tharris's Johnsen:RC [55 articles]

Abstract

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5' deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 ...

Abstract

We describe the molecular characterisation of Caenorhabditis elegans dpy-14, a gene encoding an essential cuticular collagen annotated as col-59. Expression of dpy-14 starts at the 16 E cell stage, making it the earliest-expressing collagen reported to date. SAGE data and dpy-14 promoter::GFP reporter constructs indicate that the gene is transcribed mainly during embryogenesis, specifically in ciliated neurons and hypoderm. Water permeability assays and lectin staining showed that a mutation in the DPY-14 collagen results in defects in the channels of the ...

Abstract

10.1074/jbc.M505701200 Identification of -regulatory elements and their binding proteins constitutes an important part of understanding gene function and regulation. It is well accepted that co-expressed genes tend to share transcriptional elements. However, recent findings indicate that co-expression data show poor correlation with co-regulation data even in unicellular yeast. This motivates us to experimentally explore whether it is possible that co-expressed genes are subject to differential regulatory control using the excretory cell of as an example. Excretory cell is a functional ...

Abstract

C. elegans dpy-14 encodes an essential collagen required for proper L1 cuticle formation. Study of dpy-14 promoter::GFP reporter constructs indicates that the gene is predominantly expressed during embryogenesis and supports SAGE data in revealing high levels of gene expression in both the hypodermis and ciliated neurons. Specifically, expression was observed in cells of AB lineage that have the potential to differentiate into amphids and their support cells. Furthermore, dpy-14 possesses the earliest known expression for a collagen gene.Animals carrying the dpy-14(e188) ...

Abstract

We report molecular genetic studies of three genes involved in early germ-line proliferation in Caenorhabditis elegans that lend unexpected insight into a germ-line/soma functional separation of autosomal/X-linked duplicated gene pairs. In a genetic screen for germ-line proliferation-defective mutants, we identified mutations in rpl-11.1 (L11 protein of the large ribosomal subunit), pab-1 [a poly(A)-binding protein], and glp-3/eft-3 (an elongation factor 1-alpha homolog). All three are members of autosome/X gene pairs. Consistent with a germ-line-restricted function of rpl-11.1 and pab-1, mutations in these ...

Abstract

Taking advantage of the recent availability of the whole genome sequence of Caenorhabditis briggsae, a closely related nematode to Caenorhabditis elegans, we have examined the chemosensory gene superfamily by using comparative genomic methods. We have identified a chemosensory gene family, serpentine receptor class ab (srab), which exists in both species with 25 members in C. elegans and 14 members in C. briggsae. More than 20% of these gene models are reannotated. The srab family is similar to, but distinct from, the ...

Abstract

Tissue-specific alternative pre-mRNA splicing is essential for increasing diversity of functionally different gene products. In Caenorhabditis elegans, UNC-60A and UNC-60B, nonmuscle and muscle isoforms of actin depolymerizing factor (ADF)/cofilin, are expressed by alternative splicing of unc-60 and regulate distinct actin-dependent developmental processes. We report that SUP-12, a member of a new family of RNA recognition motif (RRM) proteins, including SEB-4, regulates muscle-specific splicing of unc-60. In sup-12 mutants, expression of UNC-60B is decreased, whereas UNC-60A is up-regulated in muscle. sup-12 mutations ...

Abstract

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous developmental disorder whose molecular basis is largely unknown. Here, we show that mutations in the Caenorhabditis elegans bbs-7 and bbs-8 genes cause structural and functional defects in cilia. C. elegans BBS proteins localize predominantly at the base of cilia, and like proteins involved in intraflagellar transport (IFT), a process necessary for cilia biogenesis and maintenance, move bidirectionally along the ciliary axoneme. Importantly, we demonstrate that BBS-7 and BBS-8 are required for the normal localization/motility ...

Abstract

GFP, as a transcriptional reporter or fusion tag, is a powerful and widely used tool for elucidating temporal and spatial patterns of gene expression. When using GFP for expression profiling, information about signal termination is limited. To address this issue, a dsRed variant, Fluorescent Timer (Clontech), which has the unique property of changing its emission fluorescence peak as the chromophore matures is being employed. The protein initially fluoresces green (480nm) due to a GFP-like intermediate, which upon subsequent modification, assumes a ...

