The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

We describe a new basis for the construction of a genetic linkage map of the human genome. The basic principle of the mapping scheme is to develop, by recombinant DNA techniques, random single-copy DNA probes capable of detecting DNA sequence polymorphisms, when hybridized to restriction digests of an individual's DNA. Each of these probes will define a locus. Loci can be expanded or contracted to include more or less polymorphism by further application of recombinant DNA technology. Suitably polymorphic loci can be tested for linkage relationships in human pedigrees by established methods; and loci can be arranged into linkage groups to form a true genetic map of "DNA marker loci." Pedigrees in which inherited traits are known to be segregating can then be analyzed, making possible the mapping of the gene(s) responsible for the trait with respect to the DNA marker loci, without requiring direct access to a specified gene's DNA. For inherited diseases mapped in this way, linked DNA marker loci can be used predictively for genetic counseling.

Construction of a genetic linkage map in man using restriction fragment length polymorphisms.

Lessons from Hereditary Colorectal Cancer

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced staining for β-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

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Extension of life-span by introduction of telomerase into normal human cells

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.

https://cdr.lib.unc.edu/downloads/xk81jn53s

A second generation human haplotype map of over 3.1 million SNPs

The Saccharomyces cerevisiae proteins Tel1p and Mec1p are involved in telomere length regulation and cellular responses to DNA damage. The closest relative of these proteins is the human Ataxia Telangiectasia Mutated (ATM) protein, a wortmannin-sensitive protein kinase that primarily phosphorylates serines in an SQ motif. We constructed yeast strains containing functional epitope-tagged versions of Tel1p and Mec1p. We showed that immunoprecipitated Tel1p and Mec1p were capable of in vitro phosphorylation of the mammalian protein PHAS-I (Phosphorylated Heat and Acid Stable protein). These activities are sensitive to wortmannin. Tel1p phosphorylates serine in an SQ motif in PHAS-I. Mutations in the kinase domains of Tel1p and Mec1p result in loss of in vitro kinase activity and the in vivo phenotypes associated with the null tel1 and mec1 mutations.

https://www.pnas.org/content/pnas/97/25/13749.full.pdf

Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase

Recombination between retrotransposons as a source of chromosome rearrangements in the yeast Saccharomyces cerevisiae

The full activity of a recombination initiation site located 5' of HIS4 requires the binding of the transcription factors RAP1, BAS1, and BAS2. Two RAP1 binding sites can substitute for the wild-type initiation site. A 51-bp region of telomeric DNA inserted upstream of either HIS4 or ARG4 very strongly stimulates recombination. We suggest that the ability of transcription factors to induce recombination is a consequence of an altered chromatin structure that favors the entry of proteins that initiate recombination, rather than an effect of these factors on transcription.

Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerevisiae

We have examined meiotic and mitotic recombination between repeated genes on nonhomologous chromosomes in the yeast Saccharomyces cerevisiae . The results of these experiments can be summarized in three statements. First, gene conversion events between repeats on nonhomologous chromosomes occur frequently in meiosis. The frequency of such conversion events is only 17-fold less than the analogous frequency of conversion between genes at allelic positions on homologous chromosomes. Second, meiotic and mitotic conversion events between repeated genes on nonhomologous chromosomes are associated with reciprocal recombination to the same extent as conversion between allelic sequences. The reciprocal exchanges between the repeated genes result in chromosomal translocations. Finally, recombination between repeated genes on nonhomologous chromosomes occurs much more frequently in meiosis than in mitosis.

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Chromosomal translocations generated by high-frequency meiotic recombination between repeated yeast genes.

The region of Saccharomyces cerevisiae chromosome III located between the 5' end of the HIS4 gene and the 3' end of the adjacent BIK1 gene has a very high level of meiotic recombination. In wild-type strains, a meiosis-specific double-strand DNA break occurs in the hot spot region. This break is absent in strains in which the transcription factors Rap1p, Bas1p, and Bas2p cannot bind to the region upstream of HIS4. In strains with levels of recombination that are higher than those of the wild type, the break is found at elevated levels. The linear relationship between hot spot activity and the frequency of double-strand DNA breaks suggests that these lesions are responsible for initiating recombination at the HIS4 recombination hot spot.

Thomas D. Petes

Papers

Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase

Recombination between retrotransposons as a source of chromosome rearrangements in the yeast Saccharomyces cerevisiae

Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerevisiae

Chromosomal translocations generated by high-frequency meiotic recombination between repeated yeast genes.

Meiosis-specific double-strand dna breaks at the his4 recombination hot spot in the yeast saccharomyces cerevisiae : control in cis and trans