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Complete Coding Sequence (complete + coding_sequence)
Selected AbstractsBJ46a, a snake venom metalloproteinase inhibitorFEBS JOURNAL, Issue 10 2001Isolation, characterization, cloning, insights into its mechanism of action Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4 -reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition. [source] Prioritizing regions of candidate genes for efficient mutation screening,HUMAN MUTATION, Issue 2 2006Terry A. Braun Abstract The availability of the complete sequence of the human genome has dramatically facilitated the search for disease-causing sequence variations. In fact, the rate-limiting step has shifted from the discovery and characterization of candidate genes to the actual screening of human populations and the subsequent interpretation of observed variations. In this study we tested the hypothesis that some segments of candidate genes are more likely than others to contain disease-causing variations and that these segments can be predicted bioinformatically. A bioinformatic technique, prioritization of annotated regions (PAR), was developed to predict the likelihood that a specific coding region of a gene will harbor a disease-causing mutation based on conserved protein functional domains and protein secondary structures. This method was evaluated by using it to analyze 710 genes that collectively harbor 4,498 previously identified mutations. Nearly 50% of the genes were recognized as disease-associated after screening only 9% of the complete coding sequence. The PAR technique identified 90% of the genes as containing at least one mutation, with less than 40% of the screening resources that traditional approaches would require. These results suggest that prioritization strategies such as PAR can accelerate disease-gene identification through more efficient use of screening resources. Hum Mutat 27(2), 195,200, 2006. © 2006 Wiley-Liss, Inc. [source] Identification and function of Abdominal-A in the silkworm, Bombyx moriINSECT MOLECULAR BIOLOGY, Issue 2 2009M-H. Pan Abstract Abdominal-A (adb-A) is a key gene in the development of insects. To understand its function in the silkworm, we cloned 1193 bp of the abd-A gene of Bombyx mori (Bmabd-A), including the complete coding sequence and part of the 3, untranslated region sequence. Bmabd-A has at least three mRNA splice variants with coding sequences of lengths 1032, 1044 and 1059 bp, encoding 343, 347 and 352 amino acids, respectively. Each splice variant of Bmabd-A has three exons and differs only in second exon size. Bmabd-A was expressed at low levels in unfertilized eggs, but increased gradually in fertilized eggs after laying 22 h. Bmabd-A expression decreased in ant silkworms (newly hatched silkworms). After RNA interference for Bmabd-A, the embryos had two mutant phenotypes, either completely or partially absent abdominal feet from the third to sixth abdominal segments, suggesting that Bmabd-A is responsible for normal development of the third to sixth abdominal segments during embryonic development. [source] Expression of human tissue factor under the control of the mouse tissue factor promoter mediates normal hemostasis in knock-in miceJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2008L. A. SNYDER Summary.,Background:,Tissue factor (TF) is expressed widely at the subluminal surface of blood vessels and serves as the primary cellular initiator of the extrinsic pathway of blood coagulation. Lack of TF in mice resulted in lethality in utero, but human TF (huTF) expressed at low levels from a human minigene rescued null mice from prenatal death. Although these low-TF expressing transgenic mice developed to term, they had a significantly shorter life span and exhibited hemorrhage and fibrosis in the heart. Methods:,Human TF knock-in (TFKI) mice were generated by replacing the first two exons of the mouse (murine) TF (muTF) gene with the huTF complete coding sequence, thus placing it under the control of the endogenous muTF promoter. Results:,Expression of huTF in the TFKI mice was similar to muTF in wild-type (wt) mice. The TFKI mice showed no microscopic evidence of spontaneous hemorrhage in the heart, nor cardiac fibrosis at up to 18 months of age. Immunohistochemistry showed that huTF was expressed in cells surrounding blood vessels in TFKI mice. Coagulation activity of brain homogenates from TFKI mice was comparable with that from wt brain. Cardiac hemorrhage similar to that of the low-TF transgenic mice occurred in the TFKI mice when huTF was blocked by a neutralizing anti-huTF monoclonal antibody. Conclusion:,We generated a transgenic mouse line that expresses huTF under the control of the endogenous muTF promoter at physiological levels. Our results suggest that huTF can fully reconstitute the murine coagulation system and mediate normal hemostasis. [source] Informatic and genomic analysis of melanocyte cDNA libraries as a resource for the study of melanocyte development and functionPIGMENT CELL & MELANOMA RESEARCH, Issue 3 2007Laura L. Baxter Summary As part of the RIKEN mouse encyclopedia project, two cDNA libraries were prepared from melanocyte-derived cell lines, using techniques of full-length clone selection and subtraction/normalization