Abstract

The purpose of this study is to characterize dpy-14 , a gene that codes for a collagen type III(alpha1). This gene is presently annotated on the WormBase database as col-59 . DNA sequencing analysis of the dpy-14 canonical allele, e188 , has revealed a mutation in the third exon, at position 747. Specifically, the codon GGA, coding for Gly, is changed to AGA, a codon for Arg. The severity of the Dpy phenotype in e188 mutants increases with higher temperatures, ranging ...

Abstract

In C. elegans there are over 1000 putative GPCR genes, of which approximately 18 are predicted to code for rhodopsin-like G protein-coupled receptors (GPCRs). Heterotrimeric GTP-binding proteins mediate the response to a wide variety of neurotransmitters and hormones, all of which bind to GPCRs. I have chosen to work on M03F4.3, which is a candidate rhodopsin-like GPCR. Although M03F4.3 is believed to code for rhodopsin-like GPCR, its functions are not yet clear. To investigate its functions, a region upstream of the ...

Abstract

We have completed the analysis of lethals recovered using the duplication, sDp2. 250 essential genes have been identified in the left half of chromosome I by mutational analysis. 600 lethals were recovered after mutagenesis of 31,600 marked chromosomes balanced by sDp2. We have added 133 previously unidentified let genes, to our published collection of 117. In addition, lethal alleles of five known genes with visible mutants were recovered. The 250 essential genes are represented by 502 mutations. For 88 of the ...

Abstract

With the physical map essentially complete and the genomic sequencing well under way the challenge is to correlate biological information with genes positioned on the physical map. We have 450 mutants that map into the sDp2 region of chromosome I, which will identify or define approximately 175 essential genes. We are completing the mapping and complementation testing of these mutations, and initiating studies to characterize the defects displayed by the mutant phenotypes. The mutants are in a Dpy-5 Unc-13 genetic background ...

Abstract

Our laboratory is interested in the genetic characterization of the rol-3 gene of Caenorhabditis elegans. Worms homozygous for rol-3(e754) display left hand helical treads along the length of their bodies, left-hand helical twisted musculature, and an abnormal rolling movement1. Our interest in the rol-3 gene comes about for three reasons. First, R. C. Johnson has shown that the rol-3 locus is highly mutable, displaying a six fold increase in EMS hit frequency over the calculated average frequency for genes within the ...

Abstract

The genomic organization of the eT1 balanced region of LGV(left) has been studied extensively in our laboratory. I am attempting to correlate the genetic and physical maps in the LGV(left) region. I am using microinjection to identify the cosmids containing lethal alleles around the unc-60 locus. unc-60 was rescued using the cosmid F53E2 and we have identified a lethal allele of unc-60, s1586. A Tc1 polymorphism corresponding to a lin-40 allele was identified and was mapped by Sulston and Coulsen to ...

Abstract

Mutations in daf-7 result in constitutive formation of dauer larvae, even in abundant food. This gene has been localized by a combination of genetic and RFLP mapping, DNA transformation, and Tcl tagging. On a chromosomal walk toward fem-2, one of us (D. Pilgrim) identified a cosmid (DE9) that transformed a daf-7 mutant to wild-type when injected into the mutant germline. Cosmid DE9, which contains an insert of <30kb, was used to probe both a northern blot and a CDNA library constructed ...

Abstract

We have identified the majority (approximately 69%) of essential genes in the eTl balanced region of Linkage Group V(left). The region comprises approximately 7% of the recombinational distance in Caenorhabditis elegans. 10,900 EMS treated Fl's were screened, yielding 194 lethal mutations on LGV(left). The analysis of 166 of those lethals resulted in the identification of one deficiency (sDf56) and alleles of 74 essential genes including 38 new genes. A total of 101 genes on LGV(left) with recessive lethal alleles have now ...

Abstract

With the physical map and the genomic sequencing nearing completion the challenge is to correlate biological information with the predicted genes. The approach we have taken is to do: 1) Large scale screening for lethal mutants in balanced regions of the genome; 2) Mapping the mutants to smaller sub-regions; and 3) Rescuing of mutant phenotypes using transgenic strains containing sequenced cosmids. In collaboration with D. Janke and D. Baillie at SFU, and the C. elegans sequencing labs, we are using transgenic ...

