Targeting of endopeptidase 24.16 to different subcellular compartments by alternative promoter usage.

Endopeptidase 24.16 or mitochondrial oligopeptidase, abbreviated here as EP 24.16 (MOP), is a thiol- and metal-dependent oligopeptidase that is found in multiple intracellular compartments in mammalian cells. From an analysis of the corresponding gene, we found that the distribution of the enzyme to appropriate subcellular locations is achieved by the use of alternative sites for the initiation of transcription. The pig EP 24.16 (MOP) gene spans over 100 kilobases and is organized into 16 exons. The core protein sequence is encoded by exons 5-16 which match perfectly with exons 2-13 of the gene for endopeptidase 24.15, another member of the thimet oligopeptidase family. These two sets of 11 exons share the same splice sites, suggesting a common ancestor. Multiple species of mRNA for EP 24.16 (MOP) were detected by the 5'-rapid amplification of cDNA ends and they were shown to have been generated from a single gene by alternative choices of sites for the initiation of transcription and splicing. Two types of transcript were prepared, corresponding to transcription from distal and proximal sites. Their expression in vitro in COS-1 cells indicated that they encoded two isoforms (long and short) which differed only at their amino termini: the long form contained a cleavable mitochondrial targeting sequence and was directed to mitochondria; the short form, lacking such a signal sequence, remained in the cytosol. The complex structure of the EP 24.16 (MOP) gene thus allows, by alternative promoter usage, a fine transcriptional regulation of coordinate expression, in the different subcellular compartments, of the two isoforms arising from a single gene.

Accelerated evolution in inhibitor domains of porcine elafin family members.

Through the analysis of the porcine gene encoding the elastase inhibitor elafin, we demonstrated that there are at least three closely related members of the elafin family, and their genes have arisen by accelerated evolution. A porcine genomic DNA library was screened with a previously cloned human elafin cDNA probe, and several positive clones were obtained that can be distinguished by a combination of restriction enzymes. Sequence analysis of these clones revealed the presence of three homologous members whose genes, all consisting of three exons and two introns, are almost identical except the exon 2 sequences encoding the inhibitor domain called "WAP motif"; the intron sequences are related to each other with sequence similarities of 93-98%, whereas the exon 2 sequences exhibited only 60-77% similarities among the three members. The extreme divergence in the exon 2 sequences compared to the highly conserved intron sequences may be generated by accelerated mutations confined in a short stretch of the genes following recent duplication events of a single ancestral gene. An RNase protection assay indicated that the messages of the elafin family members are abundantly expressed in the trachea and intestine, suggesting that the most likely selective forces for the accelerated evolution are extrinsic proteinases produced by invasive microorganisms.

Mollusk genes encoding lysine tRNA (UUU) contain introns.

New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

Serological survey of hepatitis B virus (HBV) markers in out- and in-patients in a university hospital.

Of a total of 13,596 patients and 1,876 blood donors in a university hospital examined, 550 (4.1%) patients and 31 (1.7%) donors possessed hepatitis B surface antigen (HBsAg) in their blood. The higher incidence of HBsAg in the patient population than in the blood donors verified the view that medical personnel and hospitalized patients are at increased risk of acquiring HBV infection. To assess the actual hazard of the HBsAg-positive patients, we examined hepatitis Be antigen (HBeAg) and its antibody (anti-HBe) status of 228 HBsAg-positive patients and found that 39 (18%) were positive for HBeAg and 168 (74.5%) were positive for anti-HBe. This indicated that only one fifth of the HBsAg-positive patients should be drawn attention in terms of HBV transmission within a hospital.

Studies on steroids. CLXX. Separation and determination of bile acid 3-sulfates in human bile by high-performance liquid chromatography.

A method for the simultaneous determination of sulfated bile acids in human bile without prior hydrolysis and solvolysis is described. The sulfate fraction was obtained from a bile specimen by passing it through a Sep-Pak C18 cartridge, followed by group separation by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution into the 3-sulfates of unconjugated, glycine- and taurine-conjugated ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate was attained by high-performance liquid chromatography (HPLC) on an SC-02 column. Separation of these sulfates was effected when acetonitrile-0.5% ammonium carbonate (8:31, 8:26 and 8:23, v/v) was used as mobile phase. The sulfated bile acids in human bile were unequivocally identified on the basis of their behaviour in HPLC using mobile phases of various PH values. The present method proved to be applicable to the characterization and quantitation of sulfated bile acids in human bile.