Search PubMed⌕ Search

Biomedical subjects

K H Choo

Publications and source records attributed to K H Choo.

At least 109 records · Page 6Linked to original sources
PubMed1987

Genomic organization of human centromeric alpha satellite DNA: characterization of a chromosome 17 alpha satellite sequence.

We characterized a recombinant clone E7 containing a 1.6-kb Eco RI insert of human alpha satellite DNA (alpha DNA) which hybridized in situ predominantly to the centromere of chromosome 17. Three thousand copies of this sequence were detected on chromosome 17, although a lesser number of copies were also found on the centromeres of chromosomes 11, X, and the other human chromosomes, except Y. In the human genome, sequences homologous to E7 were organized principally as five major polymorphic (Pst I) forms of tandem alpha DNA repeats with molecular weights between 2.0 and 2.7 kb. We We studied the higher-order organization of these major forms using a series of 12 cosmid clones. Close linkage of the different polymorphic forms was demonstrated, with no two cosmids showing an identical linkage pattern. Six of the cosmid clones carried a considerable amount (20-25%) of nonhomologous (non-alpha) DNA, indicating that the repeat arrays are relatively frequently interrupted by other genomic DNA. In none of the cosmid inserts were the repeat arrays bound on both sides by non-alpha DNA, suggesting that short arrays are not common. However, some of the intervening non-alpha DNA sequences were relatively short, and vary in size from 6 to 24 kb. Our results suggest an irregular and complex pattern of organization of alpha DNA in the human genome.

Chromosome Mapping↗
PubMed1986

Cosmid vectors for high efficiency DNA-mediated transformation and gene amplification in mammalian cells: studies with the human growth hormone gene.

We have constructed two new recombinant cosmid vectors that can be used for direct expression and amplification of genomic DNA in mammalian cells. The vectors allow cloning of DNA fragments up to 40 kb in size. Each carries two dominant selectable markers: the bacterial neo gene and the mouse DHFR gene. In the first vector, pCV001, the neo and DHFR genes are regulated by the SV40 early promoter, and in the second, pAVCV007, by the avian sarcoma virus LTR promoter. The neo gene served as a dominant marker for the selection of transformants in all mammalian cell types, and we demonstrate here that the LTR promoter significantly improved the efficiency of DNA-mediated transformation of a human cell line. We isolated the human growth hormone genes from genomic libraries prepared in these cosmid vectors and used these recombinant cosmids for direct transfections of cultured cells. Selection of transformants in increasing concentrations of methotrexate led to the outgrowth of resistant cell populations carrying amplified copies of the DHFR marker. A 40-1000-fold coamplification of the hGH genes was observed in the different transfected cell lines, along with a corresponding increase in transcription and translation activity of the hGH gene. Gene amplification could be achieved in both DHFR deficient or normal cell lines. High level expression of a cloned gene mediated by gene amplification should facilitate characterization of DNA sequences, as well as isolation of specific gene products for biochemical, functional, and pharmacological studies.

Animals↗
PubMed1986

Vectors for expression and amplification of cDNA in mammalian cells: expression of rat phenylalanine hydroxylase.

We have constructed two recombinant plasmid vectors for direct expression and amplification of cDNA in mammalian cells. Each vector carries two dominant selectable markers (the bacterial neo gene and the mouse DHFR gene), a promoter sequence (viral LTR in pAV009/A+, and sheep metallothionein promoter in pMT010/A+), a polyadenylation signal sequence, and a Bam HI site to allow insertion of cDNA. We have used these vectors to prepare recombinant clones for the expression of rat phenylalanine hydroxylase (PH) in LTK- cells. Selection of transformants with neomycin followed by selection of the transformants in methotrexate led to a 30- to 60-fold amplification of the DHFR marker and co-amplification of the PH cDNA, with a corresponding increase in the level of PH mRNA and enzyme polypeptide. The expressed enzyme has a subunit molecular weight of 50,000 which corresponds to the W- allele of rat liver PH. PH activity was detected in the transfected cells by enzymatic measurement of the conversion of [14C]phenylalanine to [14C]tyrosine, and by growth of these cells in a tyrosine-free culture medium. Expression of rat PH in cell culture should facilitate the analysis of the biochemical properties of this enzyme.

Animals↗
PubMed1984

Characterisation and use of an intragenic polymorphic marker for detection of carriers of haemophilia B (factor IX deficiency).

