PubMed Health⌕ Search

Biomedical subjects

P Jagadeeswaran

Publications and source records attributed to P Jagadeeswaran.

At least 19 recordsLinked to original sources
PubMed1995

Age-specific regulation of clotting factor IX gene expression in normal and transgenic mice.

Factor IX (FIX), a circulating serine protease that serves as an essential component of the blood coagulation pathway, has been shown to increase with age in humans. We show here that murine FIX mRNA and activity levels also increase with age. Furthermore, one form of hemophilia B, hemophilia B Leyden, which is caused by mutations within the promoter region of the FIX gene, has a distinct age-dependent phenotype. To determine the source of the age-related increases in FIX gene expression, we have analyzed the regulation of the normal FIX gene promoter and FIX Leyden gene promoter with the +13 mutation during aging by generating transgenic mice that contain the -189 to +21 bp promoter segment ligated to a chloramphenicol acetyltransferase reporter gene. We have established that the normal FIX promoter and the Leyden promoter transgenes are expressed in a tissue-specific manner in vivo. The normal FIX promoter transgene does not show any differences in the pattern of expression with age or sex of the organism, whereas the Leyden promoter transgene showed age-dependent male-specific expression. This is the first demonstration of the FIX Leyden phenotype in a transgenic mouse model.

Age Factors↗
PubMed1992

Automated column equilibration, washing, sample loading and elution of bench-packed mini-columns.

In this paper we describe an easy and efficient way to pack mini-columns and use them in the Biomek 1000 Automated Laboratory Workstation. These procedures allow for automated column equilibration, column washing, sample loading and elution, and utilization of commercially available components. Various resins can be packed, allowing for automation of a variety of procedures in which minicolumns are used.

Automation↗
PubMed1990

Molecular defect (Gla+14----Lys) and its functional consequences in a hereditary factor X deficiency (factor X "Vorarlberg").

Factor X (FX) "Vorarlberg" is a congenital FX deficiency characterized clinically by a mild bleeding tendency. Homozygous individuals have a FX activity of less than 10% in the extrinsic system and 25% in the intrinsic system. FX antigen is 20%. Using molecular techniques, two point mutations were detected in the coding sequence of the FX Vorarlberg gene: a G----A at base pair 160 in exon II resulting in a change of Gla14 (GAA) to Lys (AAA); a G----A at base pair 424 in exon V resulting in a change from Glu102 (GAG) to Lys (AAG). The mutations abolished a TaqI restriction site in exon II and an MnlI site in exon V. To determine whether these mutations are present on one or on both alleles, restriction analyses of amplified exon II and exon V fragments were performed. Analysis of the pedigree showed that the genotype for the mutation on exon II (homozygous versus heterozygous) correlates with the severity of the phenotypic coagulation defect. We therefore conclude that the mutation in exon II is responsible for the functional defect in FX Vorarlberg. We have also purified the mutant FX protein from patient plasma. Purified FX Vorarlberg is indistinguishable from normal FX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its activity is 15% of normal FX upon activation with factor VIIa/tissue factor, 75% upon activation with factor IXa/factor VIIIa, and 100% upon activation with RVV. Activation at varying Ca2+ concentrations shows that the affinity of FX Vorarlberg for Ca2+ is decreased. Factor Xa Vorarlberg is able to convert prothrombin at a normal rate but also shows decreased affinity for Ca2+ in this interaction. Upon addition of Ca2+, FX Vorarlberg does not undergo the same conformational change as normal FX. Our data show that FX Vorarlberg has a decreased affinity for Ca2+ which impedes a normal conformational change. This leads to a decreased rate of activation by factor VIIa/tissue factor and by factor IXa. The decrease is much more marked for the extrinsic than for the intrinsic pathway.

Amino Acid Sequence↗
PubMed1990

Allelic association of human dopamine D2 receptor gene in alcoholism.

