Herman RK (1978) Genetics "Crossover suppressors and balanced recessive lethals in Caenorhabditis elegans."

Herman RK

[Genetics, 1978]

Two dominant suppressors of crossing over have been identified following X-ray treatment of the small nematode C. elegans. They suppress crossing over in linkage group II (LG II) about 100-fold and 50-fold and are both tightly linked to LG II markers. One, called C1, segregates independently of all other linkage groups and is homozygous fertile. The other is a translocation involving LG II and X. The translocation also suppresses crossing over along the right half of X and is homozygous lethal. C1 has been used as a balancer of LG II recessive lethal and sterile mutations induced by EMS. The frequencies of occurrence of lethals and steriles were approximately equal. Fourteen mutations were assigned to complementation groups and mapped. They tended to map in the same region where LG II visibles are clustered.

Suzuki N et al. (1995) International C. elegans Meeting "ISOLATION OF DAUER-CONSTITUTIVE MUTANTS ON THE UNC-31 BACKGROUND"

Ishihara T, Katsura I, Suzuki N

[International C. elegans Meeting, 1995]

Many neural mutations that don't affect dauer formation by themselves (e.g. unc-31, unc-3 ) exhibit the dauer-constitutive (Daf-c) phenotype when they are combined to form double mutants[*]. We therefore isolated such synthetic Daf-c mutants on theunc-31 background to obtain new mutations that affect the nervous system. We screened 5539 F1 progenies derived from EMS-treated unc-31 worms and isolated 44 mutants that show the Daf-c phenotype on the unc-31 background but not by themselves. We mapped one of the mutations to linkage group (LG) I, 4 to LG II, 1 to LG III, 2 to LG IV, 6 to LG V and 13 to LG X, while the other 17 mutations have not been mapped. Since some known synthetic Daf-c mutations (e.g. osm-1 ) are defective in the uptake of fluorescein dye DiO into amphids and phasmids, we checked it for 42 of the 44 mutants. Thirty-four of them were normal in the uptake of DiO, while 6 of them were completely defective, and 2 of them showed incompletely penetrant defects. [*]WBG, Vol.13, No.2, P.36; Abstract by Katsura et al. this meeting

Vijayaratnam V et al. (1995) International C. elegans Meeting "A GENETIC TOOLKIT: BALANCERS AND DEFICIENCIES"

Ho F, Edgley ML, Gawne JM, Rose AM, Vijayaratnam V

[International C. elegans Meeting, 1995]

We are building a genetic toolkit for the C. elegans genome, consisting of regional sets of overlapping balanced deficiencies whose endpoints are tied to the physical map using PCR. Initial work has focused on creating and characterizing new genetic balancers for previously unbalanced regions. For LG X left, several new balancers have been made and characterized. One of these is most likely a male-lethal duplication attached to the X chromosome, and it balances from unc-1 to dpy-3. Three new balancers have been made for the region between the right endpoint of sDp2 and unc-75 on LG I. Preliminary characterization indicates these may be useful for mutant screens. The second stage of the project is to isolate and map deficiencies. Our lab is concentrating on the right half of LG I and the right half of LG III. The inversion hIn1 balances from unc-75 to the right end of LG I, and it has been used to isolate UV and gamma ray-induced mutations. PCR mapping of these has revealed three new deficiencies around unc-101 (hDf15, hDf16, hDf17). For LG III, analysis has centered on existing sets of balanced EMS and non-EMS lethals. Here we have identified two new deficiencies, sDf110 and sDf111, in the unc-32 and unc-116 regions. This work is funded by the NIH National Center for Research Resources (NCRR).

Vijayaratnam V et al. (1996) West Coast Worm Meeting "A GENETIC TOOLKIT: BALANCERS AND DEFICIENCIES"

Vijayaratnam V, Rose AM, Edgley ML, Gawne JM, Ho F

[West Coast Worm Meeting, 1996]

We are building a genetic toolkit for the C. elegans genome, consisting of regional sets of overlapping balanced deficiencies whose endpoints are tied to the physical map using PCR. Initial work has focused on creating and characterizing new genetic balancers for previously unbalanced regions. For LG X left, several new balancers have been made and characterized. One of these is most likely a male-lethal duplication attached to the X chromosome, and it balances from unc-1 to dpy-3. Three new balancers have been made for the region between the right endpoint of sDp2 and unc-75 on LG I. Preliminary characterization indicates these may be useful for mutant screens. The second stage of the project is to isolate and map deficiencies. Our lab is concentrating on the right half of LG I and the right half of LG III. The inversion hIn1 balances from unc-75 to the right end of LG I, and it has been used to isolate UV and gamma ray-induced mutations. PCR mapping of these has revealed three new deficiencies around unc-101 (hDf15, hDf16, hDf17). For LG III, analysis has centered on existing sets of balanced EMS and non-EMS lethals. Here we have identified two new deficiencies, sDf110 and sDf111, in the unc-32 and unc-116 regions. This work is funded by the NIH National Center for Research Resources (NCRR).

