Biomedical subjects

R Barstead

Publications and source records attributed to R Barstead.

11 recordsLinked to original sources

PubMed2004

Disruption of Caenorhabditis elegans muscle structure and function caused by mutation of troponin I.

Caenorhabditis elegans strains mutant for the unc-27 gene show abnormal locomotion and muscle structure. Experiments revealed that unc-27 is one of four C. elegans troponin I genes and that three mutant alleles truncate the protein: recessive and presumed null allele e155 terminates after nine codons; semidominant su142sd eliminates the inhibitory and C-terminal regions; and semidominant su195sd abbreviates the extreme C-terminus. Assays of in vivo muscular performance at high and low loads indicated that su142sd is most deleterious, with e155 least and su195sd intermediate. Microscopy revealed in mutant muscle a prevalent disorder of dense body positioning and a less well defined sarcomeric structure, with small islands of thin filaments interspersed within the overlap region of A bands and even within the H zone. The mutants' rigid paralysis and sarcomeric disarray are consistent with unregulated contraction of the sarcomeres, in which small portions of each myofibril shorten irregularly and independently of one another, thereby distorting the disposition of filaments. The exacerbated deficits of su142sd worms are compatible with involvement in vivo of the N-terminal portion of troponin I in enhancing force production, and the severe impairment associated with su195sd highlights importance of the extreme C-terminus in the protein's inhibitory function.

Aging↗

PubMed2001

C. elegans slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor.

Robo receptors interact with ligands of the Slit family. The nematode C. elegans has one Robo receptor (SAX-3) and one Slit protein (SLT-1), which direct ventral axon guidance and guidance at the midline. In larvae, slt-1 expression in dorsal muscles repels axons to promote ventral guidance. SLT-1 acts through the SAX-3 receptor, in parallel with the ventral attractant UNC-6 (Netrin). Removing both UNC-6 and SLT-1 eliminates all ventral guidance information for some axons, revealing an underlying longitudinal guidance pathway. In the embryo, slt-1 is expressed at high levels in anterior epidermis. Embryonic expression of SLT-1 provides anterior-posterior guidance information to migrating CAN neurons. Surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3 mutants, suggesting that SAX-3 has both Slit-dependent and Slit-independent functions in development.

Animals↗

PubMed2001

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

2,2'-Dipyridyl↗

PubMed2001

Genome-wide RNAi.

In many species, double-stranded RNA can specifically and effectively silence genes. This newly discovered biological phenomenon, called RNA interference (RNAi), has practical implications for functional genomics. As shown by two recent reports, RNAi provides a rapid method to test the function of genes in the nematode Caenorhabditis elegans; most of the genes on C. elegans chromosome I and III have now been tested for RNAi phenotypes. The results validate RNAi as a powerful functional genomics tool for C. elegans, and point the way for similar large-scale studies in other species.

Animals↗

PubMed2000

Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis.

BACKGROUND: The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. RESULTS: Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure as nuclear inheritance is unaffected. CONCLUSIONS: C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor protein.

Animals↗

PubMed2000

Prolyl 4-hydroxylase is required for viability and morphogenesis in Caenorhabditis elegans.

The genome of Caenorhabditis elegans possesses two genes, dpy-18 and phy-2, that encode alpha subunits of the enzyme prolyl 4-hydroxylase. We have generated deletions within each gene to eliminate prolyl 4-hydroxylase activity from the animal. The dpy-18 mutant has an aberrant body morphology, consistent with a role of prolyl 4-hydroxylase in formation of the body cuticle. The phy-2 mutant is phenotypically wild type. However, the dpy-18; phy-2 double mutant is not viable, suggesting an essential role for prolyl 4-hydroxylase that is normally accomplished by either dpy-18 or phy-2. The effects of the double mutation were mimicked by small-molecule inhibitors of prolyl 4-hydroxylase, validating the genetic results and suggesting that C. elegans can serve as a model system for the discovery of new inhibitors.

Amino Acid Sequence↗

PubMed2000

Caenorhabditis elegans.

Transparent, free-living nematode worm. Unsegmented body plan with full set of differentiated tissues (neural, endoderm, ectoderm and muscle). Genome size approximately 97 Mb, as five autosomes and one X sex chromosome. Fully sequenced genome, which comprises approximately 20 000 predicted genes. Defined cell lineage. Has made major contribution to studies of development, cell-to-cell signalling, cell ageing and cell death processes. Large-scale gene deletion, microarray analysis of gene expression and two-hybrid protein interaction analysis projects under way. Comparative studies mainly with C. briggsae, but also with other free-living and parasitic nematodes.

Animals↗

PubMed1999

NANOS-3 and FBF proteins physically interact to control the sperm-oocyte switch in Caenorhabditis elegans.

BACKGROUND: The Caenorhabditis elegans FBF protein and its Drosophila relative, Pumilio, define a large family of eukaryotic RNA-binding proteins. By binding regulatory elements in the 3' untranslated regions (UTRs) of their cognate RNAs, FBF and Pumilio have key post-transcriptional roles in early developmental decisions. In C. elegans, FBF is required for repression of fem-3 mRNA to achieve the hermaphrodite switch from spermatogenesis to oogenesis. RESULTS: We report here that FBF and NANOS-3 (NOS-3), one of three C. elegans Nanos homologs, interact with each other in both yeast two-hybrid and in vitro assays. We have delineated the portions of each protein required for this interaction. Worms lacking nanos function were derived either by RNA-mediated interference (nos-1 and nos-2) or by use of a deletion mutant (nos-3). The roles of the three nos genes overlap during germ-line development. In certain nos-deficient animals, the hermaphrodite sperm-oocyte switch was defective, leading to the production of excess sperm and no oocytes. In other nos-deficient animals, the entire germ line died during larval development. This germ-line death did not require CED-3, a protease required for apoptosis. CONCLUSIONS: The data suggest that NOS-3 participates in the sperm-oocyte switch through its physical interaction with FBF, forming a regulatory complex that controls fem-3 mRNA. NOS-1 and NOS-2 also function in the switch, but do not interact directly with FBF. The three C. elegans nanos genes, like Drosophila nanos, are also critical for germ-line survival. We propose that this may have been the primitive function of nanos genes.

Amino Acid Sequence↗

PubMed1999

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.

Animals↗

PubMed1998

Meiotic recombination in C. elegans initiates by a conserved mechanism and is dispensable for homologous chromosome synapsis.

Chromosome segregation at meiosis I depends on pairing and crossing-over between homologs. In most eukaryotes, pairing culminates with formation of the proteinaceous synaptonemal complex (SC). In budding yeast, recombination initiates through double-strand DNA breaks (DSBs) and is thought to be essential for SC formation. Here, we examine whether this mechanism for initiating meiotic recombination is conserved, and we test the dependence of homologous chromosome synapsis on recombination in C. elegans. We find that a homolog of the yeast DSB-generating enzyme, Spo11p, is required for meiotic exchange in this metazoan, and that radiation-induced breaks partially alleviate this dependence. Thus, initiation of recombination by DSBs is apparently conserved. However, homologous synapsis is independent of recombination in the nematode, since it occurs normally in a C. elegans spo-11 null mutant.

Animals↗

PubMed1997

DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway.

Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains.

Cell Cycle Proteins↗