Cloning of the human RoDH-related short chain dehydrogenase gene and analysis of its structure

We have previously characterized the first human NAD⁺-dependent short chain dehydrogenase capable of oxidizing all-trans-retinol and androgens, and found only in the liver and skin. In a search for related human enzymes, we identified a partial open reading frame, which exhibited >60% sequence identity to human RoDH-4. The full-length cDNA for this enzyme was determined in our laboratory by 5′-RACE PCR and was found to be identical to the recently reported novel type of oxidative human 3α-hydroxysteroid dehydrogenase (3α-HSD). Analysis of the genomic structure revealed that the gene for RoDH-like 3α-HSD has four translated exons and, possibly, a fifth exon that codes for the 5′-untranslated region. The gene for RoDH-4 appears to have only four exons. The positions of exon–intron boundaries and the sizes of the protein coding regions are identical in 3α-HSD and RoDH-4. Moreover, both genes are mapped to chromosome 12q13, and are located in a close proximity to each other. Both genes appear to have satellite pseudogenes. Thus, RoDH-4 and 3α-HSD genes share similar structural organization and cluster on human chromosome 12, near the gene for 11-cis retinol dehydrogenase.     

The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron

The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by a 951?bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5′ and 3′ splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522?nt), separating the catalytic core from the 3′ splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3′ splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Structure of the chicken interferon-γ gene,and comparison to mammalian homologues

The sequence of the chicken interferon-γ (ifn-γ) gene was determined, one of the first non-mammalian cytokine gene structures to be elucidated. Initial genomic clones were amplified from chicken genomic DNA and were used to isolate a cosmid clone covering the entire gene for sequencing. The exon:intron structure of chicken ifn-γ is very similar to those of its mammalian homologues, with the exception of the third intron, which is markedly shorter in the chicken. The first exon contains both 5′ UTR and signal sequence and the first 22 aa of the mature protein. The remainder of the coding region lies in exons 2–4. Exon 4 also encodes the stop codon and the 3′ UTR, including two possible polyadenylation signals. A number of potential regulatory sequences similar to those found in mammals have been identified, in the promoter, in each intron and in the 3′ UTR. In the promoter, these include the TATAATA- and CCAT-boxes, a consensus GATA motif in the reverse orientation and a potential NF-κB binding site. Other regulatory elements identified in the promoters of mammalian ifn-γ genes are absent. Internal to the gene structure, regulatory sequences identified include elements found in the DNase I hypersensitivity region of the first intron of the human ifn-γ gene and several potential NF-κB binding sites. The 3′ UTR contains an AT-rich sequence, including nine repeats of the `instability' motif ATTTA. As in mammals, chicken ifn-γ is a single copy gene. The gene is highly conserved, with no polymorphisms yet identified using either RFLP or SSCP in the coding region. However, promoter sequence polymorphisms between different inbred lines of chickens have been identified, with possible links to disease resistance.