Johnsen RC et al. (1986) Worm Breeder's Gazette "saturation screen for lethals on linkage group V (left)."

Johnsen RC, Baillie DL

[Worm Breeder's Gazette, 1986]

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- five percent are on LG III (right). A Poisson calculation predicts that a screen of 15, 000 chromosomes should yield a mutation in ninety-five percent of the genes. We are in the process of screening another 10,900 'chromosomes' from which we have isolated 755 lethal and sterile mutations: 255 ( 36.8% ) of these are on LG V, 438 are on LG III lethal (and have been discarded) and the mutations of 62 strains have not yet been mapped. We are also screening 3000 0.07% formaldehyde treated 'chromosomes'. This should yield two results: 1) the mutation rate for 0.07% formaldehyde over a well characterized region, eT1 (III,V) and 2) a set of 20-30 short (1-2 m.u.) deficiencies on LG V (left). Breakpoints from these deficiences will divide LG V (left) into more zones than the fourteen that have already been established (see Rosenbluth et al. this issue). The mapping of the 200 plus lethals to specific zones and against known genes in those zones, will then give the distribution of essential genes along the chromosome. The mapping already done shows that they follow a non-uniform distribution.

McKim KS et al. (1987) Worm Breeder's Gazette "rearrangements of linkage group I."

Rose AM, McKim KS, Howell AM

[Worm Breeder's Gazette, 1987]

Our genetic analysis of LG I requires rearrangement breakpoints to accurately map a large number of mutations. We have been analyzing gamma radiation induced lethal mutations balanced by sDp2 and szT1(I:X) for deficiencies. So far only one deficiency (hDf6) has been found. hDf6 maps entirely within sDf4 and was isolated with szT1. We have found hDf6/hDf6/sDP2 worms are adult steriles. Three explanations that have occurred to us are: 1) the amount of gene product produced by the wild type alleles on sDp2 is not equal to the amount produced by the allele on the normal homologue, 2) sDp2 has a sterile mutation in the region deleted by hDf6, or 3) duplication of other loci results in sterility in combination with hDf6. The last possibility is not likely considering hDf6/hDf6/szT1(X) worms are viable. sT1(X) is a duplication of the left half of LG I (unc-13 to the bli-3 end) and the left end of LG X (from the lon-2 region to the unc-1 end). A second approach to isolating LG I rearrangements has been to shorten sDp2. dpy-5 dpy-5 sDp2 hermaphrodites are phenotypically wild type ( sDp2 carries the wild type alleles of dpy-5 and unc-11) and segregate only 'Wt' and DpyUnc progeny. There is no recombination between sDp2 and the normal homologues. From 16000 chromosomes treated with 1500 rads, 1 Dpy and 5 Unc F1 progeny were recovered. In a smaller experiment where 8000 chromosomes were treated with 3000 rads, 4 Dpy and 4 Unc F1 progeny were recovered. These are currently being characterized. Some events which we expected to recover in addition to shortened duplications are unlinked suppressors and point mutations or deletions within the duplication. At present, 4 of 5 analyzed have been found to be shortened derivatives of sDP2.In addition a new translocation between LG I and LG V (hT1) has been isolated. hT1(I;V) suppresses recombination from unc-42 to the left end of LG V (like eT1) and from unc-13 to the left end of LG I (like szT1).

Maduro MF et al. (1996) West Coast Worm Meeting "THE LG III DEFICIENCY eDf2 AND ASSOCIATED FREE DUPLICATION eDp6 ARE NOT MUTUALLY COMPLEMENTARY"

Maduro MF, Pilgrim DB

[West Coast Worm Meeting, 1996]

The deficiency eDf2 and the free duplication eDp6 were obtained concomitantly following acetaldehyde mutagenesis (Hodgkin, 1980). These two aberrations have been useful in mapping many LG III loci, which have demonstrated that eDp6 contains the right third of LG III and eDf2 contains the left portion. We used the eDf2 and eDp6 breakpoints as physical markers to clone unc-119, and showed that the genomic region corresponding to the unc-119 cDNA is completely absent. The gene unc-119 is the only LG III locus to not map to either eDf2 or eDp6. Our interest in these aberrations was renewed when we ran across a report by Starich et al. on the isolation of dyf-2 which maps to the far end of LG III(R) and is complemented by both eDf2 and eDp6. Using a plasmid mini-library approach, we walked across the eDf2 breakpoint, and probed the YAC grid to find that eDf2 contains sequences from the far right end of III. What is strange is that these same sequences are also present on eDp6, suggesting that eDf2 and eDp6 do not result from a simple chromosomal break, and the use of these rearrangements for mapping genes on LG III(R) should be approached with caution. The mapping evidence will be summarized and a model for the creation of eDf2 and eDp6 will be given.

