An SNP map of human chromosome 22.

The human genome sequence will provide a reference for measuring DNA sequence variation in human populations. Sequence variants are responsible for the genetic component of individuality, including complex characteristics such as disease susceptibility and drug response. Most sequence variants are single nucleotide polymorphisms (SNPs), where two alternate bases occur at one position. Comparison of any two genomes reveals around 1 SNP per kilobase. A sufficiently dense map of SNPs would allow the detection of sequence variants responsible for particular characteristics on the basis that they are associated with a specific SNP allele. Here we have evaluated large-scale sequencing approaches to obtaining SNPs, and have constructed a map of 2,730 SNPs on human chromosome 22. Most of the SNPs are within 25 kilobases of a transcribed exon, and are valuable for association studies. We have scaled up the process, detecting over 65,000 SNPs in the genome as part of The SNP Consortium programme, which is on target to build a map of 1 SNP every 5 kilobases that is integrated with the human genome sequence and that is freely available in the public domain.

The complete nucleotide sequence of chromosome 3 of Plasmodium falciparum.

Analysis of Plasmodium falciparum chromosome 3, and comparison with chromosome 2, highlights novel features of chromosome organization and gene structure. The sub-telomeric regions of chromosome 3 show a conserved order of features, including repetitive DNA sequences, members of multigene families involved in pathogenesis and antigenic variation, a number of conserved pseudogenes, and several genes of unknown function. A putative centromere has been identified that has a core region of about 2 kilobases with an extremely high (adenine + thymidine) composition and arrays of tandem repeats. We have predicted 215 protein-coding genes and two transfer RNA genes in the 1,060,106-base-pair chromosome sequence. The predicted protein-coding genes can be divided into three main classes: 52.6% are not spliced, 45.1% have a large exon with short additional 5' or 3' exons, and 2.3% have a multiple exon structure more typical of higher eukaryotes.

Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

Short-insert libraries as a method of problem solving in genome sequencing.

As the Human Genome Project moves into its sequencing phase, a serious problem has arisen. The same problem has been increasingly vexing in the closing phase of the Caenorhabditis elegans project. The difficulty lies in sequencing efficiently through certain regions in which the templates (DNA substrates for the sequencing process) form complex folded secondary structures that are inaccessible to the enzymes. The solution, however, is simply to break them up. Specifically, the offending fragments are sonicated heavily and recloned, as much smaller fragments, into pUC vector. The sequences obtained from the resulting library can subsequently be assembled, free from the effects of secondary structure, to produce high-quality, complete sequence. Because of the success and simplicity of this procedure, we have begun to use it for the sequencing of all regions in which standard primer walking has been at all difficult.

Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits.

We show that three of the eleven genes of the nematode Caenorhabditis elegans that mediate resistance to the nematocide levamisole and to other cholinergic agonists encode nicotinic acetylcholine receptor (nAChR) subunits. unc-38 encodes an alpha subunit while lev-1 and unc-29 encode non-alpha subunits. The nematode nAChR subunits show conservation of many mammalian nAChR sequence features, implying an ancient evolutionary origin of nAChR proteins. Expression in Xenopus oocytes of combinations of these subunits that include the unc-38 alpha subunit results in levamisole-induced currents that are suppressed by the nAChR antagonists mecamylamine, neosurugatoxin, and d-tubocurarine but not alpha-bungarotoxin. The mutant phenotypes reveal that unc-38 and unc-29 subunits are necessary for nAChR function, whereas the lev-1 subunit is not. An UNC-29-GFP fusion shows that UNC-29 is expressed in body and head muscles. Two dominant mutations of lev-1 result in a single amino acid substitution or addition in or near transmembrane domain 2, a region important to ion channel conductance and desensitization. The identification of viable nAChR mutants in C. elegans provides an advantageous system in which receptor expression and synaptic targeting can be manipulated and studied in vivo.

Sequence variation of the human Y chromosome.

We have generated over 100 kilobases of sequence from the nonrecombining portion of the Y chromosomes from five humans and one common chimpanzee. The human subjects were chosen to match the earliest branches of the human mitochondrial tree. The survey of 18.3 kilobases from each human detected only three sites at which substitutions were present, whereas the human and chimpanzee sequences showed 1.3% divergence. The coalescence time estimated from our Y chromosome sample is more recent than that of the mitochondrial genome. A recent coalescence time for the Y chromosome could have been caused by the selected sweep of an advantageous Y chromosome or extensive migration of human males.

