The structural organization of the gene for the human cysteine-proteinase inhibitor cystatin C was studied. Restriction-endonuclease digests of human genomic DNA hybridized with human cystatin C cDNA and genomic probes produced patterns consistent with a single cystatin C gene and, also, the presence of six closely related sequences in the human genome. A 30 kb restriction map covering the genomic region of the cystatin C gene was constructed. The positions of three polymorphic restriction sites, found at examination of digests of genomic DNA from 79 subjects, were localized in the flanking regions of the gene. The gene was cloned and the nucleotide sequence of a 7.3 kb genomic segment was determined, containing the three exons of the cystatin C structural gene as well as 1.0 kb of 5'-flanking and 2.0 kb of 3'-flanking sequences. Northern-blot experiments revealed that the cystatin C gene is expressed in every human tissue examined, including kidney, liver, pancreas, intestine, stomach, antrum, lung and placenta. The highest cystatin C expression was seen in seminal vesicles. The apparently non-tissue-specific expression of this cysteine-proteinase inhibitor gene is discussed with respect to the structure of its 5'-flanking region, which shares several features with those of housekeeping genes.

Molecular maps have been prepared of the HLA region on human chromosome 6 that includes the complement C4 and steroid 21‐hydroxylase genes (21‐OH), using DNA of individuals deficient (QO) in either of the two forms C4A or C4B. In all, 18 haplotypes with C4A QO were examined by Southern analysis and two had deletions of 28‐30 kb that included both the C4A and 21‐OHA genes. Of six C4B QO haplotypes, one had a deletion that included both the C4B and 21‐OHA genes. Thus, some of the C4 null alleles are due to deletion of the gene but the majority in this sample are not. Deletion occurred in two common haplotypes suggesting that in the population as a whole, C4A deficiency is due to deletion in about one‐half the C4A QO haplotypes. As duplication of C4A or C4B genes does occur, the possibility that unequal cross‐over could explain the C4 deletion was examined by preparing cosmid clones from the DNA of an individual typed C4A QO. A cloned genomic fragment containing the single C4B gene was isolated and found to be similar to the homologous region of a cosmid from a normal individual carrying a C4A gene. This suggests that if a cross‐over has occurred it is in a region where the two genes are identical. The biological significance of the rather frequent occurrence in the population of haplotypes with C4A or C4B deletion together with the accompanying deletion of the 21‐OHA gene is discussed.

The association between scrapie and polymorphism of the prion protein (PrP) gene was studied in the Icelandic sheep breed. Polymorphism of the three codons, 136, 154 and 171, that are important for scrapie susceptibility was determined. A BspHI restriction analysis was used to study the alleles of codons 136 and 154, while density gradient gel electrophoresis (DGGE) was used to analyse codon 171 and detect new polymorphisms. The PrP allelic variant, VRQ (amino acids at codons 136, 154 and 171), was found to be highly statistically associated with scrapie, whereas the allelic variant, AHQ, was never found in scrapie-affected animals, a finding that is statistically significant. Iceland has a few scrapie-free regions, which are a part of a quarantine network. Homozygotes for the VRQ variant were found there at a low frequency, indicating that genetic susceptibility is not enough for scrapie to develop and further evidence for the infectious nature of the disease. A comparison of PrP genotypes between sheep outside and within the scrapie-free zones revealed an increase in the AHQ allelic variant in the latter. No polymorphism was found at codon 171 in a total of 932 sheep studied, all individuals having the glutamine allele. Two novel, rare PrP alleles were found using DGGE at codons 138 and 151, i.e. S138N and R151C. Their relevance to scrapie is still unclear, but the former was found in scrapie-affected sheep as well as healthy sheep, whereas the latter was only found in healthy sheep.

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A gene library of the thermophilic eubacterium, Rkodotkermus marinus, strain 21, was prepared in pUC18 and used to transform Esckerickia coli. Of 5400 transformants, two produced halos on lichenan plates after Congo-red staining. Restriction mapping showed that the two clones shared an overlapping 1200-bp DNA fragment, which was used for DNA sequencing. Five potential methionine (Met) translational-initiation codons were identified. A putative signal peptide of 30 amino acids was identified with a hydrophobic core of nine hydrophobic amino acids. The molecular mass of the mature enzyme was estimated to be 29.7 kDa. A comparison of the primary protein sequence of P-glucanase of Rkodothermus marinus with other glycosyl hydrolases showed 38.5 % identity to the C-terminal part of the P-1,3-glucanase of Bacillus circulans and limited identity to bacterial endo-~-1,3-1,4-glucanases. The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial P-glucanases. A gene fragment of 889 bp containing the catalytic domain was overexpressed in E. coli using the pET23, T7-phage RNA polymerase system. The enzyme showed activity on lichenan, P-glucan and laminarin but not on CMC cellulose or xylan. The expressed enzyme was purified by heat treatment of the host. The enzyme had a temperature and pH optima of 85°C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80°C and have a half life of 3 h at 85°C.Rhodotkermus marinus is a thermophilic heterotrophic eubacterium, isolated from a marine alkaline hot spring in Iceland. It is an obligate aerobe and has an optimum growth at 65°C pH7.0 and 2% NaCl [l]. Analysis of a 16s ribosomal RNA gene shows that R. marinus diverges from major bacterial phyla, being most closely allied to the FlexibacterCytophaga-Bacteoides group (Andresson, 0. A., unpublished results). It produces several polysaccharide-degrading enzymes such as cellulase, xylanase and glucanase but in very low amounts.The endosperm cell walls of oat grain and barley are rich in polymers of glucose units joined by both P-1,4 and P-1,3 linkages, called P-glucans. This mixed linkage is also found in lichenan, a similar polysaccharide from the lichen Cetraria islandica [2].Endo ~-1,3-1,4-glucanases, also called lichenases hydrolyse P-1,4-glycosyl linkages adjacent to /3-1,3 linkages, in barley glucan or lichenan [2]. Endo-P-l,3-1,4-~-glucanase genes have been cloned from several members of the Bacillaceae family, such as Bacillus subtilis Enzymes. Lichenase, endo p-1,3-1,4-glycanase (EC 3.2.1.73); laminarase, endo /~'-1,3-~-glucanohydrolase (EC 3.2.1 39).Note. The novel amino acid sequence data published here has been desposited with the GenBank sequence data bank and is available under accession number U04836. ~-1,3-d-glucanohydrolases) cleave 1,3-P-glucosyl linkages, such as those found in the algal polysaccharide laminarin. Genes coding for enzymes able to hydrolyse both P-1,3-1,4-glucans and P-1,3-glucans have been cloned from Cellvibrio...

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Three anomalous balaenopterid whales, one pregnant female and two sterile males, were investigated by applying molecular approaches in order to establish their identity. The analysis showed that the whales were species hybrids between the blue and the fin whales. The female and one of the males had a blue whale mother and a fin whale father. The other male had a fin whale mother and a blue whale father. The difference between the mitochondrial cytochrome b gene of the two species suggests that they separated greater than or equal to 3.5 million years ago. The sequences of the mitochondrial control region of the blue and the fin whales differ by 7%. The difference in the mtDNA control region between three blue whale mtDNA haplotypes was less than or equal to 1%, about one tenth of the difference between the two species.