Malek Jundi et al. (2004) East Coast Worm Meeting "Cytological screening of the germ lines of sterile mutants for meiotic defects"

Monique C Zetka, Malek Jundi

[East Coast Worm Meeting, 2004]

Extensive screening for mutations in essential genes has been carried out for defined chromosomal regions on LG I (Linkage Group; Rose et al. 1980: Howell et al. 1987; Howell and Rose 1990; McKim et al. 1992; Johnsen et al. 2000), LG II (Sigurdson et al. 1984), LG IV (Rogalski et al. 1982; Rogalski and Baillie 1985; Clark et al. 1988; Clark and Baillie 1992), LG V (Rosenbluth et al. 1988; Johnsen and Baillie 1991) and LG X (Meneely and Herman 1979, 1981). A subset of the alleles in this mutant library result in sterility and are likely to have identified genes essential for the development of the gonad and the germ line. Because the germ line of the nematode presents an orderly and predictable progression of nuclei through the various stages of proliferation and meiosis, the function of the wild-type gene can be inferred by examining the DAPI-stained nuclei of sterile animals for cytologically visible defects. We are screening a collection of 138 previously uncharacterized sterile mutants originating from these analyses for the presence of univalents or chromosome fragments at diakinesis, which is a general indicator of defects in upstream processes like meiotic cell-cycle progression, chromosome pairing, synapsis, recombination, and chromosome morphogenesis. To date, we have identified four sterile mutations that result in a high frequency of univalents at diakinesis and that have associated defects in conserved meiotic processes. Supported by a grant from the Canadian Institutes of Health Research (CIHR)

Grant BD et al. (1998) East Coast Worm Meeting "Endocytosis Mutants and The Yolk Receptor in C. elegans"

Grant BD, Wallenstein E, Pedraza LG, Zhang YH, Hirsh DI

[East Coast Worm Meeting, 1998]

We are using C. elegans genetics to look for new genes involved in receptor-mediated endocytosis. We have isolated 23 mutants, in 11 complementation groups, defective in the endocytosis of YP170-GFP by oocytes. Some of these mutants are temperature sensitive lethals. We anticipate that our mutants will identify key components of the general endocytic machinery as well as gene products that specifically mediate yolk uptake. The most severe YP170-GFP uptake defective mutants identify a gene encoding a putative receptor for vitellogenins (yolk proteins) in the C. elegans oocyte. During the normal maturation of oocytes within the C. elegans germline yolk proteins (YPs), encoded by the vit genes and secreted by the intestine, are actively endocytosed from the psuedocoelomic space and stored within membrane bound compartments sometimes called yolk platelets. A vit-2::gfp transgene, encoding YP170-GFP, has allowed us to visualize this process in vivo and study its properties by fluorescence microscopy. RNAi analysis in this genetic background allowed us to test the function of potential components of the C. elegans endocytic machinery identified by the genome project. This analysis indicates that YP170 endocytosis requires clathrin coated pit components (clathrin heavy chain, adaptins, dynamin) and regulators of endosomal function (rab5, rab7, rab11), as expected from studies of lipoprotein uptake in other organisms. 	One complementation group, comprised of three alleles, appears to mutate T11F8.c, a partially sequenced member of the LRP family of lipoprotein and proteinase receptors. We are currently confirming this inference by transformation rescue and sequencing of mutant alleles. RNAi data by Bossinger et al. (1997 IWM Abstract 61) had previously indicated a possible role for T11F8.c in yolk uptake. Our results show that T11F8.c RNAi blocks YP170-GFP uptake. We are tentatively naming this gene lrp-2, in anticipation of the complete genomic sequence which is in progress at the sequencing consortium (Many thanks to Bob Waterston and Stephanie Chissoe). Immunofluorescence experiments, using polyclonal antisera we produced against intracellular or extracellular LRP-2 fusion proteins, indicate that LRP-2 is an abundant plasma membrane protein expressed specifically in oocytes. Our putative lrp-2 mutants all show an absence or mislocalization of LRP-2 in oocytes. 	We are currently engaged in a more detailed phenotypic analysis of endocytic defects in the other ten complementation groups identified in our genetic screen utilizing anti-LRP-2 antibodies and other specific antisera we have developed against general endocytic pathway components identified in our RNAi screen (see abstract by Y. Zhang et al.).

Sigurdson DC et al. (1984) Genetics "Caenorhabditis elegans deficiency mapping."

Herman RK, Spanier GJ, Sigurdson DC

[Genetics, 1984]

Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans. Complementation tests between the deficiencies and ethyl methanesulfonate-induced recessive visible, lethal and sterile mutations and between different deficiencies were used to characterize the extents of the deficiencies. Deficiency endpoints thus helped to order 36 sites within a region representing about half of the loci on LG II and extending over about 5 map units. New mutations occurring in this region can be assigned to particular segments of the map by complementation tests against a small number of deficiencies; this facilitates the assignment of single-site mutations to particular genes, as we illustrate. Five sperm-defective and five oocyte- defective LG II sterile mutants were identified and mapped. Certain deficiency-by-deficiency complementation tests allowed us to suggest that the phenotypes of null mutations at two loci represented by visible alleles are wild type and that null mutations at a third locus confer a visible phenotype. A segment of LG II that is about 12 map units long and largely devoid of identified loci seems to be greatly favored for crossing over.