Biomedical subjects
D Pilgrim
Publications and source records attributed to D Pilgrim.
A regulatory cascade of three homeobox genes, ceh-10, ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in C. elegans.
The development of the nervous system requires the coordinated activity of a variety of regulatory factors that define the individual properties of specific neuronal subtypes. We report a regulatory cascade composed of three homeodomain proteins that act to define the properties of a specific interneuron class in the nematode C. elegans. We describe a set of differentiation markers characteristic for the AIY interneuron class and show that the ceh-10 paired-type and ttx-3 LIM-type homeobox genes function to regulate all known subtype-specific features of the AIY interneurons. In contrast, the acquisition of several pan-neuronal features is unaffected in ceh-10 and ttx-3 mutants, suggesting that the activity of these homeobox genes separates pan-neuronal from subtype-specific differentiation programs. The LIM homeobox gene ttx-3 appears to play a central role in regulation of AIY differentiation. Not only are all AIY subtype characteristics lost in ttx-3 mutants, but ectopic misexpression of ttx-3 is also sufficient to induce AIY-like features in a restricted set of neurons. One of the targets of ceh-10 and ttx-3 is a novel type of homeobox gene, ceh-23. We show that ceh-23 is not required for the initial adoption of AIY differentiation characteristics, but instead is required to maintain the expression of one defined AIY differentiation feature. Finally, we demonstrate that the regulatory relationship between ceh-10, ttx-3 and ceh-23 is only partially conserved in other neurons in the nervous system. Our findings illustrate the complexity of transcriptional regulation in the nervous system and provide an example for the intricate interdependence of transcription factor action.
Caenorhabditis elegans UNC-45 is a component of muscle thick filaments and colocalizes with myosin heavy chain B, but not myosin heavy chain A.
In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B-dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.
unc-45 gene of Caenorhabditis elegans encodes a muscle-specific tetratricopeptide repeat-containing protein.
The unc-45 gene of the nematode, Caenorhabditis elegans, is essential for muscle organization and embryonic development. Genetic evidence suggests the unc-45 gene product controls muscle thick filament assembly. We report here on the determination of the gene's chromosomal location and the isolation and sequencing of its cDNA. The amino terminus of the predicted unc-45 protein contains three tandem repeats that belong in the tetratricopeptide repeat family. Tetratricopeptide motifs have been shown to be involved in protein interactions, and some of the closest homologues have chaperone-like activity. The carboxy terminus of the protein has homology with the related fungal proteins, CRO1 and She4p, which have been postulated to play a role in assembly of or interactions with a cytoplasmic myosin. We have also determined the sequence of the homologous gene from C. briggsae, which demonstrates a high level of conservation. We show that the unc-45 gene promoter can drive reporter gene expression, which is limited to muscle tissues (pharyngeal, body wall, vulval, and anal muscles), consistent with a role for the unc-45 gene in muscle development or function.
Sex and the single worm: sex determination in the nematode C. elegans.
The study of sex determination in model organisms has been especially fruitful in increasing our understanding of developmental biology, gene regulation and evolutionary mechanisms. The free living nematode, Caenorhabditis elegans, can develop as one of two sexes; male or self-fertilizing hermaphrodite. Here we discuss the progress toward a genetic and molecular understanding of that decision. Numerous genetic loci have been identified that affect sexual fate, and epistasis analysis of these genes has led to a model of a regulatory hierarchy with stepwise negative interactions. It is becoming evident that many of the genes have numerous levels of regulation. We also discuss the apparent rapid rate of evolution that many of the sex determination proteins have undergone. Protein sequences of homologues from closely related species are more divergent than homologues of proteins involved in other developmental processes. Rapid evolution of sex determination genes may be a common theme throughout the animal kingdom.
GERp95, a membrane-associated protein that belongs to a family of proteins involved in stem cell differentiation.
A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998; Cox et al., 1998; Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes.
Analysis of two cosmid clones from chromosome 4 of Drosophila melanogaster reveals two new genes amid an unusual arrangement of repeated sequences.
Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing approximately 5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus (ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met-hepatocyte growth factor receptor. The other cosmid contains only the two short 5'-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the beta-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome.
The wisdom of lay knowledge: a reply to Loughlin and Prichard.
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Molecular evolution of a sex determination protein. FEM-2 (pp2c) in Caenorhabditis.