Abstract

The developmental regulation gene lin-40 has been mapped to the left arm of chromosome V of Caenorhabditis elegans. It is an essential gene with homozygous arrest phenotypes ranging from early-mid larval to maternal effect lethal. Various alleles also dominantly affect the timing of the switch from sperm to egg production in the hermaphrodite, and thus, self brood size. Alleles that both increase and decrease the number of offspring have been observed. The locus is a complex one. The sixteen known alleles ...

Abstract

We are developing a genetic map of Caenorhabditis briggsae in order to facilitate comparative studies with Caenorhabditis elegans. The two nematode species are morphologically similar but are approximately 25 - 40 million years apart, which is about one-half the 60 - 90 million year evolutionary distance between mice and humans. Insights gained by the studies of the two nematode species should help in the studies of the two mammals. The 100 million base pair C. elegans genome is effectively completely sequenced ...

Abstract

We are continuing our attempt to saturate and map essential genes and steriles on the left half of Linkage Group V (see Johnsen and Baillie WBG Vol.9 #2). We have screened 10,900 'chromosomes', treated with 0.012M EMS. We have isolated 751 lethal and sterile mutations: 242 strictly on LGV, 451 strictly on LGIII and 29 lines with a lethal on both LGV and LGIII. All lines but the ones carrying mutations strictly on LGV have been discarded. We have begun the ...

Abstract

We have established a dose-response curve for the formaldehyde induction of recessive lethals in the region balanced by eT1(III,V). We used Moerman and Baillie's (Mutation Research, 80(1981) 273-279) method of formaldehyde mutagenesis. The lethal induction frequencies that we found were: Figure 1 We found that 65% of all the mutations lie on LGIII and 35% lie on LGV which is very similar to our EMS results, see Johnsen and Baillie WBG Vol.9 #2. There was no significant difference in the ratio ...

Abstract

Below is our current deficiency map for LGV(left). Since the last Newsletter: (1) sDf28 and sDf30 have changed (2) more mutations have been mapped and (3) more lethal gene assignments have been made. As more deletion breakpoints are mapped, the resulting new zones will aid in the task of identifying all essential genes in this region. [See Figure 1] ...

Abstract

We are attempting to saturate and map essential genes and steriles on the left half of linkage group V. This region ( 20 map units and 7% of the entire genome) is balanced by the crossover suppressor eT1( III;V) which also balances the right arm of LG III. From an initial screen of 2738 'chromosomes', treated with 0.012M EMS, we have isolated 147 eT1 balanced recessive lethal and sterile mutations. Thirty-five percent of these lie on LG V (left) and sixty- ...

Abstract

Our analysis of LGV(left) is continuing. At present 10 deletions, providing 13 different breakpoints, divide the region into 14 zones ( figure). The table shows recessive lethal mutations that have been mapped to the zones. [See Figure 1] At Cold Spring Harbor (1985) we noted that deletion mapping placed unc-34 to the left of unc-60. This has been additionally confirmed by 3-factor mapping. The two genes are 0.8 - 3.4 m.u. apart. Another gene, let-336 has now been deletion-mapped to the ...

Abstract

Spotting homozygous end deficiencies ...

Abstract

Mutations in daf-7 result in constitutive formation of dauer larvae, even in abundant food. This gene has been localized by a combination of genetic and RFLP mapping, DNA transformation, and Tc1 tagging. On a chromosomal walk toward fem-2 ,one of us (D. Pilgrim) identified a cosmid (DE9) that transformed a daf-7 mutant to wild-type when injected into the mutant germline. Cosmid DE9 was used to probe a cDNA library constructed by Stuart Kim. Twenty cDNA clones of various sizes up to ...

Abstract

Our laboratory has isolated 12 lethal alleles of the Caenorhabditis sting that the rol-3 gene performs an essential function. In an effort to locate genes whose products may interact with that of rol-3, and whose identity may shed light on the function of rol-3 itself, we have utilized the temperature sensitive homozygous lethal phenotype of rol-3(s1040) in the generation of intergenic suppressors. These suppressors have been named srl for suppressor of roller lethal. The srl alleles are all recessive suppressors of ...

Abstract

We are currently involved in the genetic analysis of the rol-3 gene of Caenorhabditis elegans. The rol-3 gene resides on the eT1 balanced region of LGV(left) within zone 18 as defined by the deficiencies sDf35 and mDf3 (1). The original rol-3 allele (e754) was EMS induced by Brenner(2). Worms homozygous for e754 display left-handed helical treads along the length of their bodies as opposed to linear ones, and have left-handed helical musculature as well. The roller mutant displays an abnormal crawl. ...

