BIMM 110 - Lectures 17-18

 TEXTBOOK:   Strachan and Read, Chapter 8, 13, 14, 19                         

SLIDES 17        SLIDES 18

Progress in this area has relied heavily on the development of molecular-genetic technology, and at this stage student should be thoroughly familiar with the techniques listed in the Introduction to the course.

We now have the complete sequence of the human genome (Science, Nature February 2001), but we have not yet identified all the genes. By mapping a mutation responsible for a disease precisely, one identifies the corresponding DNA sequence (gene) and from there one obtains the protein sequence, and from there one may deduce function, subcellular localization, etc.

 A. Definition of loci to be used in linkage studies

1. Generation of DNA probes from cDNA and genomic clones of known genes
        from cDNA or genomic libraries
        chromosome specific libraries from hybrid cells or flow sorted chromosomes
        unique anonymous DNA clones (of historical interest)
2. Loci defined by restriction fragment length polymorphisms (RFLPs)
3. Loci defined by polymorphic mini- and microsatellites (VNTRs) and their analysis by PCR-based methods

4. sequence tagged sites (STSs): a small sequence defined by oligonucleotide primers used to amplify it by PCR

 B. Mapping loci on human chromosomes

1. markers can be placed on chromosomes using somatic hybrid panels (establishment of synteny)
2. markers can be mapped subregionally by using translocations, and segregation of abnormal chromosomes in somatic cell hybrids
3. FISH (lecture 9) can map a probe on a small region of a chromosome, generally within a specific band (i.e. with a resolution of the order of a few million basepairs); with two probes that are differentially labeled one may be able to map them relative to each other if they are not too close together

        Chromosomes                            one of the most comprehensive sources
        

C. Genetic studies investigating linkage and recombination

1. classical genetic approaches involve making deliberate crosses; the usefulness for human gene mapping is severely limited:
                  long generation times
                  small number of offspring
                  no controlled matings

2. one possible approach is to consider many families in which a given trait/disease is observed AND other loci are distinguishable by RFLPs, VNTRs, SNPs (single nucleotide polymorphisms);
hence, a need for many pedigrees, with many children, and family members from more than two generations

Le Centre d'Etude du Polymorphisme Humaine (CEPH) in Paris started to accumulate cells from ~ 40 pedigrees satisfying the above criteria
DNA samples from each representative were made available to researchers

D. LOD SCORE ANALYSIS

the lod score is the logarithm of the ratio of two probabilities: the probability that a given combination of offspring will arise when the recombination frequency is q, divided by the probability for q = 0.5, i.e. there is no linkage. The loci may still be on the same chromosome, but they are so far apart that at least one chiasma will occur between them.

  

      

- the phase problem
- multi loci analysis by use of the computer
- recombination frequencies and genetic distances (1 cM = 1% recombinants)
- linkage maps for male and female meiosis
        there are sex-dependent differences in genetic distances : most notable example: the pseudoautosomal region

- present resolution and future prospects

- Web Sites and where to find up to date information

 E. genetic maps and physical maps

- Megabase mapping by pulsed field electrophoresis
        FIGE (field inversion gel electrophoresis) and CHEF

F. Yeast artificial chromosomes (YACs)

- very good for mapping by FISH (large probe), but first one needs to block hybridization of repeated DNA sequences by prehybridizing with "low Cot DNA"

- high resolution physical mapping by building YAC contigs
                      essentially a chromosome walk using YACs, i.e. with steps spanning hundreds of kb and even > 1 mb

 G. Mapping by PCR on single spermatozoa

- millions of spermatozoa are available from a male; each gamete is the product of chromosome assortment and meiotic recombination
- if the genotype of many individual gametes could be analyzed, one could obtain statistically significant results about recombination frequencies between all the genes for which the particular male individual is heterozxygous

 - PCR has been demonstrated to be capable of amplifying DNA sequences from a single haploid cell; each would have to be analyzed by sequencing or restriction mapping, etc.

