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Biomedical subjects

P Jagadeeswaran

Publications and source records attributed to P Jagadeeswaran.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1982

Interactive computer programs in sequence data analysis.

We present interactive computer programs for the analysis of nucleic acid sequences. In order to handle these programs, minimum computer experience is sufficient. The nucleotide sequence of the human gamma globin gene complex is used as an example to illustrate the data analysis.

Base Sequence↗
PubMed1981

Structural analysis of templates and RNA polymerase III transcripts of Alu family sequences interspersed among the human beta-like globin genes.

Cloned DNA fragments form the human beta-like globin genomic region can be transcribed in vitro by RNA polymerase III. We have investigated the structure of two templates and their transcripts by DNA sequencing, size fractionation of ribonuclease T1 generated oligonucleotides, and ribonuclease H digestion of RNA : DNA duplexes. The data indicate the repetitive DNA sequences, members of the Alu family of interpersed 300 bp reiterated DNA, are imbedded in both templates. The RNAs transcribed from them are composed of an entire Alu family sequence at their 5' ends linked to 3' ends of non-repetitive sequence.

Base Sequence↗
PubMed1981

Base substitution in an intervening sequence of a beta+-thalassemic human globin gene.

beta globin gene fragments from a patient with homozygous beta+-thalassemia have been cloned and subjected to restriction endonuclease, nucleotide sequence, and in vitro trancription analyses. Restriction endonuclease mapping of the cloned gene fragments revealed no deletions or other rearrangements, and transcription of the thalassemic gene appeared to be normal in vitro. However, nucleotide sequence analysis of the beta+-thalassemic gene fragments permitted identification of a single base change in the body of the small intervening sequence. This nucleotide change creates a sequence much like that of the 3' splice site of the small intervening sequence. The presence of a potential anomalous splicing site as a result of this base change suggests a mechanism for defective posttranscriptional processing of beta globin mRNA precursor molecules in beta+-thalassemia.

Base Sequence↗