Kelly WG et al. (1994) Worm Breeder's Gazette "A Defined Set of Lethal Mutations at the Right End of the LGII Cluster."

Fire A, Kelly WG

[Worm Breeder's Gazette, 1994]

A Defined Set of Lethal Mutations at the Right End of the LGII Cluster. William G. Kelly and Andrew Fire, Dept. Embryology, Carnegie Institution of Washington, Baltimore, MD. The cey-l gene encodes a DNA binding protein which exhibits specific binding to two sites in the internal enhancer of unc-54 1. cey-l is expressed in oocytes and ubiquitously in the early embryo, but later becomes restricted to mesoderm2. This suggests CEY-1 could play a role in specifying mesodermal patterns of gene expression. To elucidate the role of cey-l in normal development, we have pursued two approaches toward isolating mutations: 1) An EMS screen for visible and lethal mutations in the region of LG II where cey-l has been physically mapped; and 2) Tcl-based screens (i.e., sending off primers to Ronald Plasterk and Marianne de Vroomen). This article summarizes the current state of the EMS screens. The screens were carried out primarily by cloning wild type Fl progeny of [unc 4/mnCI males] X [mutagenized unc-4 hermaphrodites]. In these screens, 840 mnCl balanced visible and lethal mutations were recovered from 8954 mutagenized chromosomes. These mutants were then tested for complementation with mnDf90. Of the 840 candidates tested, 84 failed complementation with mnDf90; these were then tested against mnDf87 and mnDp7. In total, 7 zygotic lethal complementation groups, but no visible mutations, were found to lie within an mnDF9OimnDf77 overlap that lies outside of mnDp7.

Dunn MA et al. (1999) Worm Breeder's Gazette "A PIE-1-BASED VECTOR FOR MATERNAL EXPRESSION OF GFP FUSIONS."

Reese KJ, Seydoux GC, Dunn MA

[Worm Breeder's Gazette, 1999]

We have used sequences from the pie-1 gene to construct a vector designed to express GFP fusions in the adult germline and in early embryos. The pie-1 gene encodes a maternal RNA transcribed in the adult germline and inherited maternally in embryos (Mello et al., 1992, 1996; Tenenhaus et al., 1998). We have shown previously that an 8kb genomic fragment from the pie-1 locus is sufficient to tag PIE-1 with GFP and rescue a pie-1 mutation (Fig. 1; Dunn et al., Reese et al., 1998 East Coast Worm Meeting Abstracts). We now have modified this construct to allow expression of heterologous GFP fusions. The resulting vector (Fig. 2) contains the pie-1 promoter, the pie-1 3UTR, and the pie-1 large intron, all of which appear to be important for robust expression in the adult germline and in early embryos. We have tested this new vector in 4 configurations: as is (GFP alone), and with one of three different open-reading frames inserted at the SpeI site after the GFP: his-11 (histone H2B), C36E8.5 (beta tubulin), and act-1 (actin). These constructs were injected into N2 hermaphrodites using the Fire labs complex array injection method which permits germline expression of transgenes (Kelly et al., 1997, Genetics 146: 227-238). For all constructs, we obtained a number of lines (10% to 50% of all roller lines) with GFP expression in the germline and in early embryos. In most lines, expression was restricted to the F2 generation after injection. In all cases, the subcellular localization observed matched that expected for each fusion: throughout cells for GFP alone, on DNA for GFP:histone H2B, on centrosomes and meiotic (and more weakly mitotic) spindles for GFP:beta-tubulin, and on cell cortices and cell division remnants for GFP:actin. (In one GFP:actin line, we also saw nuclear GFP). In addition to germline expression, all lines also expressed GFP in a number of somatic cells. We are hopeful that this vector could be used by others to express additional GFP fusions in the germline and in early embryos. Anyone interested can obtain the pie-1 vector from Melanie Dunn at mdunn@jhmi.edu. Fig. 1. Fig. 2.

