Huang X-Y et al. (1988) Worm Breeder's Gazette "colocalization of nematode myofilaments and mRNAs for GAPDH and actin."

Hecht RM, Huang X-Y

[Worm Breeder's Gazette, 1988]

Glyceraldehyde-3-phosphate dehydrogenase is encoded by four genes in the nematode C. elegans. The minor isoenzyme, GAPDH-1, is encoded by two nearly identical genes namely gpd-1 and gpd-4. In agreement with the ontogeny of GAPDH-1 enzyme activity, both mRNAs were preferentially expressed in embryos which are predominantly composed of non-muscle cells GAPDH-2, a body-wall enriched isoenzyme, is encoded by a different set of nearly identical genes, gpd-2 and gpd-3. Their expression increases 10-fold during post-embryonic development. Furthermore, the mRNAs of gpd-2 and gpd-3 were exclusively observed in the body-wall muscle by in situ hybridization. By minimizing diffusion during hybridization, the gpd-2,3 mRNAs were localized to the actin-containing zones as was the GAPDH-2 isoenzyme itself. By in situ hybridization we further demonstrated that the actin genes act-1 and act-3 encoded body-wall specific mRNAs that were also localized to the thin myofilaments.

Huang X-Y et al. (1988) Worm Breeder's Gazette "a new trans-spliced leader in C elegans."

Huang X-Y, Hirsh DI

[Worm Breeder's Gazette, 1988]

While determining the 5' ends of C. elegans GAPDH mRNAs by primer extension sequencing, we found a new trans-spliced leader (SL2). The previously identified SL is present on mRNAs from three of the four C. elegans GAPDH genes. However, a new 22-nucleotide spliced leader sequence is found on mRNA from one of the four GAPDH genes. RNA Northern analysis showed that the SL2 is present on many RNAs isolated from C. elegans var. Bristol, C. elegans var. Bergerac and C. briggsae, but not on RNAs from Panagrellus redivivus and Haemonchus contortus. In this regard it differs from the original SL that is found in other nematodes. The hybridized RNAs showed a broad spectrum of different molecular weights. From the genomic Southern blot, it seems there are two clusters of SL2 genes. We have screened a C. elegans EMBL4 genomic library. Two groups of phages were isolated. Using the phage DNAs as templates, two kinds of SL2 genes have been sequenced. These two genes are nearly identical in the SL RNA region but differ in the 5' and 3' flanking regions. A consensus Sm binding site of snRNAs is present. The SL2 RNA can be folded into a secondary structure similar to that proposed for the original SL RNA. The SL2 RNA probably has a TMG cap because it can be precipitated by anti-TMG-antibody. The original SL and the new SL2 are similar; 16 out of 22 nucleotides are the same in the best alignment.

Huang X-Y et al. (1990) Worm Breeder's Gazette "CELLULAR PROTEINS BIND TO THE NEMATODE TRANS-SPLICED RNA LEADER SEQUENCE"

Hirsh DI, Huang X-Y

[Worm Breeder's Gazette, 1990]

The trans-spliced RNA leader might play a role in regulating the translation and/or stability of SL-containing mRNAs. This possibility led us to seek transacting factors that might mediate this regulatory function. Protein factors from C. elegans cells (either embryos or worms from mixed stages) that bind with high specificity to the trans- spliced RNA leader sequence (SL) have been identified. Whole cell extracts were shown by a mobility shift electrophoresis assay to form two RNA-protein complexes involving the SL1 sequence. Complex formation could be abolished by preincubation with excess, unlabeled competitor SL1-containing RNA (which is the 5' non-coding region of actin-1 mRNA fused to the firefly luciferase coding region) but not by nonspecific, control RNA that did not contain an SL1 sequence. Spliced leader binding protein-1 (SLBP1) is distinct from SLBP2. The binding of these proteins to SL1 is cap dependent though independent of the detailed structure of the cap; either monomethyl- or trimethyl guanosine cap stimulates binding. We are purifying SLBP1 and SLBP2. Future work will be focused on the characterization of these proteins and elucidation of their biological functions.

Huang X-Y et al. (1987) Worm Breeder's Gazette "localization of the glyceraldhyde-3-phosphate dehydrogenase messenger RNAs by in situ hybridization using asymmetric biotinylated probes."

