Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map.

Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as Caenorhabditis elegans. To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian island that shows a uniformly high density of polymorphisms compared with the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predicted 6,222 polymorphisms. Furthermore, 3,457 of these markers modify restriction enzyme recognition sites ('snip-SNPs') and are therefore easily detected as RFLPs. Of these, 493 were experimentally confirmed by restriction digest to produce a snip-SNP map of the worm genome. A mapping strategy using snip-SNPs and bulked segregant analysis (BSA) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. The usefulness of this approach is illustrated by the rapid mapping of the dyf-5 gene.

A survey of expressed genes in Caenorhabditis elegans.

As an adjunct to the genomic sequencing of Caenorhabditis elegans, we have investigated a representative cDNA library of 1,517 clones. A single sequence read has been obtained from the 5' end of each clone, allowing its characterization with respect to the public databases, and the clones are being localized on the genome map. The result is the identification of about 1,200 of the estimated 15,000 genes of C. elegans. More than 30% of the inferred protein sequences have significant similarity to existing sequences in the databases, providing a route towards in vivo analysis of known genes in the nematode. These clones also provide material for assessing the accuracy of predicted exons and splicing patterns and will lead to a more accurate estimate of the total number of genes in the organism than has hitherto been available.

The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men.

Deletions of the AZFc (azoospermia factor c) region of the Y chromosome are the most common known cause of spermatogenic failure. We determined the complete nucleotide sequence of AZFc by identifying and distinguishing between near-identical amplicons (massive repeat units) using an iterative mapping-sequencing process. A complex of three palindromes, the largest spanning 3 Mb with 99.97% identity between its arms, encompasses the AZFc region. The palindromes are constructed from six distinct families of amplicons, with unit lengths of 115-678 kb, and may have resulted from tandem duplication and inversion during primate evolution. The palindromic complex contains 11 families of transcription units, all expressed in testis. Deletions of AZFc that cause infertility are remarkably uniform, spanning a 3.5-Mb segment and bounded by 229-kb direct repeats that probably served as substrates for homologous recombination.

zA map for sequence analysis of the Arabidopsis thaliana genome.

Arabidopsis thaliana has emerged as a model system for studies of plant genetics and development, and its genome has been targeted for sequencing by an international consortium (the Arabidopsis Genome Initiative; http://genome-www. stanford.edu/Arabidopsis/agi.html). To support the genome-sequencing effort, we fingerprinted more than 20,000 BACs (ref. 2) from two high-quality publicly available libraries, generating an estimated 17-fold redundant coverage of the genome, and used the fingerprints to nucleate assembly of the data by computer. Subsequent manual revision of the assemblies resulted in the incorporation of 19,661 fingerprinted BACs into 169 ordered sets of overlapping clones ('contigs'), each containing at least 3 clones. These contigs are ideal for parallel selection of BACs for large-scale sequencing and have supported the generation of more than 5.8 Mb of finished genome sequence submitted to GenBank; analysis of the sequence has confirmed the integrity of contigs constructed using this fingerprint data. Placement of contigs onto chromosomes can now be performed, and is being pursued by groups involved in both sequencing and positional cloning studies. To our knowledge, these data provide the first example of whole-genome random BAC fingerprint analysis of a eucaryote, and have provided a model essential to efforts aimed at generating similar databases of fingerprint contigs to support sequencing of other complex genomes, including that of human.

An encyclopedia of mouse genes.

The laboratory mouse is the premier model system for studies of mammalian development due to the powerful classical genetic analysis possible (see also the Jackson Laboratory web site, http://www.jax.org/) and the ever-expanding collection of molecular tools. To enhance the utility of the mouse system, we initiated a program to generate a large database of expressed sequence tags (ESTs) that can provide rapid access to genes. Of particular significance was the possibility that cDNA libraries could be prepared from very early stages of development, a situation unrealized in human EST projects. We report here the development of a comprehensive database of ESTs for the mouse. The project, initiated in March 1996, has focused on 5' end sequences from directionally cloned, oligo-dT primed cDNA libraries. As of 23 October 1998, 352,040 sequences had been generated, annotated and deposited in dbEST, where they comprised 93% of the total ESTs available for mouse. EST data are versatile and have been applied to gene identification, comparative sequence analysis, comparative gene mapping and candidate disease gene identification, genome sequence annotation, microarray development and the development of gene-based map resources.

