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