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The Genes, the Whole Genes, and Nothing But the Genes Jim
KentUniversity of California Santa Cruz
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Ben Franklin - Childhood HeroHi Voltage Experiments
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A Man of High Values
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Early to bedEarly to rise
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Rock Collection
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Shell Collection
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Bottlecap Collection
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Bug Collection
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Jim Kent - Genome Scientist not to be confused with Richard
Stallman
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Modern Bug Collectionif (a = b)
if (string == something)
for (x=0; x
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Naive Biological QuestionsIs an ant an individual?Do dolphins
talk with each other?How come newts and worms can regenerate so
much better than we can?How does a plant grow from a seed, an
animal from an egg?
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Regeneration by NatureAmong vertebrates only amphibians can
regenerate limbs.The process involves dedifferentiation,
repatterning, and growth.Not likely well be able to engineer this
soon.Simpler regenerations though may be tractable and medically
quite important.
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From Egg to Adult in 3x109 BasesA single cell, the fertilized
egg, eventually differentiates into the ~300 different types of
cells that make up an adult body.With a few exceptions all of these
cells contain the full human genome, but express only a subset of
the genes.Gene expression patterns are determined largely by the
cell type, and vice versa.
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From Totempotency to SenilityHuman cells become more and more
specialized during developmentAn egg can become anything.
(Initially most of it will become placenta).Liver cells only become
liver cells.A neuron cant even reproduce.
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DNAmRNAtranscriptionfactorssplicingfactorsstructuralproteinsEnzymesstructuralsugarsstructurallipidsReceptorssignalingmoleculesEnvironment&
other cellscreationregulationPrimary Flows of Information and
Substance in Cell
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An Extreme Case of DedifferentiationThe cloning of Dolly the
sheep showed that a differentiated genome could be reset.An egg is
huge compared to a normal cell. Putting a normal cell into an egg
as Wilmut et al did, swamps out the normal cell transcription
factor and receptors with egg transcription factors and
receptors.Cloning success rate sometimes improved by passing a
nucleus through multiple eggs.
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Human Diseases Involving a Small Population of CellsParkinsons -
from the death of dopamine producing neurons in the substantia
negra. Macular degeneration - a leading cause of blindness in the
elderly.Type I Diabetes - from the death of insulin-producing cells
in the pancreas.
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Pancreas Differentiation PathwayFrom Huang Tsai, J Biomed Sci
2000:7:27-34 and Jensen et al, Diabetes 2000:49 163-176
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Cell Type DeterminantsCell type of parent cell.Interactions with
other cells.Interactions with the extracellular environment.
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Flexibility of Stem CellsIn many cases stem cells are flexible
enough that putting them into a particular tissue will cause them
to differentiate into the type of cells that make up that tissue.At
low levels bone transplanted bone marrow (blood stem cells)
develops into neuron in stroke victims!Making this happen at high
enough levels to be useful will likely require some
engineering.
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DNAmRNAtranscriptionfactorssplicingfactorsstructuralproteinsEnzymesstructuralsugarsstructurallipidsReceptorssignalingmoleculesenvironmentother
cellscreationregulationPrimary Flows of Information and Substance
in Cell
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To Understand the Body NeedThe genomeA comprehensive list of
genesGene expression dataProtein localization in
cellProtein/protein and protein/DNA interaction information. Ways
to store, display and query masses of data so human investigators
can focus on relevant bits.Many talented and hardworking human
investigators.
- Where are we now?The genome >95% complete. 98% complete in
April.A comprehensive list of genes - ~75% of coding regions.
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The GenesIdentifying genes is a prerequisite for a great deal of
other research.Expression microarraysIn situ mRNA
hybridizationProducing proteins for cellular localization
experimentsEtc.
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The Whole GenesThe full gene including the 5 and 3 UTRs are
critical forAvoiding misleading fragmentation/fusion
artifacts.Understanding mRNA targeting and stabilityFinding
transcription factor binding sitesUnderstanding the regulatory
networks that drive and maintain cell differentiation.
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Nothing But the GenesExperimental analysis is expensive.Unreal
genes can mislead:Analysis of multiple alignments to look for
active sites etc.Protein classification systems and phylogeniesOne
bogus gene can lead to another as much annotation is done via
homology.
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Methods of Identifying GenesmRNA/cDNA sequencingMicroarrays
covering entire genomeGenetics in model organismsCross species
protein homologyCross species genomic homologyHMM and other purely
computational genefinding.
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cDNA SequencingExtract RNA from cells.Use reverse transcriptase
and a poly-U primer to convert to RNA starting at poly-A
tail.Insert cDNA into vectors that grow in E. coliSequence a read
from one or both sides of insert using primers on vectorIf EST
looks to be new sequence full cDNA.Artifacts and limitations are
possible at each stage!
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Common cDNA Problems & SolutionsFor rarely expressed genes
little RNA is available.Normalize libraries. Use embryonic and
exotic tissues as mRNA source.Splicing is not instantanious, can
get retained introns.Spin out nuclei and just use cytoplasmic
mRNAAlign to genome and look for splicingReverse transcriptase
falls off before its finishedPreferentially taking larger
cDNAs.G-cap selected libraries (Sugano)Normalizing only on 5 ends
(Soares)
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More cDNA Problems & SolutionsReverse transcriptase has a
high error rate and is prone to small deletions.Compare cDNA to
genomic DNASequence multiple cDNA clonesAt a low level cell seems
to tolerate a certain degree of nonsense transcription and
splicing. Normalizing increases concentration of these as well as
of rare genes.Ignore everything thats not coding (ouch)???
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cDNA Status & Summary~10,000 cDNA sequence have been
accumulated over years by various labs working on gene families and
pathways.Riken project has ~33,000 unique cDNAs in mouse. ~11,000
of these seem to have retained introns. ~3,000 are noncoding
antisense. ~70% include initial ATGMammalian Gene Collection (MGC)
has ~15,000 human cDNAs with initial ATGs. Having to resort to
exotic libraries and RT-PCR to get more.Human refSeq has ~18,000
human cDNAs.
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Whole Genome MicroarraysPerlegen and Affymetrix are making
microarrays that cover entire non-RepeatMasker masked genome.
Results on chromosome 21 and 22 published.Based on 25-mers.Rarely
expressed genes may not stand out above background.Have to cope
with cross-hybridization issues, GC content, etc.Advantages - no
homology required, can sense lower concentrations of mRNA than
random EST sequencing.
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Cross-hybridization at WorkZoomed in on right side:
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Genetics in Model OrganismsZap hapless yeast, worms, flies, and
mice Inbreed offspring and look for twisted ones.Advantages:Works
at DNA level, so expression level doesnt matterYou get hints of
function right away.Can look for gene interactions simply by
breeding mutants.Disadvantages:Finding which DNA is mutated can
take a long time.Essential genes can be hard to find - all you see
is reduced fertility in the inbreeding stage.Genes only needed in
certain environments and duplicated genes may be missed in
screens.
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Cross Species Genome ComparisonsMutations occur more or less
randomly across genome butMutations in functional areas tend to be
weeded out by selectionIn comparing DNA across species, the
functional areas are more conserved than the nonfunctional areas in
general
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Of Fish and Mice and Men
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Comparative Genomics at BMP10
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Conservation of Gene Features Conservation pattern across 3165
mappings of human RefSeq mRNAs to the genome. A program sampled 200
evenly spaced bases across 500 bases upstream of transcription, the
5 UTR, the first coding exon, introns, middle coding exons,
introns, the 3 UTR and 500 bases after polyadenylatoin. There are
peaks of conservation at the transition from one region to
another.
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0.