Abstract

We have completed our study on the effects of 14 heterozygous LGV( left) deficiencies on recombination in adjacent regions. The deficiency chromosomes were marked with unc-46(e177) (1-3). Ten deficiencies ('Major Inhibitors') severely inhibited recombination between themselves and unc-46 (Fig. 1). In contrast, the remaining 4 deficiencies were 'Minor Inhibitors' (marked with '*' in Fig. 1). [See Figure 1] The effects of major inhibitors on recombination in different intervals was measured. Fig. 2 gives the recombination rates in terms of percent of ...

Abstract

From a Tc1 mutagenesis screen in the nT1(IV,V) balanced region using mut-4 RW7037, six lethal mutations that fail to complement lin-40 were recovered (Clark et al. In Press Genome). We have extracted DNA from the six strains containing lin-40 Tc1 mutations. We digested the DNA with EcoRI, ran it on a gel, transfered the DNA to a filter and hybridized nick translated Tc1 DNA to it. Three strains showed a common new Tc1 band. For one allele (s1351) we recovered the ...

Abstract

We are attempting to identify all the essential genes in a large genomic region. A new set of 242 strains carrying 0.012M EMS induced lethal and sterile mutations on the 23 m.u. of LGV(left) that is balanced by the translocation eT1(III:V) are being analyzed. To facilitate this work, the break points of 31 deficiencies and 1 duplication have been mapped and used to subdivide LGV(left) into 36 zones. Prior to this study 59 essential genes had been identified on LGV(left) (Rosenbluth, ...

Abstract

In order to determine factors governing meiotic pairing for recombination in C. elegans, we decided to take advantage of our LGV( left) deficiencies. In the course of their mapping, we had noticed that certain deficiencies inhibited recombination well beyond their respective breakpoints (Rosenbluth, Rogalski, Johnsen, Addison and Baillie, 1988, Genet. Res., in press). We are now systematically analyzing the degree and extent of recombination suppression caused by deleting specific regions in LGV(left). The origins of most deficiencies used are described in ...

Abstract

The duplication sDp30(V:X), on the X chromosome, carries wild type copies of dpy-11(V) and surrounding LGV genes. It arose in a strain carrying a gamma ray induced deficiency, sDf30(V), and presumably was a transposition of most (but not all) the genes deleted by the deficiency. [See Figure 1] The duplication was discovered when conflicting results were obtained from reciprocal complementation tests between the following individuals: dpy-18/dpy-18(III):unc-46 crossed to dpy-18/dpy-18(III);(unc-46) /eT1(V), - [where let-x(sy) was an allele of one of the genes ...

Abstract

A Tc1 mutagenesis screen in the nT1(IV,V) balanced region using mut- 4 RW7037 was performed (McKim et al. C. elegans Abstracts CSH 1987). They isolated 28 lethal mutations, 24 of which mapped to LGV. We substituted the balancer eT1(III,V) for nT1(IV,V). At least two of the 24 lethals on LGV balanced by nT1 were not balanced by eT1, these were not analyzed further. Of the rest: one was too sick to work with; six are still being analyzed; two define new ...

Abstract

We have analyzed a region of approximately 5.4 million base pairs for mutations, which under standard laboratory conditions result in developmental arrest, sterility, or maternal-effect lethality in Caenorhabditis elegans. Lethal mutations were isolated, maintained, and genetically manipulated as homozygotes using sDp2--a duplication of the left half of chromosome I. All of the lethals and rearrangements used in this analysis were balanced by sDp2. Relatively low doses of mutagen, (approximately 15 mM ethylmethane sulfate; EMS), were used so as to limit the ...

Abstract

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 ...

Abstract

The Caenorhabditis elegans rol-3(e754) mutation is a member of a general class of mutations affecting gross morphology, presumably through disruption of the nematode cuticle. Adult worms homozygous for rol-3(e754) exhibit rotation about their long axis associated with a left-hand twisted cuticle, musculature, gut and ventral nerve cord. Our laboratory previously isolated 12 recessive lethal alleles of rol-3. All these lethal alleles cause an arrest in development at either early or mid-larval stages, suggesting that the rol-3 gene product performs an essential ...