In a blinded experiment, we report the first allelic association of the dopamine D2 receptor gene in alcoholism. From 70 brain samples of alcoholics and nonalcoholics, DNA was digested with restriction endonucleases and probed with a clone that contained the entire 3' coding exon, the polyadenylation signal, and approximately 16.4 kilobases of noncoding 3' sequence of the human dopamine D2 receptor gene (lambda hD2G1). In the present samples, the presence of A1 allele of the dopamine D2 receptor gene correctly classified 77% of alcoholics, and its absence classified 72% of nonalcoholics. The polymorphic pattern of this receptor gene suggests that a gene that confers susceptibility to at least one form of alcoholism is located on the q22-q23 region of chromosome 11.

Alcoholism↗
PubMed1990

Synthesis of human coagulation factor XIII in yeast.

The active form of coagulation factor XIII, factor XIIIa, is a transglutaminase that covalently cross-links fibrin molecules by joining gamma-glutamyl and epsilon-lysyl primary amino groups. In this paper we report the design of a yeast expression vector called pPH3 which includes the GAL1-GAL10 promoter, multiple cloning sites, the URA3 selectable marker, the 2 mu sequences and the ampicillin-resistance gene. We have placed full-length factor XIII cDNA into the PstI site of this vector, transformed the yeast cells and expressed human coagulation factor XIII which was found to be enzymatically active based on observations that this recombinant factor XIII: (a) showed immunological identity with placental factor XIII; (b) was demonstrated to have transglutaminase activity, and (c) cross-linked fibrin in a manner identical to human placental factor XIIIa-catalyzed polymerization (gamma-gamma dimerization and alpha polymerization).

Blotting, Northern↗
PubMed1990

Human factor IXLincoln Park: a molecular characterization.

Using genomic DNA prepared from peripheral blood samples of a patient with factor IX deficiency, all eight exons as well as sequences around the splice junctions and putative promoter region of human factor IX DNA have been subjected to polymerase chain reaction (PCR) amplification and sequenced. Comparison of these sequences with normal factor IX gene sequences revealed an insertion in exon VIII that resulted in the alteration of 11 amino acids and the addition of 23 amino acids, all at the carboxy terminal of factor IX. This insertion destroys an Msp I restriction site; carrier detection and antenatal diagnosis in affected kindreds can be performed by testing for the absence of this site. This is the first characterization of a mutation in which insertion in the carboxy terminal region of factor IX causes factor IX deficiency. The genetic change in factor IX in this patient is called Factor IXLincoln Park.

Amino Acid Sequence↗
PubMed1990

A simple and easy-to-assemble device for polymerase chain reaction.

In this paper we describe an efficient polymerase chain reaction device which is easy to assemble and requires minimal investment in dedicated equipment. The polymerase chain reaction device consists of three waterbaths, three dual-head peristaltic pumps, an electronic timer and a fabricated water jacket capable of holding microcentrifuge tubes. This device has been successfully used to amplify human factor X genomic DNA in our laboratory.

Base Sequence↗
PubMed1989

Cloning and characterization of the 5' end (exon 1) of the gene encoding human factor X.

Human blood-coagulation factor X (hBCFX) is a serine protease zymogen which participates in the middle phase of blood coagulation. Recently, we and others have reported the cDNA sequences. At present, partial hBCFX gene structure is available. In this paper, we report the isolation of two genomic clones, the X-emb lambda phage clone encoding exons 1 and 2 of the hBCFX gene, and the X-cos cosmid clone encoding exons 2-8. The restriction map of the hBCFX region spans 55 kb. The gene itself was found to be 27 kb long. The sequence of the 5' region of the hBCFX gene has been determined and reveals an ATTTG pentanucleotide, which also occurs in a similar location in the genes encoding factors VII, IX, protein CC and C prothrombin, suggesting that this motif might be of importance in the regulation of these genes.

Base Sequence↗
PubMed1989

Molecular characterization of human factor XSan Antonio.

