MICROBIAL GENETICS



MICROBIAL GENETICS

Chapter 8

STRUCTURE & FUNCTION

GENETICS = Study of hereditary

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 μm)

Cell = 1 μm 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

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

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

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:

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

POINT MUTATION or BASE SUBSTITUTION

MISSENSE MUTATION: change causes a different aa to be used

NONSENSE MUTATION: nt changes results in a STOP CODON

FRAMESHIFT MUTATION

Insertion or deletion of 1 or a few bases

OFTEN creates a STOP CODON

MUTAGENS

Physical or chemical factors that cause a change in the DNA (a mutation)

CHEMICALS

BASE ANALOGS: similar structure, 2-aminopurine and 5 bromouracil

Nitrous acid

Alkylating agents

Intercalating agents

RADIATION:

IONIZING: Gamma and X-rays

NON-IONIZING: UV light (sun tanning)

GENETIC TRANSFER & RECOMBINATION

Gene transfer = movement of genetic information between organisms

In eukaryotic organisms this can occur during the fertilization of an egg

In prokaryotic organisms – this is not an essential part of the life cycle

When it does occur, DNA is transferred from a DONOR to a RECIPIENT cell

Combining genes (DNA) from two different cells = RECOMBINATION

The resulting cells = a RECOMBINATE

Bacteria have 3 ways to transfer DNA

Transformation, Transduction, and Conjugation

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

Codes for resistance to antibiotics

Two groups of genes

RTF = RESISTANCE TRANSFER FACTOR

Genes for plasmid transfer and replication

r DETERMINANT

Genes for “detoxifying” enzymes

Usually carried on transposons

Small segments of DNA which can move from one region of DNA to another region

TRANSPOSABLE GENES

TRANSPOSONS

Usually carry information for their movement and other information such as drug resistance or toxin production

“JUMPING GENES”

Chromosome to plasmid

Plasmid to plasmid

Plasmid to chromosome

Chromosome to chromosome

Within one plasmid or chromosome

DNA TRANSFER IN BACTERIA

TRANSFORMATION

Naked DNA from cell to cell

TRANSDUCTION

Transfer occurs by viral transfer

Bacteriophage

CONJUGATION

Cell to cell contact via a pili

1. TRANSFORMATION

“Naked” DNA is transferred into a cell

Susceptible to DNase degradation

1928: Frederick Griffith -DISCOVERED transformation

Technique used in laboratories

Streptococcus pneumoniae

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

DNA must have been exchanged from S to R

2. TRANSDUCTION

Transfer of genetic material by virus

Bacteriophage or PHAGE - two types of bacteriophages exist

Virulent or lytic phage:

Kills cell after infection

Virus replicates in cell using host machinery

Lyses the host cell to release progeny

Temperate of lysogenic phage

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

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”

Considered F+

The recipient cell = “female”

Considered F-

3. CONJUGATION cont’d

“MALE” or donor cell = F +

“FEMALE” or recipient cell = F -

If cross an F+ cell with an F - cell ( 2 F+ cells

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 –

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:

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

• 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

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