Somatic sex determination in Caenorhabditis elegans involves a signal transduction pathway linking a membrane receptor to a transcription factor. The fem-2 gene is central to this pathway, producing a protein phosphatase (FEM-2) of the type 2C (PP2C). FEM-2 contains a long amino terminus that is absent in canonical PP2C enzymes. The function of this domain is difficult to predict, since it shows no sequence similarity to any other known proteins or motifs. Here we report the cloning of the fem-2 homologue from Caenorhabditis briggsae (Cb-fem-2). The sequence identity is much higher than that observed for other C. briggsae homologues of C. elegans sex determination proteins. However, this level is not uniform across the entire lengths of the proteins; it is much lower in the amino termini. Thus, the two domains of the same protein are evolving at different rates, suggesting that they have different functional constraints. Consistent with this, Cb-FEM-2 is able to replace some, but not all, of the Ce-FEM-2 in vivo function. We show that removal of the amino terminus from Ce-FEM-2 has no effect on its in vitro phosphatase activity, or its ability to replace the in vivo function of a yeast PP2C enzyme, but that it is necessary for proper FEM-2 function in worms. This demonstrates that the amino terminus is not an extended catalytic domain or a direct negative regulator of phosphatase activity.
CeRep25B forms chromosome-specific minisatellite arrays in Caenorhabditis elegans.
With the completion of the Genome Sequencing Project, it is now possible to rapidly and accurately determine the frequency and position of a particular repeat sequence in the Caenorhabditis elegans genome. Several repeat sequences with a variety of characteristics have been examined and with few exceptions they show a near-random distribution throughout the genome. We characterized several genes near the left end of Chromosome III in the C. elegans genome, and found a 24-bp minisatellite repeat sequence present in the introns of two unrelated genes. This prompted a search of the databank for other occurrences of this sequence. Multiple copy arrays of this repeat are all located on the same autosome and fall in two clusters: one near the left end, and one in the central region separated by approximately 10 Mb. There are >200 copies of this repeat on the chromosome. This euchromatic repeat sequence seems unrelated to gene expression, is absent from homologous sites in a related species, is unstable in Escherichia coli, and is polymorphic between different wild isolates of C. elegans. Most CeRep25B units in the array match the consensus sequence very well, suggesting that either this repeat originated quite recently or its sequence is functionally constrained. Although chromosome-specific repeat sequences have been reported previously in many organisms, such sequences are usually structural and heterochromatic (e.g., centromeric alpha-satellite) or on the mammalian sex chromosomes. This report describes the first confirmed instance from a whole genome sequencing project of an autosomal euchromatic chromosome-specific minisatellite repeat.
Conservation of function and expression of unc-119 from two Caenorhabditis species despite divergence of non-coding DNA.
The Caenorhabditis briggsae homologue of the Caenorhabditis elegans neuronal gene unc-119 has been cloned by low-stringency hybridization. Genomic clones containing the C. briggsae gene are able to completely rescue the unc-119 phenotype in transgenic C. elegans mutants. The open reading frame (ORF) of the predicted C. briggsae cDNA is 90% identical to that of C. elegans. Although the splice donor and acceptor sites are conserved, the untranslated regions, and the introns, differ greatly. For this gene, the average intron size in C. elegans is over 600 base pairs (bp); in C. briggsae it is only 113 bp. Their upstream control regions share limited sequence similarities; however, reporter gene fusions of the two species show strongly similar expression in C. elegans. These results are consistent with the maintenance not only of the function of the unc-119 gene but also the transcriptional control of the gene through tens of millions of years of evolution.
Control of C. elegans larval development by neuronal expression of a TGF-beta homolog.
The Caenorhabditis elegans dauer larva is specialized for dispersal without growth and is formed under conditions of overcrowding and limited food. The daf-7 gene, required for transducing environmental cues that support continuous development with plentiful food, encodes a transforming growth factor-beta (TGF-beta) superfamily member. A daf-7 reporter construct is expressed in the ASI chemosensory neurons. Dauer-inducing pheromone inhibits daf-7 expression and promotes dauer formation, whereas food reactivates daf-7 expression and promotes recovery from the dauer state. When the food/pheromone ratio is high, the level of daf-7 mRNA peaks during the L1 larval stage, when commitment to non-dauer development is made.
Cross-screening: a new method to assemble clones rapidly and unambiguously into contigs.
