Senogles 2 and 3

Senogles 2

Correction for Yesterday:
Pseudoautosomal Inheritance
Erratum for yesterday’s lecture: pseudoautsomal domains include only the ends
Y linked is also holandric inheritance. No daughter carriers. Pseudoautosomal has carrier daughters.

Ways to map genome:
Complementary and tell you different things. Cytogenetic mapping today, then genetic mapping and physical mapping. Genetic is mapping by inheritance. Physical map is the sequence itself. Cytogenetic map is chromosome.
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Get this. Be able to understand what mapping CF by RFLP means.
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Chromosomes as spread here have 2 arms. Nomenclature is standard and based on Giemsa staining.
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R banding is reversed.
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position is defined unequivocally in international nomenclature.
Ideograms are based on G banding of actual chromosomes.
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Pattern FYI
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female pattern. These gross analyses are used for a 1st test for a lot of chromosomal abnormalities.
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Know that abnormalities exist. She does not care if you know details. Know they can be seen using cytogenetic methods.
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Aneuploidy
Nondisjunction can occur – chromosomes do not separate in first round- one cell gets none, one gets both chromosomes. Gametes still function and can fertilize or be fertilized. If nondisjunction at second round, normal complement for 2 cells,a diploid, and a zero complement. Know that aneuploidy happens because separation does not occur properly.
Outcome- trisomy 13,18,21 are viable, but bad. Many other trisomies are lethal. XXX not as devastating. Monosomy X is Turner’s syndrome- one of few monosomies to survive.
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Rearrangements occur with DS breaks. Repair system usually correct these breaks. Balanced- swapping pieces of DNA- no removal or deletion.
Unbalanced- duplicate or loss- serious potential effects.
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Possible changes. Squashes or FISH can detect these changes in order. Most of the time these are silent.
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Reciprocal translocations- no net loss or gain, just change in location. This is not recombination. This is translocation between different chromosomes.
May disrupt gene function if break is between promoter and gene. May be silent.
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Nonreciprocal translocation- adds chromosomal material to another chromosome.
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Tandem duplication results in repitition of segment.
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Have knowledge of FISH analysis. #1 for cytogenetic analysis. Can “paint” whole chromosome or other things listed. Human chromosome has lots of repeat sequence. SSRs are inherited markers.
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FISH probe is complementary only to a region of the chromosome. Family of probes can be made to entire chromosome.
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Whole chromosome painting- probe each chromosome with a different dye emitting at different wavelength for different color. Can see deletions, increase in chromosome size.
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Chromosome 19 has unbalanced rearrangement to attach to another chromosome.
She likes FISH analysis.
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Genetic mapping can cause problems with understanding.
Linkage analysis- tendency for alleles close together on chromosome to be transmitted together. Can map through pedigree analysis. Close together- stay together in recombination. RFLP not used much anymore. Markers are not necessarily genes- they can be followed in pedigrees. Markers allow us to map genes to chromosomes.
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1. relative to each other.
2. Polymorphic means difference among individuals.
RFLP and SSR are markers.

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cM is a genetic unit. Crossing over happens a lot, so if genes are together 1% of the time, they are close together. cM and base pairs are not equivalent. To map a disease to a chromosome, must map several markers on each chromosome for inheritance.
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Allele view. D and M could be separated by recombination. Randomly distributed- not linked.
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Look at progeny or gametes that result. Some linkage- intermediate.
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Marker A is not linked to disease phenotype, but marker B is.
Marker is not a predictor of disease per se, but happens to be linked. Not predictor. To be a predictor of disease, if you find marker very very close, can be used as diagnostic event. It may be linked to the disease gene, not associated with the disease itself.
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RFLP- anything can be used as a marker. Restriction site can be used. 1bp change creates an ECOR1 cut site. RFLP site is inherited. See it a lot in literature, but not used much anymore. Get tons of fragments. Lots of work.
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How is it done now? SSR or SRS (simple repeat sequence). Microsatellites are the repeat sequence. Repeats det by PCR and electrophoresis. Each chromosome has a different repeat number.Each individual 2 copies with different repeat sequence. Useful way to map. Marker and easier to follow than RFLPs.
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SSRs in chromosome 1 Markers are under linkage map.
39 Physical mapping
We know the sequence, but we need to be able to navigate around.
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Take part of human chromosome and fuse into rodent cell line- panel of hybrids with different parts of chromosome.PCR for gene of interest. Clonal lines .
STS are physical sites that are not simple repeat sequence. These are not polymorphic. These do not change and are the same in every human being. Cannot follow in genetic map because everyone has the same ones. One copy.
Fri- clone CF gene. Tues- inherited color vision, fragile X, Huntingdon’s disease.


