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UNIT VII – MOLECULAR GENETICS
Big Campbell – Ch 18, 19, 20Baby Campbell – Ch 10, 11, 12
UNIT OVERVIEW
• Microbial Geneticso Viruseso Bacteria
• Gene Expression in Prokaryoteso Regulation of Gene Expression
• DNA Technologyo DNA Testing Techniqueso PCRo Recombinant DNAo Extensions
I. MICROBIAL GENETICS – VIRUSES
• Discovery of Viruses First isolated by Ivanowsky in
1890s from infected tobacco leaves
Crystallized by Stanley in 1935 – proved viruses were not cells
• Not organisms; correctly referred to as particles
• Not capable of carrying out life processes without a host cell
• Parasites
I. VIRUSES, cont
I. VIRUSES, cont
• Characteristics Viral genome may be either single-stranded or double-stranded
DNA or RNA. Protein coat surrounding virus is known as a capsid made up of
protein subunits called capsomeres. Some viruses are also surrounded by a viral envelope
Typically derived from host cell membraneException is Herpes virus, synthesized from nuclear envelope of host cellAid in attachment. Envelope glycoproteins bind to receptor molecules on
host cellMost viruses that infect animals have envelope
I. VIRUSES, cont• Bacteriophages
Defense Mechanisms
I. VIRUSES, cont - Reproduction
I. VIRUSES, cont - Reproduction
Viral Entrance into Host Cell
I. VIRUSES, cont - Reproduction
1. Lytic Cycle – Results in death of host cell.
LYTIC CYCLE
I. VIRUSES, cont - ReproductionLYSOGENIC CYCLE
I. VIRUSES, cont - Retroviruses
I. VIRUSES, cont – Animal Viruses
• Coronavirus
• Filovirus
• Herpesvirus Herpes simplex I and II
Epstein-Barr virus
Varicella zoster
I. VIRUSES, cont – Animal Viruses• Influenza Virus
• Papillomavirus
• Rhinovirus
II. OTHER INFECTIOUS AGENTS
• Viroids
• Prions
III. MICROBIAL GENETICS - BACTERIA• Genetic Make-up of Bacteria
Prokaryotic Single chromosome wrapped in much
less protein than found in euks DNA concentrated in region known as
nucleoid• Reproduction
Reproduce asexually
Advantages of asexual reproduction
Disadvantages of asexual reproduction Have developed mechanisms for
genetic variability . . .
III. BACTERIA, cont• Transformation
Ability to take up DNA from surrounding environment Streptococcus pneumoniae
Used to introduce human genes into bacterial cells to produce human protein
• TransductionUse of viruses to carry bacterial genes from one host cell to another
• Conjugation “Bacterial sex” One-way transfer of a self-replication piece of DNA known as a plasmid
Donor (male) extends pilus Pulls cells togetherCytoplasmic bridge formsPlasmid is transferred
III. BACTERIA, cont
• A Closer Look at Plasmids Bacterium’s ability to produce
plasmids and form pili due to specific piece of DNA known as the F factorF factor may be integrated into
chromosome, or separate as a piece of plasmid
Contains an origin of replicationCopy of F factor may be
transferred to recipient cell; allow recipient cell to become “male”
• R Plasmids – Carry genes for enzymes that destroy antibiotics
III. BACTERIA, contA Quick Look Back at Protein Synthesis in Euks
III. BACTERIA, cont• Protein Synthesis
Similar to euks Different in that transcription & translation occur virtually simultaneously
o o
IV. REGULATION OF GENE EXPRESSION IN PROKARYOTES
• Important adaptation for bacteria
• Two basic mechanisms for metabolic controlo Regulation of Enzyme
ActivityFeedback Inhibition
o Regulation of Gene ExpressionOperons
IV. PROKARYOTE GENE EXPRESSION, cont
• Operon Modelo Operon = Promoter + Operator + all genes required for a given metabolic
pathway o Operon acts as a single transcription unito Promoter → Binding site for RNA polymeraseo Operator → “On-off” switch located either close to or within the promoter
Operator controls whether or not RNA polymerase can bind to the promoter region
Therefore operator determines whether operon genes are transcribed & translated
IV. PROKARYOTE GENE EXPRESSION, cont
• Operon Controlo Operon can be turned off by a protein known as a repressoro Repressor binds to operator and prevents attachment of RNA
polymerase to promotero Repressor is a protein controlled by a gene known as a
regulatory gene in a different location on chromosome; not part of operonExpressed continuouslyAlways a small supply of repressor protein present
IV. PROKARYOTE GENE EXPRESSION, cont
• Types of Operonso Inducible Operons
Operons that are usually off; that is, not usually transcribedCan be stimulated when a specific molecule interacts with
regulatory proteinExample is the lac Operon
o Repressible OperonsTranscription normally occursCan be inhibited when a specific molecule binds allosterically to
regulatory proteinExample is the trp Operon
IV. PROKARYOTE GENE EXPRESSION, cont Inducible Operons
• lac Operon Regulates
transcription of genes required for breakdown of lactose
Inducible – Typically off
Bacterium is metabolizing glucose, other carbs; lactose is not present
IV. PROKARYOTE GENE EXPRESSION, cont Inducible Operons
• lac Operon, cont• When lactose is available,
lactose itself binds with repressor; inactivates it by changing its shape
• Repressor cannot bind to regulator
• Therefore, RNA polymerase is able to bind to promoter; operon is “on”
• 3 enzymes required to metabolize lactose are synthesized
IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons
• Tryptophan (trp) Operon Operon controls
production of 5 enzymes required to synthesize amino acid, tryptophan when it is not available to bacterium in surrounding
Operon normally on; repressor inactive
IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons
• When tryptophan is present, it binds to the repressor of the trp operon, activating the repressor, and turning off enzyme production.
