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Genetics Part 2B

Regulation of metabolic pathways Operon: cluster of related genes with on/off switch

Three Parts:

1. Promoter – where RNA polymerase attaches

2. Operator – “on/off”, controls access of RNA poly

3. Genes – code for related enzymes in a pathway

Bacterial control of gene expression

Regulatory gene: produces repressor protein that binds to operator to block RNA poly

Repressible Operon (ON OFF) Repressible Operon

Normally ON

Anabolic (build organic molecules)

Organic molecule product acts as corepressor binds to repressor to activate it

Operon is turned OFF

Eg. trp operon

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trp operon

Inducible Operon

Normally OFF

Catabolic (break down food for energy)

Repressor is active inducer binds to and inactivates repressor

Operon is turned ON

Eg. lac operon

lac operon

• Typical human cell: only 20% of genes expressed at any given time

• Different cell types (with identical genomes) turn on different genes to carry out specific functions

• Differences between cell types is due to differential gene expression

Eukaryotic gene expression

regulated at different stages

Chromatin Structure:

• Tightly bound DNA less accessible for transcription

• DNA methylation: methyl groups added to DNA; tightly packed; transcription

• Histone acetylation: acetyl groups added to histones; loosened; transcription

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Transcription Initiation: • Control elements bind

transcription factors • Enhances gene expression

Transcription Initiation Complex

Enhancer regions bound to promoter region by activators

Regulation of mRNA:

• micro RNAs (miRNAs) and small interfering RNAs (siRNAs) can bind to mRNA and degrade it or block translation

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1. Cell Division: large # identical cells through mitosis

2. Cell Differentiation: cells become specialized in structure & function

3. Morphogenesis: “creation of form” – organism’s shape

Embryonic Development: Zygote Organism

Determination: irreversible series of events that lead to cell differentiation

• Cytoplasmic determinants: maternal substances in egg distributed unevenly in early cells of embryo

• Induction: cells triggered to differentiate

• Cell-Cell Signals: molecules produced by one cell influences neighboring cells – Eg. Growth factors

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Pattern formation: setting up the body plan (head, tail, L/R, back, front)

Morphogens: substances that establish an embryo’s axes

Homeotic genes: master control genes that control pattern formation (eg. Hox genes)

1. Proto-oncogene = stimulates cell division

2. Tumor-suppressor gene = inhibits cell division

• Mutations in these genes can lead to cancer

Control of Cell Cycle: Proto-oncogene Oncogene

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Genes involved in cancer:

• Ras gene: stimulates cell cycle (proto-oncogene)

– Mutations of ras occurs in 30% of cancers

• p53 gene: tumor-suppresor gene

– Functions: halt cell cycle for DNA repair, turn on DNA repair, activate apoptosis (cell death)

– Mutations of p53 in 50+% of cancers

• Cancer results when mutations accumulate (5-7 changes in DNA)

• Active oncogenes + loss of tumor-suppressor genes

• The longer we live, the more likely that cancer might develop

Bacteria vs. Viruses

Bacteria Virus

• Prokaryotic cell

• Most are free-living (some

parasitic)

• Relatively large size

• Antibiotics used to kill

bacteria

• Not a living cell (genes

packaged in protein shell)

• Intracellular parasite

• 1/1000 size of bacteria

• Vaccines used to prevent

viral infection

• Antiviral treatment

Viruses

• Very small (<ribosomes)

• Components = nucleic acid + capsid

– Nucleic acid: DNA or RNA (double or single-stranded)

– Capsid: protein shell

– Some viruses also have viral envelopes that surround capsid

• Limited host range (eg. human cold virus infects upper respiratory tract)

• Reproduce within host cells

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Simplified viral replicative cycle Bacteriophage

• Virus that infects bacterial cells

Lytic Cycle of T4

Phage Bacteriophage Reproduction

• Lytic Cycle:

– Use host machinery to make copies of virus

– Death of host cell by rupturing it (lysis)

– Virulent phages replicate by this method

• Lysogenic Cycle:

– Phage DNA incorporated into host DNA and replicated along with it

– Phage DNA = prophage

• Temperate Phage: uses both methods of replication

Lytic Cycle vs. Lysogenic Cycle

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Animal viruses have a membranous envelope

• Host membrane forms

around exiting virus

• Difficult for host

immune system to

detect virus

Retrovirus

• RNA virus that uses reverse transcriptase (RNA

DNA)

• Newly made viral DNA inserted into chromosome

of host

• Host transcribes viral DNA (= provirus) to make

new virus parts

• Example: HIV (Human Immunodeficiency Virus)

HIV =

Retrovirus

HIV

◦ Infects white blood cells

◦ HIV+: provirus (DNA inserted)

◦ AIDS: active viral reproduction

Vaccines

• Weakened virus or part of pathogen that

triggers immune system response

Emerging viruses = mutation of existing

viruses

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Viroids

• Small, circular RNA

molecules that infect plants

• Cause errors in regulatory

systems that control plant

growth

• Eg. coconut palms in

Philippines

Prions

• Misfolded, infectious proteins that cause

misfolding of normal proteins

• Eg. mad cow disease (BSE),Creutzfeldt-Jakob

disease (humans), scrapie (sheep)