Muscle cell attachment in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, the body wall muscles exert their force on the cuticle to generate locomotion. Interposed between the muscle cells and the cuticle are a basement membrane and a thin hypodermal cell. The latter contains bundles of filaments attached to dense plaques in the hypodermal cell membranes, which together we have called a fibrous organelle. In an effort to define the chain of molecules that anchor the muscle cells to the cuticle we have isolated five mAbs using preparations enriched in these components. Two antibodies define a 200-kD muscle antigen likely to be part of the basement membrane at the muscle/hypodermal interface. Three other antibodies probably identify elements of the fibrous organelles in the adjacent hypodermis. The mAb IFA, which reacts with mammalian intermediate filaments, also recognizes these structures. We suggest that the components recognized by these antibodies are likely to be involved in the transmission of tension from the muscle cell to the cuticle.

The Caenorhabditis elegans UNC-87 protein is essential for maintenance, but not assembly, of bodywall muscle.

Mutations in the unc-87 gene of Caenorhabditis elegans cause disorganization of the myofilament lattice in adult bodywall muscle. In order to assess the organization of specific bodywall muscle components in the absence of the unc-87 gene product, we examined the bodywall muscles of mutant animals using phalloidin and monoclonal antibodies to various muscle proteins. These studies indicated that the bodywall muscle of unc-87 embryos is initially almost wild type in its organization, but at later stages, the muscle becomes severely disorganized. To address the possibility that this disorganization is due to deterioration of the muscle as a result of contraction, we introduced into the unc-87 mutant background a mutation that decreases myosin heavy chain activity but does not substantially affect muscle structure. The improved muscle structure and motility of the double mutants are consistent with the hypothesis that at least part of the disorganization phenotype of unc-87 mutants is a consequence of the wild-type levels of force generated during muscle contraction. These results imply that the role of the unc-87 gene product is not in specifying organization but rather in serving as a structural component maintaining lattice integrity during and after contraction.

The Caenorhabditis elegans muscle-affecting gene unc-87 encodes a novel thin filament-associated protein.

Mutations in the unc-87 gene of Caenorhabditis elegans affect the structure and function of bodywall muscle, resulting in variable paralysis. We cloned the unc-87 gene by taking advantage of a transposon-induced allele of unc-87 and the correspondence of the genetic and physical maps in C. elegans. A genomic clone was isolated that alleviates the mutant phenotype when introduced into unc-87 mutants. Sequence analysis of a corresponding cDNA clone predicts a 357-amino acid, 40-kD protein that is similar to portions of the vertebrate smooth muscle proteins calponin and SM22 alpha, the Drosophila muscle protein mp20, the deduced product of the C. elegans cDNA cm7g3, and the rat neuronal protein np25. Analysis of the genomic sequence and of various transcripts represented in a cDNA library suggest that unc-87 mRNAs are subject to alternative splicing. Immunohistochemistry of wildtype and mutant animals with antibodies to an unc-87 fusion protein indicates that the gene product is localized to the I-band of bodywall muscle. Studies of the UNC-87 protein in other muscle mutants suggest that the unc-87 gene product associates with thin filaments, in a manner that does not depend on the presence of the thin filament protein tropomyosin.

Vinculin is essential for muscle function in the nematode.

Actin filaments in the body wall muscle of the nematode Caenorhabditis elegans are attached to the sarcolemma through vinculin-containing structures called dense bodies, Z-line analogues. To investigate the in vivo function of vinculin, we executed a genetic screen designed to recover mutations in the region of the nematode vinculin gene, deb-1. According to four independent criteria, two of the isolated mutants were shown to be due to alterations in the deb-1 gene. First, antibody staining showed that the mutants had reduced levels of vinculin. Second, the sequence of each mutant gene was altered from that of wild type, with one mutation altering a conserved splice sequence and the other generating a premature amber stop codon. Third, the amber mutant was suppressed by the sup-7 amber suppressor tRNA gene. Finally, injection of a cloned wild type copy of the gene rescued the mutant. Mutant animals lacking vinculin arrested development as L1 larvae. In such animals, embryonic elongation was interrupted at the twofold length, so that the mutants were shorter than wild type animals at the same stage. The mutants were paralyzed and had disorganized muscle, a phenotype consistent with the idea that vinculin is essential for muscle function in the nematode.

Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations.

By taking advantage of a lethal phenotype characteristic of Caenorhabditis elegans embryos that fail to move, we have identified 13 genes required for muscle assembly and function and discovered a new lethal class of alleles for three previously known muscle-affecting genes. By staining mutant embryos for myosin and actin we have recognized five distinct classes of genes: mutations in four genes disrupt the assembly of thick and thin filaments into the myofilament lattice as well as the polarized location of these components to the sarcolemma. Mutations in another three genes also disrupt thick and thin filament assembly, but allow proper polarization of lattice components based on the myosin heavy chain isoform that we analyzed. Another two classes of genes are defined by mutations with principal effects on thick or thin filament assembly into the lattice, but not both. The final class includes three genes in which mutations cause relatively minor defects in lattice assembly. Failure of certain mutants to stain with antibodies to specific muscle cell antigens suggest that two genes associated with severe disruptions of myofilament lattice assembly may code for components of the basement membrane and the sarcolemma that are concentrated where dense bodies (Z-line analogs) and M-lines attach to the cell membrane. Similar evidence suggests that one of the genes associated with mild effects on lattice assembly may code for tropomyosin. Many of the newly identified genes are likely to play critical roles in muscle development and function.

Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans.

Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.

Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization.

The body wall muscle cells of Caenorhabditis elegans contain an obliquely striated myofibrillar lattice that is associated with the cell membrane through two structures: an M-line analogue in the A-band and a Z-disc analogue, or dense-body, in the I-band. By using a fraction enriched in these structures as an immunogen for hybridoma production, we prepared monoclonal antibodies that identify four components of the I-band as determined by immunofluorescence and Western transfer analysis. A major constituent of the dense-body is a 107,000-D polypeptide that shares determinants with vertebrate alpha-actinin. A second dense-body constituent is a more basic and antigenically distinct 107,000-D polypeptide that is localized to a narrow domain of the dense-body at or subjacent to the plasma membrane. This basic dense-body polypeptide is also found at certain cell boundaries where thin filaments in half-bands terminate at membrane-associated structures termed attachment plaques. A third, unidentified antigen is also found closely apposed to the cell membrane in regions of not only the dense-body and attachment plaque, but also the M-line analogue. Finally, a fourth high molecular weight antigen, composed of two polypeptides of approximately 400,000-D, is localized to the I-band regions surrounding the dense-body. The attachment of the dense-body to the cell surface and the differential localization of the dense-body-associated antigens suggest a model for their organization in which the unidentified antigen is a cell surface component, and the two 107,000-D polypeptides define different cytoplasmic domains of the dense-body.

Assembly of body wall muscle and muscle cell attachment structures in Caenorhabditis elegans.

C. Elegans has four muscle quadrants that are used for locomotion. Contraction is converted to locomotion because muscle cells are anchored to the cuticle (the outer covering of the worm) by a specialized basement membrane and hemidesmosome structures in the hypodermis (a cellular syncytium that covers the worm and secretes the cuticle). To study muscle assembly, we have used antibodies to determine the spatial and temporal distribution of muscle and attachment structure components in wild-type and mutant C. elegans embryos. Myofibrillar components are first observed diffusely distributed in the muscle cells, and are expressed in some dividing cells. Later, the components accumulate at the membrane adjacent to the hypodermis where the sarcomeres will form, showing that the cells have become polarized. Assembly of muscle attachment structures is spatially and temporally coordinated with muscle assembly suggesting that important developmental signals may be passed between muscle and hypodermal cells. Analysis of embryos homozygous for mutations that affect muscle assembly show that muscle components closer to the membrane than the affected protein assemble quite well, while those further from the membrane do not. Our results suggest a model where lattice assembly is initiated at the membrane and the spatial organization of the structural elements of the muscle is dictated by membrane proximal events, not by the filament components themselves.

Myotactin, a novel hypodermal protein involved in muscle-cell adhesion in Caenorhabditis elegans.