Ketting RF et al. (1998) Worm Breeder's Gazette "Transposons In The C.elegans Germline Are Regulated At The Transcriptional Level."

van Luenen HGAM, Ketting RF, Plasterk RHA

[Worm Breeder's Gazette, 1998]

Germline transposition in C.elegans is severely repressed compared to somatic transposition. For example in Bristol N2, germline transposition is not detectable whereas transposition events in somatic cells can readily be detected. We want to understand the mechanism underlying this phenomenon. To do this, we isolated a new mutator allele, mut-7(pk204), that was generated using EMS mutagenesis, using a similar approach as described by John Collins (1,2). One important difference between the two approaches is that, in contrast to the screen by Collins et al., this screen starts with a strain that shows no transposition activity in the germline at all. We recently did another screen to get more mutator alleles, but instead of using the unc-54(r323::Tc1) allele as transposition detector we now used the unc-22(st136::Tc1) allele. We screened about 12.000 mutagenised genomes and found 46 putative mutators. They are in the process of being retested, but the majority is turning out to be real mutators. This provides us with an extensive collection of mutants, with which it will be possible to further examine transposition regulation in the germline. The mut-7(pk204) allele activates multiple transposons (Tc1, Tc3, Tc4 and Tc5), and displays a slightly higher X-chromosome non-disjunction. Both these phenotypes are increased at elevated temperatures. The mutation also causes temperature sensitive sterility. At 25C between 80 and 90% of the oocytes remain unfertilized, or die for another, unknown reason. In collaboration with Bill Kelly and Andy Fire we also looked at germline gene expression from a transgene. Simple transgenic arrays are usually silenced in the germline, even if the promoter driving the construct is normally active in the germline. We tested a transgene containing let-858 fused to GFP (3). This GFP fusion should be expresses in the nuclei of all cells, including the germline. However, the germlines of these animals stay dark. When mut-7(pk204) is introduced, the germline nuclei turn green, indicating that the transgene is not properly silenced anymore. From this we conclude that mut-7(pk204) causes improper gene expression in the germ-line. This could also lead to expression of normally silent transposase genes, resulting in transposase protein in the germ-line. As we know that transposase protein alone is sufficient to catalyze the transposition reaction (at least for Tc1 (4)), transposons will now start jumping in the germline. This model of a general derepression of germline transcription provides an explanation for the intriguing observation that one mutation could activate diverse transposons such as Tc1, Tc3, Tc4 and Tc5 (1,2).

Meyer BJ et al. (1984) Worm Breeder's Gazette "dosage compensation operates at the level of transcription in C elegans."

Meyer BJ, Champness WC

[Worm Breeder's Gazette, 1984]