to enrich for rare transcripts. End sequencing showed that these libraries display over 83% complete coding sequence at the 5, end and 96,97% complete coding sequence at the 3, end. Evaluation of the libraries, derived from B16F10Y tumor cells and melan-c cells, revealed that they contain clones for a majority of the genes previously demonstrated to function in melanocyte biology. Analysis of genomic locations for transcripts revealed that the distribution of melanocyte genes is non-random throughout the genome. Three genomic regions identified that showed significant clustering of melanocyte-expressed genes contain one or more genes previously shown to regulate melanocyte development or function. A catalog of genes expressed in these libraries is presented, providing a valuable resource of cDNA clones and sequence information that can be used for identification of new genes important for melanocyte development, function, and disease. [source] cDNA cloning, genomic structure and polymorphism of the porcine FHL3 geneANIMAL GENETICS, Issue 3 2004B. Zuo Summary LIM domain proteins are important regulators of the growth, determination and differentiation of cells. Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). In this study, we have determined the complete coding sequence of pig FHL3 which encodes a 280 amino acid protein. The coding region of the pig FHL3 gene is organized in five exons and spans an approximately 2.1-kb genomic region. Comparative sequencing of six pig breeds revealed three single nucleotide polymorphisms (SNPs) within exon 2 of which an A,G substitution at position 313 changes a codon for arginine into a codon for glycine. The substitution was situated within a PstI recognition site and developed as a PCR-RFLP marker for further use in population variation investigations and association analysis. The A/G polymorphism was segregating only in Landrace pigs. Association studies of the FHL3 polymorphism with carcass traits provided preliminary evidence that the PstI PCR-restriction fragment length polymorphism (RFLP) genotype may be associated with variation in several carcass traits of interest for pig breeding. Further investigations in more Landrace pigs are needed to confirm this. [source] Complete mutation analysis panel of the 39 human HOX genesBIRTH DEFECTS RESEARCH, Issue 2 2002Kenjiro Kosaki Background The HOX gene family consists of highly conserved transcription factors that specify the identity of the body segments along the anteroposterior axis of the embryo. Because the phenotypes of mice with targeted disruptions of Hox genes resemble some patterns of human malformations, mutations in HOX genes have been expected to be associated with a significant number of human malformations. Thus far, however, mutations have been documented in only three of the 39 human HOX genes (HOXD13, HOXA13, and HOXA11) partly because current knowledge on the complete coding sequence and genome structure is limited to only 20 of the 39 human HOX genes. Methods Taking advantage of the human and mouse draft genome sequences, we attempted to characterize the remaining 19 human HOX genes by bioinformatic analysis including phylogenetic footprinting, the probabilistic prediction method, and comparison of genomic sequences with the complete set of the human anonymous cDNA sequences. Results We were able to determine the full coding sequences of 19 HOX genes and their genome structure and successfully designed a complete set of PCR primers to amplify the entire coding region of each of the 39 HOX genes from genomic DNA. Conclusions Our results indicate the usefulness of bioinformatic analysis of the draft genome sequences for clinically oriented research projects. It is hoped that the mutation panel provided here will serve as a launchpad for a new discourse on the genetic basis of human malformations. Teratology 65:50,62, 2002. © 2002 Wiley-Liss, Inc. [source] SLA typing using the PCR-SSP method and establishment of the SLA homozygote line in pedigreed SNU miniature pigsANIMAL SCIENCE JOURNAL, Issue 2 2010Su-Cheong YEOM ABSTRACT Seoul National University (SNU) miniature pigs represent a closed colony with 24 founder pigs and a well preserved pedigree. Characterization using mRNA sequence analysis was conducted for 6 swine leukocyte antigen (SLA) loci in parental or founder pigs, and 17 defined alleles were detected. Based on these complete coding sequences, 17 sequence specific primers (SSPs) were designed for polymorphic sites. To validate the specificity of each allele SSP, the PCR-SSP was conducted with defined allele clones as templates. PCR-SSP was conducted with the hot start polymerase and touch-down PCR. The parental or found SNU miniature pigs showed overall SLA class I and II heterozygotes. Using the established PCR-SSP method, we conducted SLA typing for breeding stock including 2 pedigreed pigs and identified the novel SLA class II homozygote haplotye (DRA*0201, DRB1*0403, DQA*0102 and DQB1*0701) and 2 SLA homozygote pig lines: SLA class I Hp-3.0 and class II Hp-0.3, and SLA class I Hp-2.0 and class II Hp-0.2. We thought that our PCR-SSP SLA typing method could be applicable for new SLA homozygote line establishment by assignment and scheduled breeding. [source] | ||||||||||