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.

Alleles↗
PubMed1986

DNA sequence analysis: a procedure to find homologies among many sequences.

SEQCMP, a program that analyzes and searches for homology among multiple nucleic acid sequences, is described. The sequences are compared by the dot matrix method and the consensus sequence is derived by superimposing all the dot matrices on one another. The program is written in MBASIC and runs on IBM-PC microcomputer. It is interactive and can be used by investigators with no computer background or experience.

Base Sequence↗
PubMed1986

Isolation and characterization of human blood-coagulation factor X cDNA.

Using synthetic oligodeoxynucleotides as probes, we have isolated factor X cDNA from human liver cDNA library. We sequenced the 1430-bp cDNA which spans the coding region of the mature factor X and contains the polyadenylation signal and poly(A) tail. The amino acid (aa) sequence is in agreement with the published aa sequence. The nucleotide (nt) sequence of cDNA confirmed that factor X is synthesized and secreted as a single-chain precursor, and then converted into dimeric form by proteolytic cleavage of an internal tripeptide. From the nt sequence, it was also predicted that like other secretory proteins, human factor X is synthesized with a leader sequence (prepro-protein). The 5'-coding region of factor X cDNA is 60 and 40% homologous to the corresponding regions of factor IX and prothrombin genes, respectively. This supports the hypothesis of gene evolution by gene duplication followed by divergence.

Amino Acid Sequence↗
PubMed1986

Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA.

A retroviral shuttle vector was constructed by introducing the Escherichia coli xanthine (guanine) phosphoribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, 1053-1062]. This vector was packaged into infectious virus which then was used to infect a hypoxanthine (guanine) phosphoribosyltransferase-deficient mouse cell line. Cell lines that expressed the gpt gene were isolated, and it was found that these cells contained a single integrated copy of the vector in a proviral form. Treatment of these cell lines with either ethyl methanesulfonate or BrdUrd produced a greater than 10-fold increase in the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact gpt genes have been recovered from a number of Sgur cell lines after COS cell fusion and introduced into E. coli as part of a plasmid. The complete DNA sequences of three mutant genes have been determined. Two of the mutant genes have a single base substitution, whereas the third has a 34-base-pair deletion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is that the mutations are induced in genes integrated into mammalian chromosomes rather than in genes existing as part of autonomously replicating plasmids.

Animals↗
PubMed1985

Linkage and recombination between fragile X-linked mental retardation and the factor IX gene.

Linkage analysis on a family with fragile X-linked mental retardation was performed using a Taq 1 restriction fragment length polymorphism detected by a cloned human coagulation factor IX cDNA. Two affected brothers in this sibship were found to have different factor IX RFLP alleles, indicating a recombinational event occurred between the two genes. Our data therefore indicate that the gene responsible for fragile X-linked mental retardation is not as tightly linked to the factor IX gene as the previously published data may suggest.

Alleles↗
PubMed1984

Isolation and characterization of human factor IX cDNA: identification of Taq I polymorphism and regional assignment.

Hemophilia B or Christmas disease is an X-linked condition caused by absent or reduced levels of functional coagulation factor IX. Based upon the peptide sequence of bovine factor IX, we synthesized a 17-base pair oligonucleotide probe to screen a human liver cDNA library. A recombinant clone was identified with a 917-nucleotide insert whose sequence corresponds to 70% of the coding region of human factor IX. This factor IX cDNA was used to probe restriction endonuclease digested human DNA to identify a Taq I polymorphism associated with the genomic factor IX gene as well as to verify that there is a single copy of this gene per haploid genome. The factor IX cDNA was also used to map the locus for factor IX to a region from Xq26 to Xqter. The cloning of human factor IX cDNA and identification of a Taq I polymorphism and its regional localization will provide a means to study the molecular genetics of hemophilia B and permit linkage analysis with nearby loci.

Amino Acid Sequence↗