Graham RW et al. (1988) Worm Breeder's Gazette "analysis of the polyubiquitin gene of C elegans."

Candido EPM, Graham RW

[Worm Breeder's Gazette, 1988]

We have cloned the polyubiquitin gene of C. elegans, which we call UbiA, using a yeast ubiquitin probe and recently completed the gene sequence. A number of features are notable about the gene organization. Firstly, there are eleven 228 bp repeat units which are tandemly repeated and which each encode monomeric ubiquitin. All eleven repeats of the nematode polyubiquitin gene encode an identical amino acid sequence. This sequence is highly conserved, differing from human ubiquitin at only 1 of 76 positions and from yeast and plant ubiquitin at only two positions. A feature of UbiA which so far is unique to C. elegans is that four of the coding repeats contain a small intron which interrupts the coding region at an analogous position, i.e. splitting Gly-47 G/GA in each of the first, fourth, seventh, and tenth ubiquitin repeats. The introns are quite similar in sequence throughout their length of 50 bp, which suggests that the polyubiquitin gene arose by amplification of a tri-ubiquitin cassette. The UbiA transcript is trans spliced, much like three of the actin transcripts. There is a 3' splice acceptor sequence six nucleotides upstream of the methionine of the first ubiquitin repeat and we have been unable to detect any signal on Northern blots with sequences further upstream. S1 analyses with probes spanning the 5' flanking region protect only to the 3' splice site and don't reveal a cis leader exon. Primer extension sequencing confirmed the presence of the trans splice leader RNA on the mature UbiA mRNA. The splice leader RNA comes from chromosome 5 and UbiA has been mapped to chromosome 3 by Donna Albertson, demonstrating that trans splicing can occur between loci on different chromosomes (the trans spliced actin is also on chromosome 5). The existence of trans splicing means that the start of the UbiA primary transcript lies within the intron sequence and as such must be mapped on the unspliced gene product. Using S1 mapping and large amounts of RNA (10 g polyA+ RNA) we were able to detect the putative start site using long autoradiographic exposures. It maps 455 bp upstream of the 3' splice site. The closest thing to a TATA box is a GAATAA sequence 32 bp upstream of the start site. In the adjacent 500 bp we can find (a) two potential heat shock element (HSE) sequences (b) a stretch of cytosine rich sequence ( C15TCC) and (c) a palindrome resembling the binding site for the mammalian steroid hormone receptor. We have also looked at the expression of UbiA by Northern blot analysis. UbiA is constitutive in all life cycle stages and is expressed at essentially the same levels throughout. Surprisingly, UbiA doesn't appear to be inducible by heat shock (acute or chronic), despite the HSE's in the 5' flanking region. We also followed the expression of UbiA from L1 through lethargus/moulting and into L2 and saw no apparent regulation of transcription during the moulting process. In the next while we will be chasing down the other ubiquitin gene(s) in the worm, as well as exploring the chromatin structure of the 5' end of UbiA- 134

Graham RW et al. (1987) International C. elegans Meeting "characterization of the ubiquitin gene from C elegans."

Candido EPM, Graham RW

[International C. elegans Meeting, 1987]

Graham RW et al. (1989) International C. elegans Meeting "amplification of cDNA by the polymerase chain reaction -- cloning of homologous genes using degenerate oligonucleotides and size selection."

Graham RW, Candido EPM

[International C. elegans Meeting, 1989]

Graham RW et al. (1989) Mol Cell Biol "UbiA, the major polyubiquitin locus in Caenorhabditis elegans, has unusual structural features and is constitutively expressed."

Candido EPM, Jones D, Graham RW

[Mol Cell Biol, 1989]

Ubiquitin is a multifunctional 76-amino-acid protein which plays critical roles in many aspects of cellular metabolism. In Caenorhabditis elegans, the major source of ubiquitin RNA is the polyubiquitin locus, UbiA. UbiA is transcribed as a polycistronic mRNA which contains 11 tandem repeats of ubiquitin sequence and possesses a 2-amino-acid carboxy-terminal extension on the final repeat. The UbiA locus possesses several unusual features not seen in the ubiquitin genes of other organisms studied to date. Mature UbiA mRNA acquires a 22-nucleotide leader sequence via a trans-splicing reaction involving a 100-nucleotide splice leader RNA derived from a different chromosome. UbiA is also unique among known polyubiquitin genes in containing four cis-spliced introns within its coding sequence. Thus, UbiA is one of a small class of genes found in higher eucaryotes whose heterogeneous nuclear RNA undergoes both cis and trans splicing. The putative promoter region of UbiA contains a number of potential regulatory elements: (i) a cytosine-rich block, (ii) two sequences resembling the heat shock regulatory element, and (iii) a palindromic sequence with homology to the DNA-binding site of the mammalian steroid hormone receptor. The expression of the UbiA gene has been studied under various heat shock conditions and has been monitored during larval moulting and throughout the major stages of development. These studies indicate that the expression of the UbiA gene is not inducible by acute or chronic heat shock and does not appear to be under nutritional or developmental regulation in C. elegans.

Graham RW et al. (1988) J Biol Chem "Maturation of the major ubiquitin gene transcript in Caenorhabditis elegans involves the acquisition of a trans-spliced leader."

