Localization of the gene encoding human Factor V to chromosome 1q21–25

The gene encoding human coagulation Factor V (FV), one of the cofactors in the blood clotting process, has been mapped to chromosome 1 by both Southern hybridization to DNA from human-hamster somatic cell hybrids and in situ hybridization. The whole plasmid pUC3A containing a 1.5-kb cDNA sequence for FV was 32P-labeled for Southern analysis and 3H-labeled for in situ hybridization to metaphase chromosomes. The results localized the FV gene to the region of 1q21-25.     

The role of calcium ions in factor X activation by thrombin E192Q.

Despite considerable sequence similarities, blood coagulation serine proteases exhibit remarkable specificity with respect to which zymogen they activate. The basis for this specificity presumably involves recognition of a short sequence within the extended binding pocket of the enzyme, other interactions remote from the catalytic groove, and modulation by a definite protein cofactor. In addition, Ca2+ plays a major role in most activation processes, but, because both the enzyme and its substrate interact with Ca2+, whether Ca2+ influences the substrate, the enzyme, or both remains an open question. Thrombin is not a factor X-activating enzyme, but when Glu192, 3 residues remote from the active Ser195, is replaced with glutamine, the resultant serine protease (E192Q) becomes a bovine, but not human, factor X activator. Kinetic experiments with peptides corresponding to human and bovine factor X activating sites reveal that threonine at position P2 in human (versus a valine in bovine) accounts for the species specificity. Substitution of the threonine in P2 of the human sequence with valine allows E192Q to cleave the human peptide whereas substitution of the valine in P2 of the bovine sequence with threonine hinders E192Q catalysis. Thrombin has no high affinity Ca2+ binding sites, and E192Q proteolysis of these peptides is not altered by Ca2+. The influence of Ca2+ in E192Q-mediated factor X activation provides therefore new insights into the role of the different Ca2+ binding sites in factor X. With factor X as substrate, the addition of Ca2+ enhances Kcat 4-fold but increases Km 10-fold. When the vitamin K-dependent gamma-carboxyglutamic acid domain of factor X is removed, the Km remains high with or without Ca2+ whereas Kcat still increases upon addition of the metal ion. These results suggest that factor X undergoes two metal-dependent suggest that factor X undergoes two metal-dependent transitions that influence the presentation of the activation site to activators.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

Background  

DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM).