microbial genetics - las positas...
TRANSCRIPT
Ch 8
Microbial Genetics
© 2004 by Jones and Bartlett Publishers
• Define the terms genome and gene, and differentiate between genotype and phenotype.
• Draw a detailed segment of DNA. • Summarize the steps of bacterial DNA replication, and identify the
enzymes used in this process. • Compare and contrast the synthesis of leading and lagging strands. • Provide an overview of the relationship among DNA, RNA, and proteins. • Identify structural and functional differences between RNA and DNA. • Outline the process of transcription. • List the three types of RNA directly involved in translation. • Define the terms codon and anticodon, and recite the start codon. • Outline the process of translation. • Indicate how eukaryotic transcription and translation differ from these
processes in bacteria. • Define operon, and explain one advantage it provides to a bacterial cell. • Highlight the main parts of the lac operon • Explain the defining characteristics of a recombinant organism. • Describe the three forms of horizontal gene transfer used in bacteria. • Define the term mutation and distinguish between the different types. • Explain the importance of restriction endonucleases to genetic
engineering. • Describe in detail how to clone a gene into a bacterium and gain a desired
protein product
SLOs
Vocabulary
Genetics
Genome of cells vs. genome of viruses
Genes, 3 categories of genes
- Structural genes: _________________
- Genes that code for _______
- Regulatory genes: control gene expression
Chromosome
Haploid vs. diploid
Base pairs
Genetic code
Genotype vs. Phenotype
Fig 8.1
DNA Code Unit molecule:
___________________,
composed of:
Unit molecules covalently
linked to form a sugar-
phosphate backbone
Phosphates linked to
number 5’ (five prime)
carbon of sugar and to
number 3’ (three prime
carbon)
Compare to Fig 8.3
DNA is double helix
associated with
proteins
Strands are held
together by ___ bonds
between ____ and
____
Strands are
antiparallel
DNA Code cont.
The Bacterial DNA
Mostly single circular chromosome
Attached to plasma membrane
Chromosome length 1mm
(Cell length ? )
DNA is supercoiled
Number of genes in E. coli
Extra-chromosomal bacterial DNA: _______ (1-5% of chromosome size)
Flow of Genetic Information
DNA Replication
Collaboration of ~ 30 enzymes.
DNA polymerase initiated by RNA primer
bidirectional
leading strand: continuous DNA synthesis
lagging strand: discontinuous DNA synthesis Okazaki fragments
semiconservative
2
Elongation and Termination of the Daughter Molecules
• Speed can be 750 bases per second
• DNA pol I removes RNA primers and replaces them with DNA.
• Ligases move along the lagging strand to…..
• Mistakes in DNA replication happen ~ every 108 to 109 bases,
but most corrected by DNA polymerase III.
Replication fork Replication in 5' 3' direction
Compare to Fig 8.4
Protein Synthesis Transcription produces 3 types of RNA (?) Enzyme necessary ? Promoters and terminators
Translation produces the protein Sense codons vs. nonsense codons Anticodons
Exceptions to this pattern:
- RNA viruses convert RNA to other RNA
- Retroviruses convert RNA to DNA
A wide variety of RNAs are used to regulate gene
function
Also:
Primer RNAs in both
bacterial and
eukaryotic cells
Ribozymes: remove
unneeded sequences
from other RNAs
Fig. 8.5
Genetic code: universal and degenerate/redundant
Codons: groups of three nucleotides determining the
amino acid
Advantage of redundancy and wobble position?
More Details on Transcription
RNA polymerase binds to promotor sequence
proceeds in 5' 3' direction
stops when it reaches terminator sequence
Fig 8.7 Fig 8.7
After Transcription: Translation
Ribosomes of prokaryotes
and eukaryotes differ in size
- Bacteria: 70S (50S and
30S subunits)
- Eukaryotes: 80S (60S
and 40S subunits)
•Small subunit binds to
5‘end of mRNA.
•Large subunit supplies
enzymes for making
peptide bonds.
More Details on Translation
Nucleotide sequence of mRNA is translated into amino acid sequence of protein using “three letter words” = codons
Translation of mRNA begins at the start codon: ______
Translation ends at a stop codon: UAA, UAG, UGA
Requires various accessory molecules and 3 major components: ?
In Prokaryotes: Simultaneous transcription and translation Polyribosomes
The Translation Process in Protein Synthesis
Simultaneous Transcription and Translation
in Prokaryotes
Differences Between Eukaryotic and
Bacterial Transcription and Translation
Characteristic Bacteria Eukaryotes
Start codon Always AUG AUG, but codes for a
different form of
methionine
mRNA Can code for several
genes in a series
Only codes for one
protein
Transcription and
translation:
Occur simultaneously
in the cytoplasm
Transcription occurs in
the nucleus,
translation occurs in
the cytoplasm
Genes Exist as an
uninterrupted set of
triplets coding for a
protein
Contain introns that
do not code for
proteins and exons
that do code for
proteins. Introns must
be edited out.
Genetic Regulation of Protein Synthesis
• Genes are only expressed when needed
Example of regulation of gene expression in bacteria:
• Operons: Set of genes regulated as a single unit
– Inducible operons for catabolic enzymes. Induced
by the substrate of the enzyme(s) for which the structural
genes code
– Repressible operons for anabolic enzymes. Operon
turned off by product.
Lac Operon
DNA Recombination
– Bacteria have no sexual reproduction.
– Horizontal gene transfer allows for DNA recombination
– Recombinant: Any organism containing genes that originated in another organism
– Allows for rapid spreading of genes for drug resistance and exotoxins....
Flow of Genetic Information
Horizontal Gene
Transfer:
Any DNA transfer that
results in organisms
acquiring new genes
that did not come
from parent
organisms.
Conjugation
Plasmid and chromosomal DNA transfer via direct cell to cell contact
Gram-negative conjugation: F+ = donor cell. Contains F plasmid (factor) and produces conjugation (F) pilus (aka “sex pilus”)
Recipient cell (F– ) becomes F+
In some cells F factor integrates into chromosome Hfr cell
E.g.: R plasmids
Fig 8.11
Gram-positive conjugation:
– Opening created between two adjacent cells
– Replicated DNA passes from one cell to another
Conjugation is a conservative process. I.e.: Donor bacterium
retains (conserves) copy of genetic material being transferred.
Fig 8.11
Transformation - Capturing DNA from Solution -
DNA released by lysed cell
breaks into fragments
accepted by recipient cell
Facilitated by DNA-binding
proteins on cell wall
Competent cells are
capable of accepting DNA
Adapted for use in
recombinant DNA technology
Transduction
DNA Transfer from donor to
recipient cell with help of
bacteriophage (= transducing
phage)
Generalized vs. specialized
transduction
Many exotoxins Compare to Fig 8.12
Mutations: Changes in the Genetic Code
Driving force of evolution
May be neutral, beneficial, or harmful
Wild type vs. mutant
Spontaneous mutations: Occur in the absence of a
mutagen
•Mutagen: Physical or chemical agents inducing
mutations. (E.g.: UV light, X rays, nitrous acid)
Types of Mutations:
1. Point mutation = base substitution (silent, missense, nonsense, readthrough)
2. Frameshift mutation = Insertion or deletion of one or more nucleotide pairs
What type of mutation?
1. Nonsense mutation
2. Missense mutation
3. Silent mutation
4. Point mutation
5. Frameshift mutation
Various Point Mutations
Silent
Missense
Nonsense
TAA
Compare to Table 8.8
Radiation as a Mutagen
1. Ionizing radiation (_____ and _____)
Formation of highly reactive radicals and ions that damage nucleotides mutations.
Ds breaks of covalant bonds in backbone deletion mutations
2. UV rays lead to _________________
Chemical Mutagens
examples:
1. Nucleoside (base) analogs have altered base-pairing properties. They can be randomly incorporated into growing cells (cancer drugs)
only used by viral enzymes (e.g. AZT)
2. Frameshift mutagens such as intercalating agents (e.g.:, aflatoxin, ethidium bromide)
Distortion due to
intercalating
agent will lead to
one or more
base-pairs
inserted or
deleted during
replication.
Potent
carcinogens!
Intercalation
• DNA Pol has proofreading capacity DNA
repair of replication mistakes
• The cell has additional systems for finding and
repairing DNA that has been damaged.
• Photolyases for UV damage repair. Light repair
enzymes separate thymine dimers using energy
from visible light
• Nucleotide
excision repair
repairs all mutations
Repair of Mutations
Compare to Fig 8.15
Genetic Engineering
Manipulation and change of the genome using biotechnology
Restriction Endonucleases (REs) = Molecular scissors Recognize and clip at palindromes specific cuts!
Bunt ends vs. sticky ends
Destroy bacteriophage DNA in bacterial cells
Staggered symmetrical
cuts leave short tails
called “sticky ends”
Adhesive tails will base-
pair with complementary
tails on other DNA
fragments or plasmids
Site of cleavage EcoRI
Restriction Endonuclease,
aka Restriction Enzymes
Restriction fragments: pieces of DNA
produced by restriction endonucleases
Role of Restriction Enzymes in Making
Recombinant DNA Molecules
Compare to
Fig 8.16
Additional Important Enzymes
for Genetic Engineering
• Ligase,
– seals (ligates) sticky ends together
– used in final step of splicing genes into plasmids and
chromosomes
• Reverse transcriptase
- Role in nature?
- Converts RNA into DNA to make cDNA
• cDNA
- Made from mRNA, tRNA, and rRNA
- Used to synthesize eukaryotic genes from mRNA
transcripts. Advantage vs. using DNA directly?
Recombinant DNA Technology
• Intent to remove DNA from one organism
and combine it with that of a different
organism
• Bacteria are genetically engineered to mass
produce:
– Hormones
– Enzymes
– Vaccines
Vectors Also known as cloning vectors.
Must be
Small and easy to manipulate.
________ & _________ serve as vectors.
self-replicating
large quantities
When they carry “insert”:
= Recombinant DNA molecules
Introduce foreign DNA
(desired gene) into host cells
Shuttle vectors can exist
in several different species.
... One of most commonly used vectors:
Compare to Fig 8.18
Gene Cloning Requires 2 Main
Ingredients
1. ____________
2. ____________ how do you get this?
Various Ways of Obtaining Gene of Interest
1. DNA removed from cells and separated into
fragments by REs
2. Gene synthesized from isolated mRNA transcripts
using RT
3. Gene amplified using PCR (See lab)
Recombinant vector is then inserted into
host cell
Blue and White Screening Method for Selecting a Clone (or Recombinant DNA Molecule)
Direct selection of engineered vector via antibiotic-resistance markers (ampR) on plasmid vectors.
Vector also contains-galactosidase gene for blue-white screening
Desired gene is inserted into the -galactosidase gene site gene inactivated
Not in book
1) Plasmid cloning Three possible outcomes:
1. Bacteria lack vector _______________
2. Bacterial clones contain vector without the new gene colony type? _______________
3. Bacterial clones contain recombinant vector resistant to Ampicillin and
unable to hydrolyze X-gal colony type? _______________
2) Selecting Recombinant Bacteria
Which type of
colonies do you
want?
a)White
b)Blue
c) I don’t want
any
Making a Gene Product
E. coli: prokaryotic workhorse of biotech. Easily grown and genomics well understood. Disadvantage: Cells must be lysed to get product release of ______
Yeast: Saccharomyces cerevisiae is eukaryotic workhorse of biotechnology. Advantage: Continuous secretion of gene product.
Mammalian cells: May express eukaryotic genes easily. Disadvantage: Harder to grow.
Plant cells: Easy to grow. May express eukaryotic genes easily.
Some Therapeutic Applications of Recombinant DNA
Technology
1. Pharmaceutical applications, e.g.: Insulin production
2. Subunit vaccines
3. DNA vaccines
4. Gene therapy to replace defective or missing genes
Case File: A Body Attacking Itself
Who will present?
Inside the Clinic: Using
Recombinant DNA to Produce
Insulin (covered by teacher)
PCR and Gel Electrophoresis
covered in lab