Senogles 3

Assignment is posted.
Today we talk about positional cloning. Text reference is to cystic fibrosis (CF). Specific references are not required reading. Presented because it is good science and illustrative of how you do the technique.
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Functional common until 1990s. Purified protein, followed activity as you purified, got to homogeneity, sequenced, got protein sequence, made oligonucleotide, screened library, got gene. You knew what you were looking for and what the activity was.
Now we look on the basis of where it maps on the chromosome with very little protein information. Positional cloning- map by genetics, then clone.
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Knowing the DNA sequence does not help because you do not know what you are looking for.
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2 types of mapping: see slide
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10% codes for exons. Small part of genome.
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Localize by linkage analysis position of disease gene you are interested in. Want to localize to greatest extent we can. Yacs and BACs can handle large pieces of DNA by cloning.
If you go to human genome project, is good resource. Can click on a region and see all the known genes in the region.
The two markers were well known.
Linkage disequilibrium- had a mutation in a chromosome. As goes through successive generations, some markers closely segregate with it. B1 stays with CF. It is in linkage disequilibrium. Suggests a founder chromosome with that marker.
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High resolution map. Things with D are markers, polymorphic, inherited. Closer you can narrow it, less DNA you have to sequence.
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Trying to minimize brute-force sequencing.
CpG islands tend to be islands of methylated CG that are close to promoters in coding regions. If it cross hybridizes, it is conserved and may be the candidate gene.
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Want to isolate large fragments first. Large fragment- Mb.
14 Question: Disease- how to clone it out?
Chromosome walking- series overlapping clones. Cut up DNA. Make series of restriction fragments. Pull out and sequence along. Laborious.
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Chromosome jumping- maximize potential hits by jumping to places from which you can sequence. Covers maximum amount of distance and bypasses unsequenceable stretches of DNA.
Circularized- A and B become close. Can sequence short segment between A and B to find the sequence of site B. Can jump, do not plan to go anywhere. You don’t really care what was between A and B to begin. Can make jumping library, and make a library of sites to walk from. Jumping randomly, then sequence from positions in both directions. Increases chances you will hit something interesting.
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Series of jumps and where sequence from are shown.
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Arcs are jumps, horizontal lines are sequence. Roman numerals indicate exons.
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How do we know we have sequenced something interesting? Look for an ORF.
2 types of candidate genes.
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Used 2 criteria for candidate genes:
Conserved in phylogenetic tree if important for viability. Used strategy for trying to clone further. Looked for CpG islands at 5’end of genes.
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CpGs tend to fall close to beginning of genes. Housekeeping genes do not often have unmethylated CpG islands, but class 2 proteins do.
23 Found cross-hybridized exons. They probed a zooblot.
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Found exon1 with ORF. Screened library and got 24 exons. He had sequence of something.
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Found 12 TM domains.
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Evidence-Does mutation correlate with disease? Is function disrupted?
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Sequencing ladder shown. CTT was deleted. Was an in-frame deletion. Segregated with disease.
Did Southern blot. Most of homozygotes had mutation.
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Ahead 10 years- CFTR protein can have lots of mutations. II most common. Protein never gets to cell surface.
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Point of exercise is to understand positional cloning (need a method to identify candidate exons- looking for kinase-can look for homology with other kinase sequence, or can express in culture and look for activity). Once you have CDNA, you have to give evidence that your putative candidate gene does in fact cause the disease. Do not have to know names of markers.