One of our approaches to studying dosage compensation in the nematode has been to establish a biochemical assay for the expression of X-linked genes. This has involved isolating genes from the X chromosome that are transcriptionally active and using the DNA as probes to measure mRNA levels in XO males and XX hermaphrodites. The expectation was that the level of mRNA from X-linked genes would be equal in the two sexes (when normalized to mRNA from autosomal genes) if a dosage compensation mechanism were operational. In general, such an assay is a powerful tool to demonstrate the involvement of various genes in either dosage compensation or the mechanism of assessing the ratio of X chromosomes to autosomes, the presumed basis for setting dosage compensation. In particular, this assay has the potential to determine directly whether mutations in the genes, dpy-21, dpy-23, dpy-27, sult in phenotypes dependent on the X dosage, affect the level of X-linked gene expression. Cloning of Transcriptionally Active X-Specific Genes. At the time this work was begun no methods were available for cloning nematode genes identified solely by genetic criteria. Thus we devised such a strategy in order to clone genes on X. Our approach involved differentially probing a nematode cDNA library with total genomic DNA from males (haploid for X) and hermaphrodites homozygous for a duplication of the right end of the X chromosome (mnDp27) (tetraploid for this region). cDNA clones exhibiting a four-fold difference in intensity between the hybridizations were isolated and analyzed more completely to prove their linkage to the X chromosome. Two cDNA libraries, each containing five million independent cDNA clones, were constructed using the vectors lambda gt10 and lambda gt11. We chose to construct cDNA, rather than genomic, libraries to avoid the problem that in probing a genomic library, probe sequences containing repetitive but highly dispersed DNA as well as unique DNA would preferentially hybridize to clones containing copies of the repeated sequences. This would interfere with the sensitive quantitation required by our method. cDNA libraries contain much less repetitive DNA, and using them also guarantees that selected clones represent transcriptionally active genes. These libraries were screened as indicated above and the clones with the desired properties were isolated. These clones were proven to represent cDNA from the region of the genetic duplication of X as follows: The clones were used to probe a Southern blot of genomic DNA from males (1X), hermaphrodites (2X), hermaphrodites heterozygous for a deficiency of the region, mnDf42, ( 1X), and hermaphrodites homozygous for the duplication of the region, mnDp27, (4X). A single band of restricted DNA was observed in each lane, with intensities in the expected ratio 1:2:1:4. (As a control to ensure that equal amounts of the different DNAs were in the lanes, the filters were probed simultaneously with a cDNA clone of actin gene) . Among the clones that passed this test, one (which was homologous to five separate isolates) was selected and shown genetically to map to the X chromosome. First, a clone-specific restriction fragment length polymorphism was identified between Bristol and Bergerac. Then Bristol-Bergerac hybrids homozygous for one or the other X chromosome were constructed. In each set of hybrids probed, only one of the two restriction fragment patterns was observed and the pattern corresponded completely with the strain from which the X chromosome was derived. (With this X-specific clone in hand, it was most efficient to obtain further clones by 'walking' to adjacent regions.) In addition, we felt it was important to confirm any conclusions reached about dosage compensation by using clones from different regions of the X chromosome. Thus, when X-specific clones were isolated by other laboratories, we incorporated their use into our assay. In particular, we have used transcriptionally active DNA from genomic 'walks' in the region surrounding the amber suppressor tRNA gene, sup-7, (R. Waterston) and from the region around a locus on the left end of X encoding yolk proteins (T. Blumenthal). As autosomal controls, we have used a gene-specific probe for actin- 1 on chromosome V( Kraus and Hirsh) and a transcriptionally active sequence found in a 'walk' in the region of the amber suppressor tRNA gene, sup-5 on III (R. Waterston). Results of Biochemical Analysis. The various X-specific and autosomal DNA sequences were used to probe Northern blots of RNA from adult wild-type hermaphrodites (XX animals), adult wild-type males (XO animals) and adult dpy-21 hermaphrodites (X animals). Mutations in dpy-21 appear to produce wild-type XO animals but morphologically dumpy XX animals. By genetic criteria XX dpy-21 animals behave as if they express their X chromosomes to the level expected of animals with three X chromosomes. Specifically, in a dpy-21 background triplo-X animals are dead. (J. Hodgkin [1983] Molec. Gen. Genet. 192 452- 458)}. Our results were: 1. The phenomenon of dosage compensation exists at the level of transcription in the nematode. Specifically, mRNA levels transcribed from X-specific sequences were found to be identical in XX and XO animals (normalized to mRNA levels associated with autosomal genes). 2. dpy-21 disrupts proper dosage compensation. Specifically, the level of X-transcription in dpy-21 hermaphrodites was found to be three-fold higher than in wild-type hermaphrodites, again controlling for autosomal mRNA levels. These studies are now being extended by the inclusion of more X- specific and autosomal probes and are being used to investigate mRNA levels in males and in hermaphrodites containing mutations in dpy-21, dpy-23, dpy-27, als containing double and triple combinations of these mutations. With this information we hope to sort out the patterns of interactions among these genes which are excellent candidates for being involved in dosage compensation.