Genomic organization of major sperm protein genes and pseudogenes in the nematode Caenorhabditis elegans.

The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3' untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3' untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.

Preparation of large numbers of plasmid DNA samples in microtiter plates by the alkaline lysis method.

A protocol is described for the growth and preparation of plasmid DNAs from small culture volumes (250 microliters) and utilizing standard 96-well plates. Several hundred plasmids can be prepared simultaneously, yielding sufficient DNA for subsequent analysis by restriction digestion and gel electrophoresis. This protocol may be useful for rapid screening of clones arising in recombinant DNA work such as site-directed mutagenesis, oligonucleotide cassette cloning, deletion analysis, etc. The technique was initially developed to meet our requirement to provide large numbers of cosmid DNAs for restriction enzyme fingerprint analyses in genome mapping projects.

Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promoters within the insert: effect on DNA yield and cloned insert frequency.

Transcription terminators have been included in a phage-lambda-replicon-based cosmid vector, Lorist2, to insulate vector genes against transcriptional interference from cloned insert DNA. DNA yields of recombinant clones containing Escherichia coli genomic DNA inserts are more even for Lorist2 than with its progenitor LoristB. However, the terminators provide only a partial reduction in the over-representation of r X DNA-containing clones generally observed in cosmid libraries of Caenorhabditis elegans DNA, suggesting that causes other than transcriptional readthrough into the vector contribute to this problem.

The rDNA of C. elegans: sequence and structure.

We have sequenced one complete rDNA tandem repeat from the nematode C. elegans. By comparative analysis we derive secondary structures for the 18s, 5.8s, and 26s rRNA molecules, and comment on other important features of the sequence. We also present the sequence of a junction between the rDNA and non-ribosomal DNA. Finally, we use our data to quantify the evolutionary relationships among several organisms currently studied in developmental biology.

The neural circuit for touch sensitivity in Caenorhabditis elegans.

The neural pathways for touch-induced movement in Caenorhabditis elegans contain six touch receptors, five pairs of interneurons, and 69 motor neurons. The synaptic relationships among these cells have been deduced from reconstructions from serial section electron micrographs, and the roles of the cells were assessed by examining the behavior of animals after selective killing of precursors of the cells by laser microsurgery. This analysis revealed that there are two pathways for touch-mediated movement for anterior touch (through the AVD and AVB interneurons) and a single pathway for posterior touch (via the PVC interneurons). The anterior touch circuitry changes in two ways as the animal matures. First, there is the formation of a neural network of touch cells as the three anterior touch cells become coupled by gap junctions. Second, there is the addition of the AVB pathway to the pre-existing AVD pathway. The touch cells also synapse onto many cells that are probably not involved in the generation of movement. Such synapses suggest that stimulation of these receptors may modify a number of behaviors.

The embryonic cell lineage of the nematode Caenorhabditis elegans.

The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.

Induction of neuronal branching in Caenorhabditis elegans.

The two postembryonic touch receptor neurons in the nematode Caenorhabditis elegans arise from essentially identical cell lineages and have the same ultrastructural features. The cells are found in different positions in the animal, however, and differ in neuronal branching, connectivity, and function. These structural and functional differences are not seen when cells are placed in similar positions by mutation or laser-induced damage. Thus, some, but probably not all, of the differentiated properties of these cells are a consequence of their cellular environment.

Mutations affecting programmed cell deaths in the nematode Caenorhabditis elegans.

Mutations in two nonessential genes specifically block the phagocytosis of cells programmed to die during development. With few exceptions, these cells still die, suggesting that, in nematodes, engulfment is not necessary for most programmed deaths. Instead, these deaths appear to occur by cell suicide.

Serotonin and octopamine in the nematode Caenorhabditis elegans.

The biogenic amines serotonin and octopamine are present in the nematode Caenorhabditis elegans. Serotonin, detected histochemically in whole mounts, is localized in two pharyngeal neurons that appear to be neurosecretory. Octopamine, identified radioenzymatically in crude extracts, probably is also localized in a few neurons. Exogenous serotonin and octopamine elicit specific and opposite behavioral responses in Caenorhabditis elegans, suggesting that these compounds function physiologically as antagonists.