Lecture

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Lecture 12Genomania (cont.)Wed., March 5, 2002
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• EST Expressed sequence tags. These are simply
  STS derived from cDNA (from polyA mRNA)
• -ESTs can be aligned on the contig map thus
  nailing down a GENE to the physical map.
• -Note From ESTs one can derive ORFs to look
  into function and expression of gene
• -ESTs make great reagents to probe the
  transcriptome of the cell (more later)
• CLONING of THE Y CHROMOSOME IS DESCRIBEDPLS.
  READ OVER. Successful cloning of larger genomes
  is based upon STS/contig alignments.
• GENOME PROJECTS Two strategies employed.
• WHOLE GENOME SHOTGUN SEQUENCING Random
  shearing/sampling of genome, sequence all, feed
  into computer reconstruct contigs into large
  scaffolds of sequence and order the position of
  the reads. Then map to chromosomes using
  conventional mapping methods.
• BAC by BAC approach Make a library of
  overlapping BACs grow and characterize each
  clone. Rather ineffiecient

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• Genome Maps for Genetic Analyses
• Maps as starting points for gene isolation and
  functional genomics.

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• Gene Isolation by positional cloning Example of
  the gene for CF.
• Gene localized on Chm 7 (linkage/marker data).
  Chm 7 flow sorted out to make a library.
• With other markers the gene thought to be in
  1.5cM segment (still large ca. 1.5 megabases or
  million bp). Needed to walk across vast stretch
  of uncharted DNA
• Relied heavily on chromosome walking/jumping.
  Recallwalking

Chm. Ends are used to probe library to obtain
additional overlapping (contiguous) clones that
walk you into new loci.
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Related to Chromosome Walking Jumping.
-Advantage Can rapidly traverse genome
segments containing repeat DNA sites that may be
difficult to clone

• Large frags. Cloned by RE partials (original
  library)
• Cut and circularize inserts, to bring ends
  together, re-cut and make new library of the
  junction fragments JL jumping library
• Screen JL with clone having one end (starting
  point) of sequence of interest. When clone is
  found, recover opposite end from this clone and
  rescreen the JL
• This jumps you to the next segment (5 and 3)
• Gives bidirectional search of linked regions
  that can be recovered, sequenced and ORFed by
  computers and bioinformaticsNEXT SLIDE

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• Once you get the region (megabases) mapped and
  sequenced.now start a gene hunt
• Promoters often decorated with CpG islands (a
  motif search can find this)
• Look for ORFs, homologies to start/stop signals,
  poly A sites, splice junctions etc.
• Sequence the cDNA clone with normal and CF
  patient. Look for differences (mutation
  found)should be conserved among many different
  patients or normal controls.
• Correct the defect in cell culture by
  transformation with WT gene (verification)
• GENE THERAPY based rescue?
• READ OVER orphan genes and the candidate gene
  approach to gene isolation and review
  quantitative trait loci (QTL).
• QTL phenotypic variation is due to or
  allelic effects and is complex (vs. simple).

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• Functional Genomics How are we going to analyze
  the HG? How can we mine data?
• Characterize the proteome by ORF hunts
• Computers search out ORFs based 6 reading frames
  that display AUG initiation codon and a stop
  codon (any ORF 100 residues is a hit).
• Many ORFs will be novel. How to learn more about
  a new ORF?
• Computer homology searches to known (conserved)
  genes.
• Learn more about gene function from this
  comparison
• Location, orientation and clustering of genes in
  the proteome is important information for a given
  genome.
• Facilitates mapping new genes
• Examine significance of chromosomal
  translocations.
• D. Example of yeast proteome.
• -Genetic landscape of chm 3
• BLUE shows clones used in sequencing.
• GREEN, previously known gene from phenotype
• ORANGE, ORFs that are likely to be genes
  (examine with knock outs note that many of these
  will not have any phenotype)

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PtnPtn interactions in vivo are important. (Key
factors tend to aggregate together in vivo if
they are related in control or function) ENTER
Yeast 2 hybrid system to find interactive
partners in vivo. BASIS if GAL4 activation
domain and GAL4 DNA binding domain come together,
the GAL4 promoter will be activated and reporter
gene will be elevated.
A Bait gene you are trying to find its
interactive partners
Mix Bait with expression library and identify
clones that have active LACZ gene.
This is a screening method to search for clones
that show high level of LACZ activity. Positives
indicate a physical interaction between the bait
gene and a new target.
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Gene array analsyses DNA array methods dissect
out gene expression programs to learn about a
given biological process. Example Comparing
normal cells with tumor cells. Here is how it
works Isolate total polyA RNA (mRNA) from
cells being analyzed. Convert to cDNA using
reverse transcriptase and radioactive or
fluorescent tagged nucleotide. Hybridize to
oligos on filters, slides or some other chip
medium. Quantify signals as or between
controls. In theory Could set up every exon
from human genome onto array slides -Estimated
31,000 genes x average of 7 exons/gene -Total
217,000 exons to interrogate -each oligo probe
has 60 nucleotides -each residue 0.05 to
0.12. -Total coverage 1.5million for all
probes gets pricey! Want more infor? See
www.microarrays.org web site.
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Robotic Multiple tips deliver microdroplets
(ul) of known volumes (oligos/cDNAs) onto
specific positions (addresses) on positively
charged surfaces. -Possibly Place entire
transcriptome on slide. -Prepare mRNA from
source and reference fluorescent labels and
hybridize Goal to identify regulatory circuits or
large networks of coordinate regulation.
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Red Active genesGreen Inactive.
Red highest levels of expression at that
addressBlue Lowest level
Look for patterns and clusters
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The Transcriptional Program in the Response of
Human Fibroblasts to Serum Vishwanath R. Iyer,
Michael B. Eisen, Douglas T. Ross, Greg Schuler,
Troy Moore, Jeffrey C. F. Lee, Jeffrey M. Trent,
Louis M. Staudt, James Hudson Jr., Mark S.
Boguski, Deval Lashkari, Dari Shalon, David
Botstein, Patrick O. Brown The temporal
program of gene expression during a model
physiological response of human cells, the
response of fibroblasts to serum, was explored
with a complementary DNA microarray representing
about 8600 different human genes. Genes could be
clustered into groups on the basis of their
temporal patterns of expression in this program.
Many features of the transcriptional program
appeared to be related to the physiology of wound
repair, suggesting that fibroblasts play a larger
and richer role in this complex multicellular
response than had previously been appreciated.
Science 283 83-87 (1999)
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