EUKARYOTIC GENE EXPRESSION

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EUKARYOTIC GENE EXPRESSION
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• DNA PACKING
• Histones
• Nucleosomes (1st)
• 30nm fibers (2nd)
• Looped domains (3rd)
• Heterochromatin
• not transcribed,
• metaphase chromatid
• Euchromatin open,
• actively transcribed
• Chromosome location specific

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GENOME ORGANIZATION - DNA LEVEL

• Repetitive DNA (tandem repeats)
• 10-15 in mammals
• short sequences (10) repeated in series,
  100,000s times
• Fragile X 100s instead of 30 triplet repeats
• Huntingtons CAG repeats
• of repeats correlates with severity and age of
  onset.
• Regular, mini micro satellites
• Interspersed Repetitive DNA
• Scattered, 25-40, similar but not identical
• Alu elements family of sequences 5 primate
  genome, about 300 bp, many transcribed most
  transposons

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Telomeres centromeres
Useful in fingerprinting
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Chromosome puff
MULTIGENE FAMILY Collection of similar or
identical genes
Salamander RNA 3 kinds of rRNA after
processing, S-sedimentation Rates due to
differences in density
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NON-IDENTICAL MULTIGENE FAMILY 2 related families
that code for globins
4 subunits, 2a 2ß
Nonfunctional, very similar to functional genes
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• Gene Amplification the temporary increase in the
  number of copies of a gene
• rRNA in amphibians
• rRNA in developing ovum, large of ribosomes ?
  burst of protein synthesis after fertilization
• Extra copies cannot replicate are broken down
• Selective Gene Loss occurs in certain insects,
  whole chromosomes or parts of chromosomes may be
  lost early in development.

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REARRANGEMENTS

• Transposons can prevent normal functioning, may
  ? or ?production, or be activated, 10 of human
  genome
• Retrotransposons use an RNA intermediate
• Immunoglobulin Genes code for antibodies, genes
  become rearranged as immune cells differentiate

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Retrotransposon Movement like retrovirus
reproduction, can populate the genome in huge
numbers
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DNA Rearrangementmaturation of an immunoglobulin
gene
The joining of V, J C regions of DNA in random
combinations ? enormous variety of
antibody-producing lymphocytes

• Hundreds of V regions
• Several Junction regions
• 1-2 Constant regions

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• CONTROL OF GENE EXPRESSION
• Cellular differentiation divergence in form
  function as cells become specialized during
  development
• 3-5 expressed at any given time (liver vs skin)
• DNA Methylation genes not expressed, methyl
  group (CH3) attached
• Barr body heavy methylation, inactive X,
  heterochromatin
• Genomic Imprinting turning off alleles
• Histone Acetylation acetyl groups on histone
  a.a. causing shape change, grip DNA less tightly,
  easier access to genes for transcription

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OPPORTUNITIES FOR CONTROL OF GENE EXPRESSION

1. DNA packing, methylation, acetylation
2. Transcription (most important)
3. RNA Processing
4. Transport to cytoplasm
5. Degradation of mRNA
6. Translation
7. Cleavage, Chemical modification, Transport to
   cellular destination
8. Degradation of protein

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TRANSCRIPTIONAL CONTROLS

• Transcription factors
• Equivalent to repressors activators
• Bind to specific sites (TATA box)
• May be near promoter
• Optimum binding 50 ish factors
• read DNA without unzipping to find appropriate
  gene (zinc fingers leucine zippers)
• Promoter, Enhancer, Suppressor sequences

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Transcription initiation controlled by
transcription factors (proteins) that interact w/
DNA each other. Typical Eukaryotic Gene
promoter, terminator, distal proximal control
elements (key to high levels of transcription)
Promoter regions bind to RNA polymerases I,II,III
(tRNAs)
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Model for enhancer action
Activator proteins bind to enhancer sequences
DNA bends, activators closer to promoter
Protein-binding domains attach to transcription
factors, form active transcription initiation
complex on promoter
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DNA Binding Domain 3-D part of a transcription
factor that binds to DNA
Found in many regulatory proteins
a helix ß sheet held by zinc atom
2 a helices w/ spaced leucines coil
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POST-TRANSCRIPTIONAL CONTROLS