Graham RW, Candido EPM, Van Doren K, Bektesh SL

[J Biol Chem, 1988]

Recently, studies on the 5'-ends of actin mRNAs in the nematode, Caenorhabditis elegans, have demonstrated that three of the four mature actin transcripts contain a 22-nucleotide leader sequence which is acquired by trans-splicing from a novel 100-nucleotide RNA. In the course of our studies of the ubiquitin genes in C. elegans (R. W. Graham and E. P. M. Candido, manuscript in preparation) we were led to the possibility that the major ubiquitin transcript in this organism might also contain a trans-spliced leader sequence. The 5'- noncoding region of the major ubiquitin gene (UbiA) contains a 3'- splice consensus sequence six nucleotides upstream of the initiation codon; however, sequencing of a further 1.6 kilobases upstream failed to reveal the existence of any potential 5'-splice sites in the correct relationship to known promoter elements. Furthermore, S1 and Northern blot analyses using probes complementary to upstream regions failed to detect the ubiquitin transcript. Primer extension experiments on total RNA using an oligonucleotide which hybridized to the ubiquitin transcript downstream of the 3'-splice site demonstrated the existence of a 22-nucleotide leader sequence not present in the ubiquitin gene 5'-region. Thus we show here that the 2500-nucleotide mRNA encoding a polyubiquitin precursor protein contains a 22-nucleotide leader sequence identical to that found in several C. elegans actin mRNAs and which is most likely acquired by a trans-splicing mechanism. In addition we have localized the site of transcript initiation for UbiA by S1 nuclease mapping.

Graham RW et al. (1990) Worm Breeder's Gazette "Rapid Amplification and Analysis of Ubiquitin cDNA Sequences from C. elegans, C. briggsae etc. etc. using Versatile Degenerate Ubiquitin Primers"

Candido EPM, Graham RW

[Worm Breeder's Gazette, 1990]

Based on Northern analysis with 3'-end specific probes we have determined that C. elegans must possess at least two classes of ubiquitin genes. We have cloned the major ubiquitin gene, UbiA, which encodes worm polyubiquitin, and have undertaken various schemes for the isolation of the non-UbiA member(s) of the ubiquitin family. Unfortunately, exhaustive screening of available C. elegans cDNA and genomic libraries and attempts to directly clone size-fractionated genomic DNA have led only to the reisolation of UbiA clones ad nauseum.

Friedman LC et al. (1996) Midwest Worm Meeting "mog-3 ETC."

Friedman LC, Kimble JE

[Midwest Worm Meeting, 1996]

The fem-3 gene is regulated post-transcriptionally to effect the switch from spermatogenesis to oogenesis (Barton et al., 1987; Ahringer and Kimble, 1991). The mog genes are excellent candidates for encoding trans-acting regulators of fem-3 to achieve this switch (Graham and Kimble, 1993; Graham et al., 1993; Gallegos and Kimble, unpublished). We will describe our initial efforts to clone mog-3 and to characterize it genetically.

Jones D et al. (1989) DNA "Differential regulation of closely related members of the hsp16 gene family in Caenorhabditis elegans."

Graham RW, Jones D, Dixon DK, Candido EPM

[DNA, 1989]

The heat-inducible genes encoding 16-kD heat shock polypeptides in Caenorhabditis elegans are found at two separate loci, one containing the 16-1 and 16-48 genes (locus A), and the other, the 16-2 and 16-41 genes (locus B). Despite the highly conserved structures of these genes and their promoters, the B locus produces up to sevenfold more mRNA during heat induction than does the A locus. Since there are two copies of the 16-1 and 16-48 genes at the A locus, the discrepancy in mRNA production is actually as high as 14:1 on a per gene basis. Measurements of the rate of hsp16 mRNA decay during recovery from a heat shock suggest that this difference is not caused by differential mRNA stability; furthermore, nuclear runon experiments yield rates of transcription for the 16-1/48 locus that are approximately threefold higher than those from the 16-2/41 locus. The higher levels of mRNA from the 16-2/41 locus, particularly at longer induction times, seem to be due to a marked difference in the temporal pattern of mRNA production from the two loci. While both loci are transiently activated by a heat shock, the 16-1 and 16-48 genes of the A locus are down-regulated to a lower transcription rate sooner than the genes from the B locus.

Jones D et al. (1995) Dev Biol "A portable regulatory element directs specific expression of the Caenorhabditis elegans ubiquitin gene ubq-2 in the somatic gonad."

Jones D, Stringham-Durovic EG, Graham RW, Candido EPM

[Dev Biol, 1995]

The Caenorhabditis elegans ubq-2 gene encodes a fusion of ubiquitin and a 52-amino-acid ribosomal protein. This single copy gene is both cis- and trans-spliced. It is expressed in all life stages of the worm and its transcript abundance is unaffected by heat stress. Transgenic analysis shows that expression of ubq-2 is regulated by an upstream promoter and a downstream element. The downstream element is required for ubq-2 promoter activity in embryos and in cells of the somatic gonad, including the distal tip cells, sheath cells, spermathecal cells, and cells of the uterus. The gonad-specific activity of the downstream regulator is transferable to a stress gene promoter such that heat-inducible expression of the transgene occurs in the somatic gonad. Stress-inducible beta-galactosidase expression in the gonad does not occur in all life stages, but is initiated late in the second or early in the third larval stage, when differentiation of gonadal tissues begins. Expression of a beta-galactosidase fusion protein from constructs containing the downstream ubq-2 regulator causes abnormalities of the gonad and embryonic lethality. Gonad abnormalities include arrested development and general disorganization. These abnormalities may be related to the overexpression of ubiquitin in the gonad. (C) 1995 Academic Press, Inc.

Candido EPM et al. (1989) Genome "Structure, organization, and expression of the 16-kDa heat shock gene family of Caenorhabditis elegans."

Dixon DK, Candido EPM, Jones D, Russnak RH, Kay RJ, Graham RW

[Genome, 1989]

Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in mouse alpha-crystallin gene.(ABSTRACT TRUNCATED AT 250 WORDS)