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Chapter 19.

Control of Eukaryotic Genome

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The BIG Questions…How are genes turned on & off in

eukaryotes?

How do cells with the same genesdifferentiate to perform completelydifferent, specialized functions?

How have new genes evolved?

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Prokaryote vs. eukaryote genome Prokaryotes

small size of genome circular molecule of naked DNA most of DNA codes for protein or RNA

small amount of non-coding DNA regulatory sequences promoters, operators

prokaryotes use operons to regulate gene transcription, howevereukaryotes do not.

since transcription & translation are fairly simultaneous there is littleopportunity to regulate gene expression after transcription, so controlof genes in prokaryotes really has to be done by turning transcriptionon or off.

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Prokaryote vs. eukaryote genome Eukaryotes

much greater size of genome DNA packaged in chromatin fibers

regulates access to DNA by RNA polymerase cell specialization

need to turn on & off large numbers of genes most of DNA does not code for protein or RNA

97% in humans = “junk DNA”

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Points of control The control of gene expression

can occur at any step in thepathway from gene tofunctional protein unpacking DNA transcription mRNA processing mRNA degradation translation protein processing protein degradation

Turning on Gene movie

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Gene expression Specialization

each cell of a multicellular eukaryoteexpresses only a small fraction of its genes

Development some gene expression must be controlled on

a long-term basis for cellular differentiation &specialization during development

Responding to organism’s needs cells of multicellular organisms must

continually turn certain genes on & off inresponse to signals from their external &internal environment

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DNA packingCurrent modelfor stages ofDNA coiling &folding culminating

in condensedmetaphasechromosome

nucleosomes “beads on a string” 1st level of DNA packing histone proteins have high proportion of positively charged amino

acids (arginine & lysine) bind tightly to negatively charged DNA

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Nucleosomes “Beads on a string”

1st level of DNA packing histone proteins have high proportion

of positively charged amino acids(arginine & lysine) bind tightly to negatively charged DNA

DNA packing movie

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DNA packing & methylation Chromatin modifications affect the

availability of genes for transcription packing DNA into compact form serves

to regulate transcription heterochromatin is compact & not transcribed less compacted euchromatin may be

transcribed DNA methylation inactivates genes = off

attachment of methyl groups (–CH3) to DNAbases (cytosine) after DNA is synthesized nearly permanent suppression of genes ex. the inactivated mammalian X

chromosome

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Histone acetylation Histone acetylation activates genes = on

attachment of acetyl groups (–COCH3) tocertain amino acids of histone proteins when histones are acetylated they change shape &

grip DNA less tightly = unwinding DNA transcription proteins have easier access to genes

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Chromatin structure Chromatin structure is part of gene

regulation heterochromatin

highly compact not transcribed found in regions of few genes

euchromatin “true” chromatin transcribed found in regions of

many genes

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Heterochromatin Heterochromatin

highly compact not transcribed (inactive genes)

increased methylation of histones & cytosines decreased acetylation of histones

found in regions of few genes centromeres telomeres other “junk” DNA

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Euchromatin Euchromatin

loosely packed in loops of 30nm fibers near nuclear pores

transcribed “active” genes decreased methylation of histones & cytosines increased acetylation of histones

found in regions of many genes

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Transcription initiation Transcription factors

control proteins which interact with DNA & witheach other

initiation complex only when complete complex has assembled can RNA

polymerase move along DNA template to make mRNA

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Model for Enhancer action

Enhancer DNA sequences distant control sequences

Activator proteins bind to enhancer sequence &

stimulates gene transcriptionSilencer proteins

bind to enhancer sequence &block gene transcription

Much of molecular biology research is trying to understand this: theregulation of transcription

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Post transcriptional control Alternative RNA splicing

variable processing of exons creates afamily of proteins

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Regulation of mRNA degradation Life span of mRNA determines pattern

of protein synthesis mRNA can last from hours to weeks

RNA processing movie

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Control of translation Block initiation stage

regulatory proteins attach to 5’ end ofmRNA prevent attachment of ribosomal subunits

& initiator tRNA block translation of mRNA to protein

Control of translation movie

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Protein processing & degradation Protein processing

folding, cleaving, adding sugar groups,targeting for transport

Protein degradation ubiquitin tagging & proteosome

degradation

Protein processing movie

The cell limits the lifetimes of normal proteins by selectivedegradation. Many proteins, such as the cyclins involved in regulatingthe cell cycle, must be relatively short-lived.

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Cancers = cell cycle genes Cancer results from genetic changes

that affect the cell cycle proto-oncogenes

normal cellular genes code for proteins thatstimulate normal cell growth & division

oncogenes mutations that alter proto-oncogenes cause

them to become cancer-causing genes

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Proto-oncogenes & Oncogenes Genetic changes that can turn

proto-oncogenes into oncogenes removing repression of genes

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Cancers = failures of regulation Cancer cells have escaped cell cycle

controls do not respond normally to the body’s

control mechanisms divide excessively & invade other tissues if unchecked, they can kill the organism

growth &metastasis ofmalignantbreast tumor

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Effects of signal pathways

Signaling pathways that regulate cell growth: Both stimulatory &inhibitory pathways regulate the cell cycle. Cancer can result fromaberrations in either.

