Cloning, mapping and expression of UBL3, a novel ubiquitin-like gene
Introduction
The human genome project and the accompanying model organism projects have made it posssible not only to identify many new genes, but also to compare them between the different organisms and find homologues of novel genes by data base searching(Collins and Galas, 1993). Although sequence conservation between human and mouse protein sequences is usually too high to yield information about the importance of residues or structures, this is a useful strategy for species related more distantly to humans like flies and nematodes. Furthermore, if genes are evolutionarily conserved between humans, mice, flies, nematodes and yeasts, knowledge of their function and regulation in one of these organisms can provide clues to such information in all the others. Genetic and biochemical analyses of conserved genes in lower organisms have contributed to the understanding of their human homologues (Tugendreich et al., 1994) and their mode of action in human disease (Foury, 1997). Comparison of the nematode gene complement to that of Saccharomyces cerevisiae (S. cerevisiae) is providing insight into both the common necessary elements to make a eukaryotic cell and which genes may only be required in multicellular animals (Chervitz et al., 1998). We describe the cloning of a novel gene, UBL3, which is conserved from man to nematodes but does not have an obvious homologue in S. cerevisiae or among the available Schizosaccharomyces pombe (S. pombe) genes. In doing so, we have made extensive use of gene, Expressed Sequence Tag (EST) and genome databases. This illustrates how searching databases has not only replaced low stringency DNA complementary to RNA (cDNA) library screening but also far extended its sensitivity since the UBL3 genes are unlikely to have cross-hybridized at the nucleotide level.
Section snippets
Isolation and sequencing of cDNA clones
The D. melanogaster UBL3 gene was identified by screening a λgt11 maternal D. melanogaster cDNA library with a 2.3-kb EcoRI genomic fragment from the D. melanogasterX chromosome close to the 3′ end of the Myb gene (data not shown). Two overlapping cDNA clones were isolated and sequenced. The full cDNA sequence for D. melanogaster UBL3 was used in a TBLASTX search against entries in the expressed sequence tag (EST) database at GenEmblBank. A single human EST clone (Accession No. T82438) was
Isolation and sequence characterization of the Drosophila melanogaster UBL3 gene
In the course of following up a screen for lethal mutations (T. Kidd, D. Ish-Horowicz, A. Katzen, unpublished, and see Section 4), a cDNA clone for the D. melanogaster UBL3 gene was isolated from a maternal cDNA library using a genomic probe. A 1.2-kb cDNA clone was isolated and sequenced (Accession No. AF044219). There are two potential start codons at positions 85 and 764 with an open reading frame (ORF) extending to nucleotide (nt) 1127 potentially coding for proteins of 121 or 347 amino
Discussion
This paper reports the cloning of the ubiquitin-like gene UBL3, which is highly conserved between man, mouse, drosophila and nematodes. UBL3 was mapped to human chromosome 13q12–13 by radiation hybrid mapping, the mouse homologue by FISH to the telomeric end of mouse chromosome 5 within a region of conserved gene order with human chromosome 13q (DeBry and Seldin, 1996).
Fig. 1 shows the alignment of the predicted UBL3 protein sequence from man, mouse, Drosophila and several nematodes. A very
Acknowledgements
We thank Jill Williams and Denise Sheer for the mouse mapping by FISH. We are also grateful to Alisa Katzen for the genomic fragment used to isolate the Drosophila UBL3 cDNA, Phil Jones and Fiona Watt for the human keratinocyte stem cell cDNA library and Ian Goldsmith and colleagues for oligonucleotide synthesis. We thank Paul Freemont for informative discussions and helpful comments on the manuscript. The GeneBridge-4 radiation hybrid mapping panels, monochromosomal hybrid mapping panels and
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Present address: Institute of Human Genetics, Harvard Medical School, Boston, MA 02115, USA.
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Present address: Department of Cell and Molecular Biology, University of California at Berkeley, Berkeley, CA 94720, USA.