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.

/pdf/extension-of-life-span-by-introduction-of-telomerase-into-4ucjiz7vl1.pdf

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

Genomic deletions and point mutations induced in Saccharomyces cerevisiae by the trinucleotide repeats (GAA·TTC) associated with Friedreich's ataxia.

Genetic instability at palindromes and spaced inverted repeats (IRs) leads to chromosome rearrangements. Perfect palindromes and IRs with short spacers can extrude as cruciforms or fold into hairpins on the lagging strand during replication. Cruciform resolution produces double-strand breaks (DSBs) with hairpin-capped ends, and Mre11p and Sae2p are required to cleave the hairpin tips to facilitate homologous recombination. Fragile site 2 (FS2) is a naturally occurring IR in Saccharomyces cerevisiae composed of a pair of Ty1 elements separated by ∼280 bp. Our results suggest that FS2 forms a hairpin, rather than a cruciform, during replication in cells with low levels of DNA polymerase. Cleavage of this hairpin results in a recombinogenic DSB. We show that DSB formation at FS2 does not require Mre11p, Sae2p, Rad1p, Slx4p, Pso2p, Exo1p, Mus81p, Yen1p, or Rad27p. Also, repair of DSBs by homologous recombination is efficient in mre11 and sae2 mutants. Homologous recombination is impaired at FS2 in rad52 mutants and most aberrations reflect either joining of two broken chromosomes in a “half crossover” or telomere capping of the break. In support of hairpin formation precipitating DSBs at FS2, two telomere-capped deletions had a breakpoint near the center of the IR. In summary, Mre11p and Sae2p are not required for DSB formation at FS2 or the subsequent repair of these DSBs.

https://www.genetics.org/content/genetics/183/2/423.full.pdf

Chromosome aberrations resulting from double-strand DNA breaks at a naturally occurring yeast fragile site composed of inverted ty elements are independent of Mre11p and Sae2p

Centromeres are the structural elements of eukaryotic chromosomes that hold sister chromatids together and to which spindle tubules connect during cell division. Centromeres have been shown to suppress meiotic recombination in some systems. In this study yeast strains genetically marked within and flanking a centromere, were used to demonstrate that gene conversion (nonreciprocal recombination) tracts in mitosis can enter into and extend through the centromere.

Mitotic recombination within the centromere of a yeast chromosome

Meiotic recombination in yeast is associated with heteroduplex formation. Heteroduplexes formed between nonidentical DNA strands contain DNA mismatches, and most DNA mismatches in wild-type strains are efficiently corrected. Although some patterns of mismatch correction result in non-Mendelian segregation of the heterozygous marker (gene conversion), one predicted pattern of correction (restoration-type repair) results in normal Mendelian segregation. Using a yeast strain in which a marker leading to a well-repaired mismatch is flanked by markers that lead to poorly repaired mismatches, we present direct evidence for restoration-type repair in yeast. In addition, we find that the frequency of tetrads with conversion-type repair is higher for a marker at the 5' end of the HIS4 gene than for a marker in the middle of the gene. These results suggest that the ratio of conversion-type to restoration-type repair may be important in generating gradients of gene conversion (polarity gradients).

https://www.genetics.org/content/genetics/149/4/1693.full.pdf

Conversion-type and restoration-type repair of DNA mismatches formed during meiotic recombination in Saccharomyces cerevisiae.

Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA. These genes are arranged in a single tandem array of 100 repeats. Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene. Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA. The coding sequence of this gene is different fro-n that of the "normal" 5S gene in three positions located at the 3' end of the gene.

Thomas D. Petes

Papers

Genomic deletions and point mutations induced in Saccharomyces cerevisiae by the trinucleotide repeats (GAA·TTC) associated with Friedreich's ataxia.

Chromosome aberrations resulting from double-strand DNA breaks at a naturally occurring yeast fragile site composed of inverted ty elements are independent of Mre11p and Sae2p

Mitotic recombination within the centromere of a yeast chromosome

Conversion-type and restoration-type repair of DNA mismatches formed during meiotic recombination in Saccharomyces cerevisiae.

Tandemly arranged variant 5S ribosomal RNA genes In the yeast Saccharomyces cerevisiae