- however, one is pushing the limit here, and this procedure has not become routine

 H. Genetic maps and evolution

- many linkage groups are preserved in species not too far apart in a phylogenetic tree, and thus mapping and linkage data from one species can yield information about the same genes in other species.

- some of the interesting chromosome rearrangements (and hence changes in linkage and synteny) can give valuable clues about

                               speciation
                               phylogenetic relationships and relative dates

example: the evolution of the X and Y chromosomes and sex determination in mammals.

The sequencing of the Human Genome was "completed" in February 2001. Editing, corrections and annotations are in progress and may take years
Some regions of chromosomes (heterochromatin, centromeres with long alpha-satellite segments) have not even been sequenced in detail.

The latest update and many links can be found at

Human Genome Resources    or at the National Human Genome Research Institute  

Human Genome Organization (HUGO) Ethics Committee
STATEMENT ON BENEFIT-SHARING
April 9, 2000

A. Introduction
The HUGO Ethics Committee subscribes to the following four principles (presented in the HUGO Statement on the Principled Conduct of Genetic Research (1996)) [See also SCIENCE 290:49 (2000)]

• Recognition that the human genome is part of the common heritage of humanity
• Adherence to international norms of human rights
• Respect for the values, traditions, culture, and integrity of participants
• Acceptance and upholding of human dignity and freedom.

 A. Historical

 - Barlow, D.P., and Lehrach, H. (1987). Genetics by gel electrophoresis: the impact of pulsed field gel electrophoresis on mammalian genetics. Trends in Genet. 3, 167-171.
- Bellanne-Chantelot, C., Barillot, E., Lacroix, B., Le Paslier, D., and Cohen, D. (1991). A test case for physical mapping of the human genome by repetitive sequence fingerprints: Construction of a physical map of a 420 kb YAC subcloned into cosmids. Nucleic Acids Res. 19, 505-510.
- Boehnke, M., Arnheim, N., Li, H., Collins, F.S. (1989) Fine-structue mapping of human chromosomes using the polymerase chain reaction on single sperm: experimental design considerations. Am.J.Hum.Genet. 45:21-32.
- Botstein, D., White, R.L., Skolnick, M., and Davis, R.W. (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32, 314-331.
- Brown, S.D.M., Brockdorff, N., Cavanna, J.S., Fisher, E.M.C., Greenfield, A.J., Lyon, M.F., and Nasir, J. (1988). The long-range mapping of mammalian chromosomes. Curr. Top. Microbiol. Immunol. 137, 3-12.
- Brown, W.R.A., and Bird, A.P. (1986). Long-range restriction site mapping of mammalian DNA. Nature 322, 477-481.
- Brownstein, B.H., Silverman, G.A., Little, R.D., Burke, D.T., - Korsmeyer, S.J., Schlessinger, D., and Olson, M.V. (1989). Isolation of single-copy human genes from a library of yeast artificial chromosome clones. Science 244, 1348-1351.
- Burke, D.T., Carle, G.F., and Olson, M.V. (1987). Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236, 806-812.
- Carle, G.F., and Olson, M.V. (1984). Separation of chromosomal DNA molecules from yeast by orthogonal field alternation electrophoresis. Nucl. Acids Res. 12, 5647-5664.
- Carle, G.F., Frank, M., and Olson, M.V. (1986). Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science 232, 65-68.
- Chu, G., Vollrath, D., and Davis, R.W. (1986). Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234, 1582-1585.
- Cox, D.R., Burmeister, M., Price, E.R., Kim, S., and Myers, R.M. (1990). Radiation hybrid mapping: A somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science 250, 245-250.
- Donis-Keller, H., and et.al. (1987). A genetic linkage map of the human genome. Cell 51, 319-337.
- Drayna, D., Davies, K., Hartley, D., Mandel, J.-L., Camarino, G., Williamson, R., and White, R. (1984). Genetic mapping of the human X chromosome by using restriction fragment length polymorphisms. Proc. Natl. Acad. Sci. USA 81, 2836-2839.
- Gardiner, K., Laas, W., and Patterson, D. (1986). Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis. Somat. Cell Molec. Genet. 12, 185-195.
- Jordan, B.R. (1988). Megabase methods: a quantum jump in recombinant DNA techniques. Bioessays 8, 140-145.
- Kidd, K.K. (1990). Chromosome plotbooks and diskettes available from the human gene mapping library. Am. J. Med. Genet. 37, 292
- Krumlauf, R., Jeanpierre, M., and Young, B.D. (1982). Construction and characterization of genomic libraries from specific human chromosomes. Proc. Natl. Acad. Sci. USA 79, 2971-2975.
- Lander, E.S., and Botstein, D. (1986). Strategies for studying heterogeneous genetic traits in humans by using a linkage map of restriction fragment length polymorphisms. Proc. Natl. Acad. Sci. USA 83, 7353-7357.
- Li, H., Gyllenstern U.B., Cui, X., Erlichj, H.A., and Arnheim, N. (1988). Amplification and analysis of DA sequences in single human sperm and diploid cells. Nature 335:414-417.
- Lichter, P., Tang, C.-J.C., Call, K., Hermanson, G., Evans, G.A., Housman, D., and Ward, D.C. (1990). High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 64-69.
- Olson, M., Hood, L., Cantor, C., and Botstein, D. (1989). A common language for physical mapping of the human genome. Science 245, 1434-1435.
- Riethman, H.C., Moyzis, R.K., Meyne, J., Burke, D.T., and Olson, M.V. (1989). Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector. Proc. Natl. Acad. Sci. USA 86, 6240-6244.
- Schwartz, D.C., and Cantor, C.R. (1984). Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67-75.
- Stephens, J.C., Cavanaugh, M.L., Gradie, M.I., Mador, M.L., and Kidd, K.K. (1990). Mapping the human genome: Current status. Science 250, 237-244.
- Vollrath, D., Davis, R.W., Connelly, C., and Hieter, P. (1988). Physical mapping of large DNA by chromosome fragmentation. Proc. Natl. Acad. Sci. USA 85, 6027-6031.
- Wallace, M.R., Fountain, J.W., Brereton, A.M., and Collins, F.S. (1989). Direct construction of a chromosome-specific NotI linking library from flow-sorted chromosomes. Nucleic Acids Res. 17, 1665-1678.
- White, R., and Lalouel, J.-M. (1988). Sets of linked genetic markers for human chromosomes. Annu. Rev. Genet. 22, 259-279.
-Mandel, J.-L., Monaco, A.P., Nelson, D.L., Schlessinger, D., and Willard, H. (1992b). Genome analysis and the human X chromosome. Science 258, 103-109.
-NIH-CEPH Collab Mapping Group, (1992). A comprehensive genetic linkage map of the human genome. Science 258, 67-102.
-Risch, N. (1992). Genetic linkage: Interpreting lod scores. Science 255, 803-804.

 B. Recent

 - Cooperative Human Linkage Center, Murray, J.C., Buetow, K.H., Weber, J.L., Ludwigsen, S., Scherpbier-Heddema, T., Manion, F., Quillen, J., Sheffield, V.C., Sunden, S., Duyk, G.M., Weissenbach, J., Gyapay, G., Dib, C., Morrisette, J., Lathrop, G.M., Vignal, A., White, R., Matsunami, N., Gerken, S., Melis, R., Albertsen, H., Plaetke, R., and Odelberg, S. (1994). A comprehensive human linkage map with centimorgan density. Science 265, 2049-2070.
- Schmitt, K., Lazzeroni, L.C., Foote, S., Vollrath, D., Fisher, E.M.C., Goradia, T.M., lange, K., Page, D.C., and Arnheim, N. (1994). Multipoint linkage map of the human pseudoautosomal region, based on single-sperm typing: Do double crossovers occur during male meiosis. Am. J. Hum. Genet. 55, 423-430.
- Gyapay, G., J. Morissette, A. Vignal, C. Dib, C. Fizames, P. Millasseau, S. Marc, G. Bernardi, M. Lathrop, and J. Weissenbach. 1994. The 1993-94 Généthon human genetic linkage map. Nature Genet. 7 Suppl.246