfer and replication
r DETERMINANT
• Genes for “detoxifying” enzymes
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Usually carried on transposons
Small segments of DNA which can move from one region of
DNA to another region
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TRANSPOSABLE GENES
TRANSPOSONS
• Usually carry information for their movement and other
information such as drug resistance or toxin production
• “JUMPING GENES”
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Chromosome to plasmid
Plasmid to plasmid
Plasmid to chromosome
Chromosome to chromosome
Within one plasmid or chromosome
DNA TRANSFER IN BACTERIA
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TRANSFORMATION
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Naked DNA from cell to cell
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Transfer occurs by viral transfer
Bacteriophage
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Cell to cell contact via a pili
TRANSDUCTION
CONJUGATION
1. TRANSFORMATION
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“Naked” DNA is transferred into a cell
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Susceptible to DNase degradation
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Technique used in laboratories
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ROUGH (R) strain (not virulent) --> mice - -> LIVE
SMOOTH (S) strain (virulent) -- > mice --> DIE
Heat killed S strain --> mice -- > LIVE
Heat killed S strain + R strain -- > mice -- > DIE
• Recovered colonies of S strain from dead mice
1928: Frederick Griffith -DISCOVERED transformation
Streptococcus pneumoniae
DNA must have been exchanged from S to R
2. TRANSDUCTION
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Transfer of genetic material by virus
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Bacteriophage or PHAGE - two types of bacteriophages exist
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Kills cell after infection
Virus replicates in cell using host machinery
Lyses the host cell to release progeny
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Virus enters cell & integrates into host DNA (PROPHAGE)
Can exist in quiescent form while integrated in host genome
Virus then replicates with the cell
It can excise out & leaves to infect another cell
Virulent or lytic phage:
Temperate of lysogenic phage
2. TRANSDUCTION cont’d
• GENERALIZED TRANSDUCTION
– Occurs during the lytic cycle of viruses
– Random packaging of bacterial genes and proteins into virus
– These “generalized” DNA can be carried to a new host
• SPECIALIZED TRANSDUCTION
– Temperate phage: incorporates into host’s chromosome
– Must exist as a prophage
– Can spontaneously revert to LYTIC and excise out of the host DNA
• May include some of the host’s DNA  new phage
– These new “specialized” phage  carried to a new host
3. CONJUGATION
• Genetic exchange occurring through direct cell-cell contact
– Mediated by a plasmid
• Requires the presence of a SEX PILUS
– Encoded for by a fertility plasmid called the F factor
• The F plasmid contains information to code for conjugal
transfer and for autonomous replication
• The donor cell carrying the F plasmid = “male”
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Considered F+
• The recipient cell = “female”
– Considered F-
3. CONJUGATION cont’d
or donor cell = F +
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“FEMALE” or recipient cell = F • If cross an F+ cell with an F - cell  2 F+ cells
• “MALE”
– F + transfers F factor (plasmid) to F – There is simultaneous replication and transfer of the plasmid
• If F factor is incorporated into cell’s chromosome
– Hfr = High frequency recombinant
– This cell has the transfer information but it is integrated in host cell’s DNA
• If we cross an HFr with an F – we get Hfr + F
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– Because usually only a portion of F factor is transferred
RECOMBINANT DNA & BIOTECHNOLOGY
Chapter 9
RECOMBINANT DNA
• Artificial manipulation of genes
• Genetic engineering
• BIOTECHNOLOGY: all industrial applications of biological
systems or processes
– Now includes industrial application of genetic engineering
• 1970s RESTRICTION ENDONUCLEASES
– Cut the DNA at specific sites in the DNA
– Leaves BLUNT or STAGGERED “STICKY” ENDS in the DNA that can
bind to other pieces of DNA cut with the same endonuclease
WHY?
• Express a gene of interest
– Human insulin
– Blood clotting factors
• Vaccine development
– Hepatitis B vaccine
• Genetically modify an organism
– Pesticide resistant plants
– Oil-eating bacteria
• Rapidly produce a large number of copies of a gene
• IN VITRO : in glass ie in a test tube
CLONING VECTORS
• Used to transfer a gene(s) from one organism to another
• Self-replicating DNA molecule used as a carrier to
transmit/insert a gene into a cell
– Plasmids
– Bacteriophages
• TRANSFORMATION - “naked” DNA
• ELECTROPORATION - uses protoplasts
• MICROINJECTION - into animal cells
PCR: POLYMERASE CHAIN REACTION
• 1980s: Kary Mullis
• Use of DNA polymerase to make a large number of copies of a
DNA template in vitro
• One DNA template --> billions of copies within a few hours
• Process involves alternating cycles of heat denaturation and
replication
• Heat denaturation = heating ds DNA to 98°C to separate the
strands
• Replication = amplification step
PCR: THE PROCESS
• Heat denature DNA template
• Must supply the following:
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– A supply of the 4 nt (ACTG)
– DNA polymerase - enzyme needed, must be stable at high temperatures
– Needs small DNA primers
Lower temperature to 60C --> primers anneal
Amplification step = synthesis of DNA copies
Heat denature - templates / copies come apart
START OVER
SOUTHERN BLOTTING
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Technique developed in 1975 by Southern
Used to identify and separate pieces of DNA by gel electrophoresis
DNA is cut by enzymes
Fragments are separated by gel electrophoresis
Fragments are transferred to nitrocellulose paper
Filter is exposed to radioactive gene of interest
Expose the filter to x-ray and identify the gene of interest