Hecht RM, Huang X-Y

[Worm Breeder's Gazette, 1987]

Specific asymmetric DNA probes for the GAPDH genes in C. elegans have been made by chain elongation with specific primers in the presence of biotinylated nucleotide analogues. The biotinylated probes were used to in situ hybridize embryo and adult worm squashes. The results showed that GAPDH mRNAs of the two nearly identical copies (Ia and Ib) are present in all cells; whereas GAPDH mRNAs of the two tandem direct repeats (IIa and IIb) are preferentially located in body- wall muscle cells. This confirms the notion that Genes Ia and Ib code for the GAPDH-1 isoenzyme; genes IIa and IIb code for the GAPDH-2 isoenzyme. Also the developmental expression analysis showed that genes Ia and Ib mRNAs are enriched in embryos while genes IIa and IIb mRNAs increase during postembryonic development. These results are expected based on our earlier ontogenetic study of GAPDH-1 and GAPDH-2 isoenzyme expression.

Huang X-Y et al. (1992) Worm Breeder's Gazette "Do Newly Formed Somatic Telomeres in Ascaris lumbricoides Exert a Position Effect on Nearby Located Genes?"

Tobler H, Huang X-Y, Mueller F

[Worm Breeder's Gazette, 1992]

Telomeres, the ends of eukaryotic chromosomes, are essential for the stable maintenance and replication of linear chromosomes in eukaryotic cells. They are thought to occupy unique locations within the nucleus, typically being associated with the nuclear envelope. Frequently, they show heterochromatic structures. It has been demonstrated that PolII genes, if placed near the ends of chromosomes in Saccharomyces cerevisiae, undergo a position effect(1). The telomeres of the somatic chromosomes in Ascaris lumbricoides are of particular interest since they are newly formed during chromatin diminution. In the course of this process, which takes place in all presomatic cells of the early embryo of this parasitic nematode, developmentally regulated chromosomal breakage occurs within a specific, 2-3 kb long region, and is followed by the addition of many repeats of the telomeric sequence TTAGGC(2). Recently, we found a gene to be located within only 10 kb of such a de novo formed somatic telomere. Our partial sequence data indicate that it may encode a protein which contains a GTP-binding domain. The gene is expressed in oocytes and early embryonic stages but not in the intestine of adults, as shown by preliminary Northern blot experiments. Its expression pattern in cells before and after the elimination process is now analyzed in order to see whether the newly formed Ascaris somatic telomere exerts a position effect on this nearby located gene.

Huang Y-J et al. (1994) Worm Breeder's Gazette "From Ascaris to C. elegans: A Way to Study Gene Structure and Function"

Tobler H, Huang Y-J, Mueller F

[Worm Breeder's Gazette, 1994]

FROM ASCARIS TO C. ELEGANS: A WAY TO STUDY GENE STRUCTURE AND FUNCTION Huang Y-J., Tobler H. and Muller F., Institute of Zoology, University of Pribourg, Perolles, CH-1700 Fribourg, Switzerland

Meneely PM et al. (1984) Worm Breeder's Gazette "genes regulating X-chromosome expression."

Wood WB, Meneely PM

[Worm Breeder's Gazette, 1984]

In the previous newsletter, we described the use of X-linked hypomorphs to provide a genetic assay for X-chromosome expression. We have used them to examine the effects of four mutants on X-chromosome expression. Mutations in two autosomal genes, dpy-21 V and dpy-26 IV, lead to abnormally high expression of the X, based on their ability to suppress the hypomorphs. Three alleles of dpy-21 do not differ appreciably in suppression, whereas the maternal (and presumed null) allele of dpy-26, n199 is a much stronger suppressor of the hypomorphs than the non-maternal allele ct14. We also reported that the X-linked gene dpy-22 is an apparent enhancer of the hypomorphs. None of the three dpy genes affects autosomal hypomorphs or null alleles of X- linked genes. From this, we postulate that dpy-21 and dpy-26 are negative regulators of X-expression and that dpy-22 is a positive regulator. We had shown previously that duplications of the X can interact with dpy-21; in particular, a dpy-21 XO animal with a large X duplication may be a Dpy intersex. There are at least two possible explanations for this: 1) the duplication turns on dpy-21 in some manner, for example by supplying an X-linked inducer; or 2) The duplication competes with the normal X for something that represses X-chromosome expression, and this effect is more evident when dpy-21 is mutant. To distinguish these hypotheses, we looked at the effect of the large X- duplication mnDp25 on a hypomorph it does not cover, lin-15(n767), in dpy-21+ strains. The duplication suppresses the hypomorph, albeit somewhat weakly, suggesting that X-duplications increase overall X expression by titrating an X repressor. One candidate for this postulated X repressor is dpy-26. Thus, the dpy-21 product may not interact with the X at all. We are testing other X-duplications, both singly and in combinations, to see if this effect is limited to mnDp25. We are also asking how dpy-26(null) XO animals are affected by X duplications.

Deak P et al. (1980) Worm Breeder's Gazette "bal-X-1 chromosome."