Diseases caused by prions

• Prions act slowly – incubation period of at least 10

years before symptoms develop

• Prions are virtually indestructible (cannot be

denatured by heating)

• No known cure for prion diseases

Tools of Genetic Engineering

Restriction enzymes (restriction endonucleases): used to cut strands of DNA at specific locations (restriction sites)

Restriction Fragments: have at least 1 sticky end (single-stranded end)

DNA ligase: joins DNA fragments

Cloning vector: carries the DNA sequence to be cloned (eg. bacterial plasmid)

Using a restriction

enzyme (RE) and

DNA ligase to

make recombinant

DNA

Gene Cloning

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Applications of Gene Cloning

PCR (Polymerase Chain

Reaction): amplify (copy)

piece of DNA without use

of cells

Gel Electrophoresis: used to separate DNA molecules on

basis of size and charge using an electrical current (DNA

+ pole)

Gel Electrophoresis: used to separate DNA molecules on

basis of size and charge using an electrical current (DNA

+ pole)

Cloning Organisms

• Nuclear transplantation: nucleus of egg is removed and replaced with nucleus of body cell

Nuclear Transplantation

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Problems with Reproductive Cloning

• Cloned embryos exhibited various defects

• DNA of fully differentiated cell have epigenetic changes

Stem Cells

• Stem cells: can reproduce itself indefinitely and

produce other specialized cells

– Zygote = totipotent (any type of cell)

– Embryonic stem cells = pluripotent (many cell types)

– Adult stem cells = multipotent (a few cell types) or

induced pluripotent, iPS (forced to be pluripotent)

Embryonic

vs. Adult

stem cells

Applications of DNA Technology

1. Diagnosis of disease – identify alleles, viral DNA

2. Gene therapy – alter afflicted genes

3. Production of pharmaceuticals

4. Forensic applications – DNA profiling

5. Environmental cleanup – use microorganisms

6. Agricultural applications - GMOs

Gene therapy using a retroviral vector “Pharm” animal: produce human protein

secreted in milk for medical use

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DNA Fingerprinting RFLPs (“rif-lips”)

• Restriction Fragment Length Polymorphism

• Cut DNA with different restriction enzymes

• Each person has different #s of DNA fragments created

• Analyze DNA samples on a gel for disease diagnosis

• Outdated method of DNA profiling (required a quarter-sized sample of blood)

RFLPs – Disease Diagnosis

STR Analysis

• STR = Short Tandem Repeats

• Non-coding DNA has regions with

sequences (2-5 base length) that are repeated

• Each person has different # of repeats at

different locations (loci)

• Current method of DNA fingerprinting used

– only need 20 cells for analysis

STR Analysis STR Analysis

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Human DNA

• 3 billion base pairs

• ~20,000 genes

• Only 1.5% codes for proteins (or RNA)

• Repetitive DNA: sequences present in multiple copies

Transposable Elements

Make up 75% of repetitive DNA

Can be moved from one location to another in genome

Discovered by Barbara McClintock – corn breeding experiments

2 Types:

Transposons

Retrotransposons

Transposons

Moves within genome via DNA intermediate

“cut & paste” or “copy & paste” mechanisms

Requires enzyme transposase

Retrotransposons

Move by means of RNA intermediate

Leaves copy at original site

Involves enzyme reverse transcriptase

Multigene Families

• Collections of 2 or more identical or very similar genes

• Eg. hemoglobin: -globin and -globin gene families

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Transpositions chromosomal rearrangements Transposable elements contribute to evolution

• Promote recombination, disrupt genes or control elements, & carry genes to new locations

• May be harmful or lethal, but can also have small beneficial effects

• Provides raw material for natural selection

Evolutionary Development (Evo-devo)

• Compares developmental processes to understand how changes can lead to evolution of organisms

Homeotic genes: master regulatory genes

• Control placement and spatial organization of body parts

Homeobox: widely conserved 180-nucleotide sequence within homeotic (Hox) genes

• Found in many groups (fungi, animals, plants)

• Hints at relatedness between all life forms

Conservation of homeotic genes

Genetic Diversity in Prokaryotes

Factors:

1.Rapid reproduction (binary fission)

2.Mutations – errors in replication

3.Genetic recombination

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Genetic Recombination in Bacteria

1. Transformation: uptake of foreign DNA from surroundings

2. Transduction: viruses transfer genes between prokaryotes

3. Conjugation: DNA transferred from one to another

Transformation • Uptake of foreign DNA from surroundings

• Observed by Griffith (bacteria & mice)

Plasmids

Small ring of DNA that carries a few genes

Replicates separately from bacterial chromosome

Can carry genes for antibiotic resistance

Used frequently in genetic engineering for gene cloning

AP Bio Lab 6A - Transformation Using plasmids and bacteria in genetic engineering

Transduction

• Viruses (bacteriophages) carry bacterial genes from one host cell to another

• Recombine DNA of donor and recipient cell

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Conjugation

• One cell donates DNA to another

• Donors cell extends a sex pilus (“mating bridge”) through which DNA is transferred

• Requires the presence of a piece of DNA called the F factor to produce the pilus