• mRNA processing mG cap? poly A tail?
• Alternative splicing same primary transcript,
  different introns exons
• mRNA degradation prokaryotic mRNAs last a few
  minutes eukaryotic- hours, can be days even
  weeks (hemoglobin)
• Translation inhibited by masked mRNA prior to
  fertilization (activated in embryo)
• Protein processing degradation

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ALTERNATIVE mRNA SPLICING
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PROTEIN DEGRADATION
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EUKARYOTIC vs PROKARYOTIC

• Genes spread out
• 1 promoter 1 gene
• Many introns
• Processing
• 2 copies of DNA (2n)
• Paired, rod shaped chrom.
• 3 polymerases
• Nucleus, separation of transcription/translation

• Operons
• 1 promotermultiple genes
• Lack introns
• No processing
• 1 copy of DNA (n)
• Single circular chrom.
• 1 polymerase
• No nucleus, simultaneous transcription/translation
  

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CANCER

• Mutations in genes that regulate growth
  division, chemical carcinogens, physical mutagens
  (X-rays, viruses)
• Oncogenes cancer causing
• Proto-oncogenes normal gene ? oncogene
• Tumor-suppressor genes prevent uncontrolled
  division
• ras gene proto-oncogene
• p53 gene tumor suppressor gene

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Proto-oncogenes ? Oncogenes
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• ras gene
• G protein
• Relays growth signal
• Result stimulation of cell cycle

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p53 transcription factor, activates p21,
product blocks CDKs
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MODEL DEVELOPMENT OF COLORECTAL CANCER
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DNA TECHNOLOGY
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TERMINOLOGY Recombinant DNA DNA in which genes
from two different sources are combined in vitro
into the same molecule. Genetic engineering
direct manipulation of genes for practical
purposes. Biotechnology manipulation of
organisms or their components to make useful
products. Gene cloning method for preparing
well defined, gene sized pieces of DNA in
multiple identical copies.
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BACTERIAL PLASMIDS FOR CLONING
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• RESTRICTION ENZYMES
• Cut up foreign DNA.
• Endonucleases
• RESTRICTION SITE
• Recognition sequence
• Usually symmetrical
• RESTRICTION FRAGMENT
• Piece of DNA cut by specific enzyme
• STICKY END
• Single strand end of restriction fragment

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Cloning a human gene in a bacterial plasmid
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USING A NUCLEIC ACID PROBE TO IDENTIFY A CLONED
GENE Cloned gene of interest on a plasmid, probe
is short length of radioactive single stranded
DNA complimentary to part of gene. 2) Result
single stranded DNA stuck to filter paper 3)
Probe DNA hybridizes with complimentary DNA on
filter 4) Filter laid on photographic film,
radioactive areas expose film (autoradiography)
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MAKING COMPLEMENTARY DNA (cDNA) FOR A EUKARYOTIC
GENE
Expression vector - Cloning vector w/ prokaryotic
promoter upstream of restriction site where
eukaryotic gene can be inserted. cDNA made in
vitro using mRNA template reverse transcriptase
(DNA w/o introns)
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TERMINOLOGY
Yeast artificial chromosomes (YACs) vectors
that combine the essentials of a eukaryotic
chromosome ( origin for replication, centromere,
2 telomeres) w/ foreign DNA. Electroporation
application of electric pulse to solution
containing cells creating temporary hole in
membrane allowing DNA to enter. Genomic library
complete set of thousands of recombinant plasmid
clones, each carrying copies of a particular
segment from the initial genome. cDNA library
library containing a collection of genes
(represents only part of a cells genome only
the genes that were transcribed in the starting
cells)
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Shown - 3 of the thousands of books in the
library. Each is a bacterial clone containing
one particular variety of foreign genome fragment
in its recombinant plasmid
The same 3 foreign genome fragments in a phage
library
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PCR

• Polymerase chain reaction
• Technique to quickly amplify (copy many times) a
  piece of DNA w/o using cells
• Start w/ double stranded DNA (target), add to
  polymerase, nucleotide supply primers
• 5 minutes per cycle

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DNA ANALYSIS GENOMICS Genomics the
study of whole sets of genes and their
interactions Gel electrophoresis separates
macromolecules (nucleic acids or proteins) based
on size, electrical charge and other physical
properties. Southern blotting hybridization
technique that enables researchers to determine
the presence of certain nucleotide Sequences in
a sample of DNA. Restriction fragment length
polymorphisms RFLPs - differences in DNA sequence
on homologous chromosomes that can result in
different restriction fragment patterns.
Scattered abundantly throughout genomes
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GEL ELECTROPHORESIS
Negatively charged DNA migrates toward positive
electrode. Longer fragments travel more slowly
DNA samples arranged in bands along a lane
according to size. Shorter fragments travel
farthest
3 DNA samples placed in wells. Electrodes
attached voltage applied
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Using restriction fragment patterns to
distinguish DNA from different alleles

