Cloning of the rat steroid sulfatase (Sts) gene
- UCSF School of Medicine, San Francisco, CA (United States); and others
Although the human STS gene has been cloned and characterized in detail, several attempts to clone its mouse homologue, using either anti-human STS antibodies or human STS cDNA probes, have failed, suggesting a substantial divergence between these genes. On the other hand, partial amino-terminus sequence from purified rat liver STS is very similar to its human counterpart. In addition, a GKWHLG domains, a putative active site of the enzyme, has been shown to be conserved among all the sulfatases. Thus, we used a degenerate-primer RT-PCR approach to amplify a 319 bp fragment from rat liver cDNA, whose sequence revealed an ORF about 70% similar to human STS cDNA. This fragment was used to screen a rat liver cDNA library resulting in the isolation of 5 clones. A total of 2.5 kb sequence information has been generated, and the predicted amino acid sequence from the ORF 5{prime}-end agreed with the published amino terminus of purified rat STS. Comparison of the protein coding region between the rat and human genes showed 66% homology both at the DNA and the protein levels. Northern hybridization showed two main Sts transcripts of 2.6 and 3.2 kb in all tissues studied. STS activity was conferred to STS(-) A9 cells upon transfection with the rat Sts expression construct, indicating the authenticity of the cloned cDNA. Using the exon-intron organization of the human STS gene, we designed primers flanking the putative intron insertion sites in the rat cDNA to amplify intron sequences from rat genomic DNA and determined its sequence organization. The overall structures of the rat Sts and human STS are very similar, except that the insertion site for intron 1 in the rat is 26 bp upstream for that in the human. However, the rat Sts is only 9.5 kb long while the human STS spans over 146 kb. FISH experiments showed characteristic hybridization signals on the X chromosome, but not on the Y chromosome, indicating that Sts is not pseudoautosomal in the rat.
- OSTI ID:
- 133850
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-0584
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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