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.

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A simple method for displaying the hydropathic character of a protein

We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

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DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data.

The complete sequence of the 16,569-base pair human mitochondrial genome is presented. The genes for the 12S and 16S rRNAs, 22 tRNAs, cytochrome c oxidase subunits I, II and III, ATPase subunit 6, cytochrome b and eight other predicted protein coding genes have been located. The sequence shows extreme economy in that the genes have none or only a few noncoding bases between them, and in many cases the termination codons are not coded in the DNA but are created post-transcriptionally by polyadenylation of the mRNAs.

Sequence and organization of the human mitochondrial genome

The complete DNA sequences of both the human and the bovine mitochondrial genomes have been determined recently (Anderson et al. 1981; S. Anderson et al., in prep.). Because these two genomes exhibit a high degree of homology and virtually identical organization, they can be interpreted as a unit, allowing a more comprehensive understanding of mammalian mitochondrial gene expression and gene-product structure. The sequences of the human and bovine mtDNAs can serve to link up several important bodies of evidence. For example, the precise mapping of human mitochondrial transcripts (Ojala et al. 1980), combined with the amino acid sequences of bovine proteins such as cytochrome oxidase subunit II (COII) (Steffens and Buse 1979), enables one to predict confidently that the large open reading frames conserved in both the human and the bovine mitochondrial genomes (Anderson et al. 1981) (Fig. 1) are functional protein-coding genes. Similarly, the mapping and charging data for human mitochondrial tRNAs (Angerer et al. 1976; Lynch and Attardi 1976) and the sequence data for bovine mitochondrial tRNAs (Roe et al., this volume; B. A. Roe et al., in prep.) enable one to locate precisely the positions of the tRNA genes in the two genomes. Finally, with two complete mammalian mitochondrial genome sequences it is possible to examine the pattern of mtDNA sequence conservation during mammalian evolution. Such a comparative study can yield information about the relative importance of various structural features found in macromolecules coded in the mitochondrial genome. The human mitochondrial genetic system has been found to...

Comparison of the Human and Bovine Mitochondrial Genomes

Many methods exist for genotyping--revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping--determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a 'tiling' approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches.

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An efficient method for multi-locus molecular haplotyping

We have made a high-resolution HAPPY map of chromosome 6 of Dictyostelium discoideum consisting of 300 sequence-tagged sites with an average spacing of 14 kb along the approximately 4-Mb chromosome. The majority of the marker sequences were derived from randomly chosen clones from four different chromosome 6-enriched plasmid libraries or from subclones of YACs previously mapped to chromosome 6. The map appears to span the entire chromosome, although marker density is greater in some regions than in others and is lowest within the telomeric region. Our map largely supports previous gene-based maps of this chromosome but reveals a number of errors in the physical map. In addition, we find that a high proportion of the plasmid sequences derived from gel-enriched chromosome 6 (and that form the basis of a chromosome-specific sequencing project) originates from other chromosomes.

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A high-resolution HAPPY map of Dictyostelium discoideum chromosome 6.

A small proportion (0.1-0.5%) of the total DNA content of native Chinese hamster metaphase chromosomes is protected from nucleolytic degradation following the removal of histones by extraction with either 0.2 N HCl or 2 M NaCl, and remains attached to the nonhistone protein core. Acid extraction followed by DNase I digestion leads to small fragments of 10-30 bases. Salt extraction followed by micrococcal nuclease digestion gives approx. 140 b.p. fragments which are undistinguishable in size from nucleosome core DNA fragments. Furthermore, DNase I treatment of salt extracted chromosomes gives DNA fragments containing single strands which are multiples of 10 bases in length, again characteristic of the nucleosome structure. Reassociation kinetics using the 32P-labelled 140 b.p. fragments as probes suggests they are enriched for rapidly reassociating sequences.

A partial characterization of DNA fragments protected from nuclease degradation in histone depleted metaphase chromosomes of the Chinese hamster

Four RNA polymerase II promoters have been mapped in the DNA sequence of the EcoRI-H and -Dhet fragments of B95-8 Epstein-Barr virus RNAs transcribed from three of these promoters are dramatically induced by treatment of B95-8 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) The other promoter is active with or without TPA treatment of the cells and is thus active in the latent virus cycle Deletion mapping suggests that DNA sequence homologies between some of the promoters lie in the same region as essential upstream promoter elements

Alan T. Bankier

Papers

Comparison of the Human and Bovine Mitochondrial Genomes

An efficient method for multi-locus molecular haplotyping

A high-resolution HAPPY map of Dictyostelium discoideum chromosome 6.

A partial characterization of DNA fragments protected from nuclease degradation in histone depleted metaphase chromosomes of the Chinese hamster

Latent and lytic cycle promoters of Epstein-Barr virus.