1. 2 homologous segments w/ different alleles
2. Electrophoresis separates the fragments allele
   1has 3 fragments, allele 2 has 2
3. Addition of binding dye, fragments fluoresce
   pink shown 6 samples cut w/ a restriction enzyme

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RESTRICTION FRAGMENT ANALYSIS BY SOUTHERN BLOTTING
DNA denatured transferred to paper
Radioactivity exposes film, image forms bands
w/ DNA base-pairs w/ probe
Probe complimentary to gene of interest
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• MAPPING GENOMES AT THE DNA LEVEL
• Human Genome Project effort to map the entire
  human nucleotide sequence for each chromosome.
• Genetic (Linkage) Mapping construction of a
  linkage map using various genetic markers.
• Physical Mapping Ordering DNA Fragments
• chromosome walking make fragments that overlap,
  then use probes of the ends to find the overlaps
• Bacterial artificial chromosome (BAC) artificial
  version of a bacterial chromosome that can carry
  inserts of 100,000 500,000 base pairs
• DNA Sequencing determining the nucleotide
  sequence of a DNA segment or an entire genome. 3
  sequencing methods
• Alternative Approaches to Whole-Genome Sequencing

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Chromosome walking

• Prepare probe to match 3 end
• Cut starting DNA w/ 2 restriction
• enzymes clone fragments
• 3) Use probe 1 to screen library II
• for DNA fragments that overlap the
• known gene
• 4) Isolate DNA from tagged clone,
• prepare probe 2 to match 3 end of
• that segment.
• 5) Use probe 2 to screen library I for
• an overlapping fragment farther along
• 6) Repeat 45 with new probes
• Alternating libraries to walk down DNA
• 7) Result DNA map w/ series of
• known markers (sequences) in a
• Known order separated by known
• distances

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SANGER METHOD

• 4 portions each incubated w/
• primer
• DNA polymerase
• 4 deoxyribonucleoside triphosphates dATP, dGTP,
  dCTP, dTTP
• Different one of the 4 nucleotides in modified
  dideoxy (dd) form

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• 4 portions each incubated w/
• primer
• DNA polymerase
• 4 deoxyribonucleoside triphosphates dATP, dGTP,
  dCTP, dTTP
• Different one of the 4 nucleotides in modified
  dideoxy (dd) form

Synthesis of new strands begins at primer
continues until a dideoxyribonucleotide is
incorporated, which prevents further synthesis.
SANGER METHOD
SANGER METHOD
Eventually, a set of labeled strands of various
lengths is generated.
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SANGER METHOD
New strands separated by electrophoresis
Sequence can be read from bands on autoradiograph
and original template sequence deduced. Longest
fragment ends with a ddG, so G must be the last
base in the sequence
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ALTNERATIVE STRATEGIES FOR SEQUENCING AN ENTIRE
GENOME - The arrangement of DNA fragments in
order depends on their having overlapping regions
Cut DNA of chromosome into small fragments
Clone fragments in plasmid or phage
vectors Sequence fragments
Assemble overall sequence
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DNA microarray assay for gene expression
Researcher simultaneously test all the genes
expressed in particular tissue for hybridization
with an array of short DNA sequences representing
thousands of genes. Fluorescence intensity
indicates relative amount of mRNA in tissue.
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Practical Applications of DNA Technology

• Diagnosis of Diseases PCR labeled probes
• Human Gene Therapy replacing defective genes
• Pharmaceutical Products vectors, vaccines
• Forensics - DNA fingerprinting, simple tandem
  repeats
• Environmental Uses mining, sewage treatment,
  detoxifying microbes (oil spills etc.)
• Agricultural Uses transgenic organisms, pharm
  animals, Ti plasmid, herbicide resistant crops
• Genetically Modified Organisms safety ethical
  questions

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Using RFLP markers to detect presence of disease
causing alleles
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Gene Therapy
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DNA Fingerprinting
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Using the Ti plasmid as a vector for genetic
engineering