• Tryptophan acts as a co-repressor, a
molecule that works with a repressor protein to switch an operon off.
IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons
IV. PROKARYOTE GENE EXPRESSION, cont
• Positive Gene Regulationo In addition to repressors, some operons are also under the control of
proteins known as activatorso Essentially the opposite of repressors o They “turn up” an operon by making it easier for RNA polymerase to bind to
DNA, therefore facilitating transcription of operon geneso In the lac operon . . .
If both glucose and lactose are available, bacterium utilizes glucose until its supplies are depleted
As glucose ↓, concentration of cyclic AMP (cAMP) ↑Increase in cAMP triggers release of activator protein known as CAP;
CAP binds to promoter, facilitates binding of RNA polymerase to promoter of operon to enhance synthesis of enzymes of lac operon
When glucose concentration is high, decrease in cAMP results in decrease in CAP → RNA polymerase has very low affinity for lac operon promoter so lactose metabolism does not occur
IV. PROKARYOTE GENE EXPRESSION, cont
DNA TECHNOLOGY & GENOMICS
V. TECHNIQUES IN DNA TECHNOLOGY• Restriction Enzymes
o Used by bacteria to “chop up” viral DNA
o Bacterial DNA protected by _________o Very specific
Each enzyme recognizes a particular nucleotide sequence
Called a restriction sequence or restriction site
Palindromic Cuts made at specific points May create “sticky ends”
o Used in gel electrophoresis o Also used to form recombinant DNA
Fragments may be pasted together with DNA ligase to form recombinant DNA
V. TECHNIQUES, cont
• Polymerase Chain Reaction (PCR)o In vitro method of
amplifying small amounts of DNA DNA is heated to
separate the double helix.
Mixture is allowed to cool, DNA primers attach to target
Heat-stable polymerase is used to extend the primers in the 5’–3’ direction.
V. TECHNIQUES, cont
• Gel Electrophoresiso Separates DNA
fragments based on sizeo Restriction fragment
analysis DNA treated with
restriction enzymes Resulting fragments
migrate based on size Produce a pattern
characteristic of original DNA and restriction enzyme used
V. TECHNIQUES, cont
• Southern Blotting Designed by Dr.
Southern Detects particular
DNA sequences• Northern Blotting
Detects particular mRNA sequences
• Western Blotting Used to detect
proteins
VI. EXTENSIONS IN DNA TECHNOLOGY • Recombinant DNA
DNA containing nucleotides from other sources
Process utilizes restriction enzymes that make jagged cuts in DNA; creates sticky ends
When DNA from different sources treated with same restriction enzyme, sticky ends “mix & match”
Often use reporter genes to determine success; for example, ampicillin resistance
VI. EXTENSIONS, cont
• cDNA - complementary DNAo Procedure for “cloning DNA” that uses mRNA, reverse transcriptaseo
• STRs – short tandem repeatso Short segments of DNA that are highly repetitive, polymorphico Repeat patterns are inheritedo Useful for identifying individuals
• SNPs – single nucleotide polymorphismso Single base-pair that shows variation in a significant % of populationo SNPs that alter the fragment length following exposure to restriction
enzymes called RFLPs (restriction fragment length polymorphisms)o Genetic markers
VI. EXTENSIONS, cont
• DNA Microarray Assayso AKA DNA
Chipso Test used to
determine gene function, gene interactions
o May be used to determine agressiveness of cancers, method of treatment, etc
VI. EXTENSIONS, cont• Gene Cloning
o Process of preparing multiple copies of a particular segment of DNAo Requires host and vectoro Hosts
Initially done using bacterial cells Now eukaryotic hosts are used
YeastPlants
o Vector Should have 4 characteristics
Ability to replicate independently of host cell DNARecognition sequenceReporter geneSmall size
Possible vectors includePlasmidsVirusesYAC = Yeast Artificial Chromosome
VI. EXTENSIONS, cont• Gene Cloning
Use of plasmid as vector
Plasmid isolated from bacterial cell
Foreign DNA inserted into plasmid
Plasmid returned to bacterial cell; described as recombinant bacterium
Foreign gene is cloned as bacteria reproduce
Common bacterium used for plants is Agrobacterium tumefactiens
VI. EXTENSIONS, cont
A CLOSER LOOK AT
GENECLONING
VI. EXTENSIONS, cont
• Reproductive Cloning Nuclear Transplantation Process of using unfertilized
egg cell & replacing nucleus with DNA
In 1997, scientists were able to produce first reproductive clone, “Dolly”, by culturing somatic cells in a nutrient-poor medium to de-differentiate them and force them back to totipotency.
Reproductive cloning in animals has enjoyed limited success.
VI. EXTENSIONS, cont• Gene Silencing
o Knockout GenesUse of genetic recombination to create an inactive , “knocked out” geneBased on principal of homologous recombination
Mutated allele introduced into embryonic stem cellsForms chimeras
Often used in mice to study gene expressiono RNAi
Based on principal of microRNASmall-interfering RNA (siRNA) synthesized complementary to mRNABase-pairing occursTranslation is blockedHas been used to block production of growth factors in certain cancers
VII. GENOMICS• Human Genome Project
International government effort begun in 1990 Goals
o identify all the approximately 20,000-25,000 genes in human DNA, o determine the sequences of the 3 billion chemical base pairs that make
up human DNA, o store this information in databases, o improve tools for data analysis, o transfer related technologies to the private sector, and o address the ethical, legal, and social issues (ELSI) that may arise from the
project. Celera Genomics
o Shotgun sequencing Completed early and under-budget in 2003 Genomics has given rise to proteonomics