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Multi-step model for cancer Multiple mutations underlie the development

of cancer several changes must occur at DNA level for

cell to become fully cancerous including at least 1 active oncogene & mutation or

loss of several tumor-suppressor genes telomerase is often activated

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p53 gene “Guardian of the Genome”

the “anti-cancer gene” after DNA damage is detected, p53

initiates: DNA repair growth arrest apoptosis

almost all cancershave mutations in p53

The cancer-fighting p53 molecule has a commanding presenceinside our cells, coordinating the actions of more than 60 genes toprevent a damaged cell from developing into a malignant tumor.The p53 molecule detects DNA damage or other deadly stress in thecell and either commands the cell to stop growing so the damagecan be repaired or enacts the cell's suicide plan (apoptosis).Though p53 gives instructions to more than 60 genes, not all of thegenes are turned on every time p53 is active. Some genes are onlyturned on when p53 stops cell growth; others are only turned onwhen p53 starts apoptosis, the cell's suicide program.

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P53 re-enforces G2 checkpoint= “tumor suppressor” protein

Mutant p53 can no longer bind DNAtherefore p21 protein is not triggeredas 'stop signal' for cell division = cellsdivide uncontrollably & form tumors

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Repetitive DNARepetitive DNA & other noncoding sequencesaccount for most of eukaryotic DNA

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Genetic disorders of repeats Fragile X syndrome

most common form ofinherited mental retardation

defect in X chromosome mutation of FMR1 gene causing many

repeats of CGG triplet in promoter region 200+ copies normal = 6-40 CGG repeats

FMR1 gene not expressed & protein (FMRP) not produced function of FMR1 protein unknown binds RNA

FMR1 = Fragile X Mental Retardation geneFMRP = Fragile X Mental Retardation Protein

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Fragile X syndrome The more triplet repeats

there are on the Xchromosome, the moreseverely affected theindividual will be mutation causes

increased number ofrepeats (expansion) witheach generation

A summary of existing research conducted by the Centers forDisease Control and Prevention in 2001 estimated thatapproximately one in 3,500 to 8,900 males is affected by the fullmutation of the FMR1 gene, and that one in 1,000 males has thepremutation form of the FMR1 gene. This study also estimated thatone in 250 to 500 females in the general population has thepremutation. Another study6 estimated that one in 4,000 females isaffected by the full mutation.

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Huntington’s Disease Rare autosomal dominant degenerative

neurological disease 1st described in 1872 by Dr. Huntington most common in white Europeans 1st symptoms at age 30-50

death comes ~12 years after onset Mutation on chromosome 4

CAG repeats 40-100+ copies normal = 11-30 CAG repeats CAG codes for glutamine amino acid

When the HD gene was found, researchers discovered that HDbelongs to a newly discovered family of diseases called "tripletrepeat" diseases. A gene's DNA bears coding molecules which aretranslated into specific amino acids, the building blocks of proteins.In the HD gene, the coding molecules cytosine, adenine, andguanine (CAG) - the triplet - repeat in a stretch more than they do inthe normal gene. Normally the CAG sequence repeats between 11and 30 times. People with HD may have CAG repeated between 36and 125 times. The onset of the disease generally is in the 30s and40s (with a range of age 2 to 82), but more than 60 repeats of CAGoften are associated with an onset of HD before age 20. With themajority of people who have fewer than 60 repeats, there is greatvariability. A person can have 40 CAG repeats in his or her DNAsequence and have onset at age 17 or 70. On average, however, afeature dubbed "genetic anticipation" occurs. Each time the unstableDNA is passed on to offspring, the affected person experiences anearlier onset.

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Huntington’s disease Abnormal (huntingtin) protein produced

chain of charged glutamines in protein bonds tightly to brain protein, HAP-1

Woody Guthrie

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Families of genes Human globin gene family

evolved from duplication of commonancestral globin gene

Different versions areexpressed at differenttimes in developmentallowing hemoglobin tofunction throughout lifeof developing animal

This evolution occurred long ago. All vertebrateshave multiple globin genes & most mammals havethe same set of globin genes.Different versions of each globin subunit areexpressed at different times in development, allowinghemoglobin to function effectively in the changingenvironment of the developing animal.

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Interspersed repetitive DNA Repetitive DNA is spread throughout

genome interspersed repetitive DNA make up

25-40% of mammalian genome in humans, at least 5% of genome is

made of a family of similar sequencescalled, Alu elements 300 bases long Alu is an example of a "jumping gene" –

a transposon DNA sequence that"reproduces" by copying itself & insertinginto new chromosome locations

5% of genome = millions of copies of this sequence!Alu doesn’t seem to be active anymore. No useful function toorganism.Used to study population genetics = relatedness of groups of people

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Rearrangements in the genome Transposons

transposable genetic element piece of DNA that can move from one

location to another in cell’s genome

One gene of an insertion sequence codes for transposase, which catalyzes thetransposon’s movement. The inverted repeats, about 20 to 40 nucleotide pairs long, arebackward, upside-down versions of each oth. In transposition, transposase moleculesbind to the inverted repeats & catalyze the cutting & resealing of DNA required forinsertion of the transposon at a target site.

One gene of an insertion sequence codes for transposase, whichcatalyzes the transposon’s movement. The inverted repeats, about20 to 40 nucleotide pairs long, are backward, upside-down versionsof each other. In transposition, transposase molecules bind to theinverted repeats & catalyze the cutting & resealing of DNA required forinsertion of the transposon at a target site.

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TransposonsInsertion oftransposonsequence in newposition in genome

insertion sequencescause mutationswhen they happen toland within thecoding sequence of agene or within a DNAregion that regulatesgene expression

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Transposons Barbara McClintock

discovered 1st transposons in Zeamays (corn) in 1947

1947|1983

She found that transposons were responsible for a variety of types ofgene mutations, usually insertions deletions and translocations

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Retransposons Transposons actually make up over 50% of the

corn (maize) genome & 10% of the humangenome.

Most of thesetransposons areretrotransposons,transposable elementsthat move within agenome by means ofRNA intermediate,transcript of theretrotransposon DNA