We have developed a new procedure that relies on an array of cross-hybridization tests to order a set of random clones into a contig. The method, called cross-screening, uses each clone as a target and its end sequences as probes, in a matrix of reciprocal cross-hybridization tests performed on a single blot. The relationships among the clones are determined rapidly from the pairwise tests, allowing clone order to be determined directly. We have applied this technique to DNAs from a set of overlapping lambda clones from Drosophila chromosome 4. The location and orientation of each clone derived from the cross-screening data was that expected from the map assembled from overlapping restriction sites and chromosomal walking. The procedure provided additional information on a previously unknown, internally repeated DNA sequence. To demonstrate the general utility of the procedure, we have applied it to a previously described clone set within a contig in region 22q12 of human chromosome 22. The correct relative position and orientation of these clones were derived from the cross-screening data without knowledge of, or reference to, any nucleotide sequence or restriction site analysis of the DNA concerned. The cross-screening procedure is fast, economical, and robust and allows clone overlaps to be determined efficiently, with minimal interference from repeated DNA sequences. This new procedure is specifically designed for small groups of overlapping clones (tens to hundreds) and should facilitate the ordering of subclone libraries derived from small chromosomes or the large cloned inserts carried in YAC, BAC, and P1 vectors.
The pros and cons of immunisation.
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The C. elegans sex-determining gene fem-2 encodes a putative protein phosphatase.
The genetic and molecular analysis of genes involved in the regulation of sex determination in Caenorhabditis elegans suggests that the gene fem-2 plays an important role in regulating a pathway transducing a non-cell-autonomous signal to a nuclear transcription factor. The wild-type fem-2 gene was cloned by identifying sequences from the C. elegans physical map that could restore normal Fem-2 function to homozygous mutant fem-2 transgenic animals. cDNA sequences mapping to the minimal rescuing region correspond to an open reading frame with a sequence similar to protein phosphatase 2C enzymes from systems as diverse as yeast, humans, and plants, but the alignments suggest that FEM-2 falls into a separate class of proteins than the canonical homologues. Several fem-2 mutant alleles were sequenced, and the mutations are predicted to cause protein changes consistent with their observed phenotypes, such as missense mutations in conditional alleles, and a nonsense mutation in a predicted null allele. This is the first evidence implicating phosphorylation and/or dephosphorylation as a control mechanism in C. elegans sex determination.
Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system.
A spontaneous mutation affecting locomotion of the nematode Caenorhabditis elegans has been mapped to a new gene, unc-119. Phenotypic characterization of the mutants suggests the defect does not lie in the musculature and that the animals also have defects in feeding behavior and chemosensation. unc-119 has been physically mapped relative to a previously identified chromosomal break in linkage group III, and DNA clones covering the region can rescue the mutant phenotype in transgenic animals. Three more alleles at the locus, with identical phenotypes, have been induced and characterized, all of which are putative null alleles. The predicted UNC-119 protein has no significant similarity to other known proteins. Expression of an unc-119/lacZ fusion in transgenic animals is seen in many neurons, suggesting that the unc-119 mutant phenotype is due to a defect in the nervous system.
Mass childhood immunization: some ethical doubts for primary health care workers.
The mass childhood immunization programme has traditionally been viewed as a safe and effective preventative measure by health promoters, primary health care professionals and governments. This consensus has meant that immunization has rarely been viewed as ethically problematic. A number of recent changes in the context of the delivery of health care, particularly the emphasis on consumerism and the effect of the marketization of services, makes timely an examination of ethical, social and political issues. This article examines four main grounds for problematizing the mass childhood immunization programme. These are: clinical research evidence about the safety and efficacy of vaccines; the masking of wider social and political determinants of ill health; the contradictory strictures about collective and individual rights in relation to immunization; and the uniqueness of childhood immunization as a physical intrusion into a healthy body. The implications of these ethical issues are discussed in relation to informed consent and the need for a 'greenfield' review that includes the views of dissenting parents, lawyers and moral philosophers, as well as health professionals.
Comparison of neurologic changes in 'successfully aging' persons vs the total aging population.
OBJECTIVE: To determine age-related changes in neurologic function in the general population. DESIGN: To administer a neurologic examination to participants in such a way that it is possible to calculate the proportion of elderly persons in the population with each abnormal finding and the proportion of persons with each finding but without evidence of the medical and neurologic diseases likely to produce neurologic abnormalities (eg, stroke and diabetes). SETTING: Individuals were selected from a community-dwelling population. PARTICIPANTS: A stratified random sample of 467 persons aged 65 years and older were evaluated. RESULTS: Many neurologic abnormalities are increasingly common with advancing age and are present in a substantial portion of the elderly population. The prevalence of abnormal neurologic findings not attributable to disease, however, is substantially lower, typically one half to one third the total prevalence. Moreover, the increase with age in the prevalence of abnormal neurologic findings not attributable to major disease varies substantially among the different measures. CONCLUSIONS: Primitive reflexes and measures of gait show statistically significant increases with age in multiple measurement domains, suggesting a selective age-related vulnerability.