DNA from 33 healthy White subjects was analysed with a 2 X 5 kilobase subgenomic DNA probe derived from the gene for coagulation factor IX, containing the exon "d" region of that gene. Intragenic Taq I restriction-fragment length polymorphism was revealed, with allelic frequencies estimated at 0 X 65 and 0 X 35 (SE = 0 X 06), also detectable by a cDNA probe. The genomic DNA probe is technically superior to the cDNA probe and has been used in three families with haemophilia B (factor IX deficiency). The polymorphic marker segregates with the deleterious mutation, allowing the identification or exclusion of carriers. The allelic frequencies of the Taq I polymorphism are virtually ideal. Therefore, such a polymorphism should be helpful both in genetic counselling of approximately 40% of affected families and in prenatal diagnosis.

Alleles↗
PubMed1984

The gene structure of human anti-haemophilic factor IX.

The mRNA sequence of the human intrinsic clotting factor IX (Christmas factor) has been completed and is 2802 residues long, including a 29 residue long 5' non-coding and a 1390 residue long 3' non-coding region, but excluding the poly(A) tail. The factor IX gene is approximately 34 kb long and we define, by the sequencing of 5280 residues, the presumed promoter region, all eight exons, and some intron and flanking sequence. Introns account for 92% of the gene length and the longest is estimated to be 10 100 residues. Exons conform roughly to previously designated protein regions, but the catalytic region of the protein is coded by two separate exons. This differs from the arrangement in the other characterized serine protease genes which are further subdivided in this region.

Amino Acid Sequence↗
PubMed1984

Assignment of the haemophilia B (factor IX) locus to the q26-qter region of the X chromosome.

Two independent approaches have provided an assignment for the factor IX (Haemophilia B) locus to Xq26-qter, the distal region of the X chromosome long arm. Cloned DNA sequences of the gene were hybridized to restriction enzyme digests of DNA prepared from a series of somatic cell hybrids containing different X-chromosomal regions and also in situ to prepared metaphase chromosomes.

Animals↗
PubMed1982

Molecular cloning of the gene for human anti-haemophilic factor IX.

A functional deficiency of factor IX, one of the coagulation factors involved in blood clotting, leads to the bleeding disorder known as Christmas disease, or haemophilia B. Both this disease and haemophilia A (factor VIII (C) deficiency) are X chromosome-linked and together occur at a frequency of approximately 1 in 10,000 males. The molecular basis for the functional alteration of factor IX in Christmas disease is not clearly understood. As a first step towards the elucidation of the molecular events involved, we have attempted molecular cloning of the factor IX gene. We used a bovine factor IX cDNA clone, isolated using synthetic oligonucleotides as probes, to screen a cloned human gene library. Here we report the isolation and partial characterization of a lambda recombinant phage containing the human factor IX gene.

Bacteriophage lambda↗
PubMed1981

Comparative studies of four monoclonal antibodies to phenylalanine hydroxylase exhibiting different properties with respect to substrate-dependence, species-specificity and a range of effects on enzyme activity.

Four monoclonal antibodies to phenylalanine hydroxylase are described. Two are inhibitory (PH alpha 1-1 and PH alpha 2-1-1 antibodies), one is stimulatory (B5-1 antibody) and one has no effect on enzyme activity (PH alpha 3-0 antibody). Their properties are compared. Two antibodies (PH alpha 1-1 and B5-1 antibodies) bind primate and rodent phenylalanine hydroxylase, whereas the other two (PH alpha 2-1-1 and PH alpha 3-0 antibodies) bind only the primate enzyme. The binding of PH alpha 1-1 antibody to phenylalanine hydroxylase is dependent on substrate phenylalanine, whereas the binding of the others is not influenced by phenylalanine. Affinity adsorbents prepared from the four antibodies purified phenylalanine hydroxylase substantially (greater than 80% purity) in one step, except for a PH alpha 3-0 antibody--Sepharose column, which behaved anomalously. Two previous publications described the isolation and preliminary characterization of B5 and PH alpha 1-1 antibodies. PH alpha 2-1-1 and PH alpha 3-0 antibodies are reported for the first time.

Animals↗
PubMed1981

Heterogeneity of the molecular defect in human dihydropteridine reductase deficiency.

Radioimmunoassay, immunoprecipitation, affinity chromatography and two-dimensional gel electrophoresis were used to test cultured cells from three families with dihydropteridine reductase deficiency for a catalytically incompetent product of the mutant gene. No mutant enzyme was detected in one dihydropteridine reductase-deficient homozygote or in her parents. A second homozygote and both her parents had easily detectable concentrations of inactive mutant enzyme. In a third family one parent fitted into each of these categories.

Cells, Cultured↗
PubMed1981

Molecular and immunological comparison of human dihydropteridine reductase in liver, cultured fibroblasts and continuous lymphoid cells.