Deak P, Fodor A

[Worm Breeder's Gazette, 1980]

The isolation and first characterization about a balancer chromosome temporarily called Bal-X-1 was presented at the Cold Spring Harbor meeting. This chromosome when heterozygous with an X chromosome marked by dpy-8 and unc-3 suppress crossover on this region. Characterizing the Bal-X-1 further on, we found, that Bal-X-1 viable only in heterozygous or hemizygous form. Bal-X-1 has been marked with lon-2(e678) allele. Bal-X-1 (lon) males are perfectly normal and fertile. Hermaphrodites heterozygous for Bal-X-1 always segregate males about 8-20% frequencies. The males are XO. Bal-X animals homozygous for her-1(e1520) (kindly provided by Jonathan Hodgin) are viable but almost completely sterile. Even if they lay some eggs, their oldest progeny has never been larger than L1, which die soon. Bal-X-1 can balance the following mutants: mn-110 (BH); let-2(e1470) (A.F.); let-15(e1471) (A.F.). We are going on to learn, whether Bal-X-1 is a translocation. In these experiments, we use stocks of double homozygous for two markers ( one in the medium and one in one of the ends the autosome) of the different autosomes and measure the recombination between them. If Bal-X-1 is a translocation like Dp-1 (BH) we expect, that the recombination frequency will be changed on the autosome being involved.

Wood WB et al. (1985) Worm Breeder's Gazette "suppression of dpy-22 male inviability by X duplications."

Wood WB, Meneely PM

[Worm Breeder's Gazette, 1985]

XO males carrying the dpy-22(e652) X mutation generally do not survive to adulthood; those that do are small and sickly. We have postulated that the low viability is due to apparent under-expression of the X chromosome caused by this mutation (Meneely and Wood, 1985 C. elegans Meeting Abstracts, p. 90; Meneely et al., in preparation). Presence of X duplications generally appears to increase overall X expression (Meneely and Wood, 1983, Genetics 106:29; Meneely, this issue). To determine how these two effects interact, we have begun testing the viability of dpy-22 XO males that carry X duplications derived from either the maternal or the paternal parent. In N2(male) x dpy-22(hermaphrodite) crosses, the progeny male ratio (PMR: identifiable Dpy progeny males to total outcross progeny) ranges from about 10% to 30%. For the initial duplication experiments we used mnDp25 (X;I), a duplication of about 25% of the X including the unc-3 locus but not the dpy-22 locus. The cross mnDp25/+;unc-3/0(male) x dpy-22 rodite) gave PMR of 16% +- 5% (39/244 in two experiments), compared to 19% +- 6% (31/165) in the control cross N2(male) x dpy-22 maphrodite). Therefore, introduction of this duplication paternally has no significant effect. To test the effect of introducing it maternally, we crossed N2(male) x mnDp25;unc-20 unc-3(hermaphrodite) (rather than the simpler mnDp25;dpy-22 maphrodite), which turned out to have unexpectedly low viability). We obtained a PMR of 46% +- 5% (158/341), compared to 5% +- 2% (21/389) in the control cross N2( male) x unc-20 unc-3(hermaphrodite). Similar results have been obtained in preliminary experiments with mnDp8 (-15% of X) and mnDp9 (-25% of X), neither of which include the dpy-22 locus. Further experiments with these and additional duplications are in progress. So far it appears that X duplications not including the dpy- 22 locus can suppress the inviability of dpy-22 XO males if supplied from the maternal parent, but not if supplied from the paternal parent, consistent with a maternal effect of X duplications on early X- chromosome expression in the embryo.

Herman RK et al. (1980) Worm Breeder's Gazette "Dominant Him Mutants"

Kari CK, Herman RK

[Worm Breeder's Gazette, 1980]

While screening for chromosome rearrangements that will act as crossover suppressors, we have picked up several X-ray-induced male- throwing mutants, some of which we have begun to study. We now have in stock ten independent mutants that have been backcrossed three times. They segregate about 10-40% males. The mutations responsible for male-throwing are all X-linked and dominant and are all transmissible by males. Some mutations are in fact translocations; for example, mnT2(II;X) heterozygotes throw 39% males, about half of which carry mnT2. The mnT2-bearing males can transmit either mnT2 or a half-translocation, which fails to complement unc-1 X (but does complement dpy-3, unc-6, ecessive lethal, but which still promotes male-throwing. Other dominant Him mutations appear to reside solely on the X. Some of the mutations (including mnT2) greatly suppress recombination on the X--as measured by the widely-spaced markers dpy-3 and unc-3--but others seem to have little effect on X recombination. We do not know whether or not any of the mutant X chromosomes are mitotically unstable. We have only a few mutations in homozygotes; the homozygotes in hand also segregate males at high frequency.