In C. elegans, assembly of hypodermal hemidesmosome-like structures called fibrous organelles is temporally and spatially coordinated with the assembly of the muscle contractile apparatus, suggesting that signals are exchanged between these cell types to position fibrous organelles correctly. Myotactin, a protein recognized by monoclonal antibody MH46, is a candidate for such a signaling molecule. The antigen, although expressed by hypodermis, first reflects the pattern of muscle elements and only later reflects the pattern of fibrous organelles. Confocal microscopy shows that in adult worms myotactin and fibrous organelles show coincident localization. Further, cell ablation studies show the bodywall muscle cells are necessary for normal myotactin distribution. To investigate myotactin's role in muscle-hypodermal signaling, we characterized the myotactin locus molecularly and genetically. Myotactin is a novel transmembrane protein of approximately 500 kd. The extracellular domain contains at least 32 fibronectin type III repeats and the cytoplasmic domain contains unique sequence. In mutants lacking myotactin, muscle cells detach when embryonic muscle contraction begins. Later in development, fibrous organelles become delocalized and are not restricted to regions of the hypodermis previously contacted by muscle. These results suggest myotactin helps maintain the association between the muscle contractile apparatus and hypodermal fibrous organelles.

Movement of cortical actin patches in yeast.

In yeast, actin forms patches associated with the plasma membrane. Patch distribution correlates with polarized growth during the cell cycle and in response to external stimuli. Using green fluorescent protein fused to capping protein to image actin patches in living cells, we find that patches move rapidly and over long distances. Even patches in clusters, such as at the incipient bud site, show movement. Patches move independently of one another and generally over small distances in a local area, but they can also move larger distances, including through the mother-bud neck. Changes in patch polarization occur quickly through the cell cycle. These observations provide important new parameters for a molecular analysis of the regulation and function of actin.

Antigen competition: a paradox.

Immunization of mice with pig erythrocytes caused impairment of the antibody response to subsequent immunization with sheep erythrocytes, a phenomenon called "antigen competition." Paradoxically, spleen cells from mice previously injected with pig erythrocytes produced an increased response when immunized in vitro with sheep erythrocytes. Augmentation of the in vitro response is due to an increase in one of the interacting cell types. "Antigen competition" is not due to competition for cells. Cell transfer experiments provided evidence that "antigen competition" observed in animals is the result of a humoral factor, presumably antibody, present in the animal but eliminated during preparation of cells for culture.

The nematode Caenorhabditis elegans and its genome.

Over the past two decades, the small soil nematode Caenorhabditis elegans has become established as a major model system for the study of a great variety of problems in biology and medicine. One of its most significant advantages is its simplicity, both in anatomy and in genomic organization. The entire haploid genetic content amounts to 100 million base pairs of DNA, about 1/30 the size of the human value. As a result, C. elegans has also provided a pilot system for the construction of physical maps of larger animal and plant genomes, and subsequently for the complete sequencing of those genomes. By mid-1995, approximately one-fifth of the complete DNA sequence of this animal had been determined. Caenorhabditis elegans provides a test bed not only for the development and application of mapping and sequencing technologies, but also for the interpretation and use of complete sequence information. This article reviews the progress so far toward a realizable goal--the total description of the genome of a simple animal.

Interaction between a putative mechanosensory membrane channel and a collagen.

The degenerin family of proteins in Caenorhabditis elegans is homologous to subunits of the mammalian amiloride-sensitive epithelial sodium channels. Mutations in nematode degenerins cause cell death, probably because of defects in channel function. Genetic evidence was obtained that the unc-105 gene product represents a degenerin homolog affecting C. elegans muscles and that this putative channel interacts with type IV collagen in the extracellular matrix underlying the muscle cell. This interaction may serve as a mechanism of stretch-activated muscle contraction, and this system could provide a molecular model for the activation of mechanosensitive ion channels.

Ancient conserved regions in new gene sequences and the protein databases.

Sets of new gene sequences from human, nematode, and yeast were compared with each other and with a set of Escherichia coli genes in order to detect ancient evolutionarily conserved regions (ACRs) in the encoded proteins. Nearly all of the ACRs so identified were found to be homologous to sequences in the protein databases. This suggests that currently known proteins may already include representatives of most ACRs and that new sequences not similar to any database sequence are unlikely to contain ACRs. Preliminary analyses indicate that moderately expressed genes may be more likely to contain ACRs than rarely expressed genes. It is estimated that there are fewer than 900 ACRs in all.

Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans.

Caenorhabditis elegans has 12 tRNA(UGGTrp) genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrp gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNA(UAGTrp) molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressers against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNA(Trp) gene family were differentially regulated in a tissue- or development stage-specific manner.