An antiserum was raised in a rabbit against highly purified human liver dihydropteridine reductase (EC 1.6.99.7). Dihydropteridine reductase from human liver, in human cultured fibroblasts and in continuous lymphoid cells all showed identical antigenic properties. The structural characteristics of the reductase from these three sources were further compared by the use of high-precision two-dimensional polyacrylamide-gel electrophoresis. The enzyme from radiolabelled fibroblasts and continuous lymphoid cells was isolated by immunoprecipitation or by affinity chromatography and compared with the purified liver enzyme. Two major polypeptide species were resolved, and polypeptides from all three sources co-migrated identically. Indirect evidence is presented indicating that one of the polypeptide species may have been derived from the other via a post-translational modification. These results support the concept that the same structural gene(s) encodes for dihydropteridine reductase in human liver, fibroblasts and lymphocytes.

Cells, Cultured↗
PubMed1981

Double-label reductive methylation of tissue proteins for precision two-dimensional polyacrylamide-gel electrophoretic analysis.

Reductive methylation has been used to radioactively label crude-extract proteins with 3H or 14C. The procedure achieved good isotope incorporation and resolution of proteins on two-dimensional polyacrylamide gels. It allows high-precision comparison of tissue samples by double-labelling and should facilitate the study of tissue proteins by two-dimensional gel electrophoresis.

Animals↗
PubMed1981

Two-dimensional polyacrylamide gel analysis of fibroblast polypeptides: discussion of its relevance for inherited diseases.

Two-dimensional polyacrylamide gel electrophoresis has been used to resolve several hundred polypeptides from six different human fibroblast cell lines. Two of the cell lines were from normals while the other four were from patients with diseases of the type known to be due to single gene mutations. Less than 2% of the polypeptides from the different cell lines were found to differ in their electrophoretic mobility. The existence of only a small degree of polypeptide variability in pairwise comparison between cell lines suggests the possibility of employing two-dimensional gel electrophoretic comparison as a direct approach for the recognition of pathogenetically important polypeptide alterations in genetic diseases.

Cell Line↗
PubMed1980

Isolation and characterization of a myeloma--spleen-cell hybrid producing antibody to phenylalanine hydroxylase.

Application of the technique of myeloma--spleen-cell fusion [Kohler & Milstein (1975) Nature (London) 256, 495--497] has allowed the isolation of a cell colony that produced a monoclonal antibody against monkey liver phenylalanine hydroxylase. The antibody exhibited cross-reactivity against hepatic phenylalanine hydroxylase from other mammalian species, including human, rat and mouse. Cross-reactivity was established by (a) enzyme-inhibition assay, (b) double-immunodiffusion reaction, and (c) two-dimensional polyacrylamide-gel-electrophoretic analysis of immunoprecipitate. The various properties of the monoclonal antibody and its use in the study of mammalian phenylalanine hydroxylase are presented.

Animals↗
PubMed1980

Genetics of mammalian phenylalanine hydroxylase system. IV. Evidence of phenylalanine hydroxylase in a cultured human hepatoma cell line.

We report here te identification of a cultured human hepatoma cell line which possesses an active phenylalanine hydroxylase system. Phenylalanine hydroxylation was established by growth of cells in a tyrosine-free medium and by the ability of a cell-free extract to convert [14C]phenylalanine to [14C]tyrosine in an enzyme assay system. This enzyme activity was abolished by the presence in the assay system of p-chlorophenylalanine but no significant effect on the activity was observed with 3-iodotyrosine and 6-fluorotryptophan. Use of antisera against pure monkey or human liver phenylalanine hydroxylase has detected a cross-reacting material in this cell line which is antigenically identical to the human liver enzyme. Phenylalanine hydroxylase purified from this cell line by affinity chromatography revealed a multimeric molecular weight (estimated 275,000) and subunit molecular weights (estimated 50,000 and 49,000) which are similar to those of phenylalanine hydroxylase purified from a normal human liver. This cell line should be a useful tool for the study of the human phenylalanine hydroxylase system.

Animals↗
PubMed1980

Observations indicating the nature of the mutation in phenylketonuria.

Rabbit antiserum was prepared against purified normal human liver phenylalanine hydroxylase. This was used to test for cross-reacting material in crude extracts of livers from patients with classical phenylketonuria (PKU); the samples from patients were two livers obtained at autopsy and a needle biopsy core. None of these enzymically inactive livers contained detectable cross-reacting material capable of neutralizing antibody activity in double immunodiffusion and/or enzyme inhibition experiments. In an earlier study, no phenylalanine hydroxylase was found in the two autopsy PKU livers by an affinity chromatography method or by the use of a specific antiserum raised against purified monkey liver phenylalanine hydroxylase (Choo et al., 1979a). This evidence suggests that mutations in th PKU patients studied may be regulatory in nature.

Antigen-Antibody Complex↗