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K B Choo

Publications and source records attributed to K B Choo.

At least 37 records · Page 2Linked to original sources
PubMed1995

Genotyping of hepatitis D virus by restriction-fragment length polymorphism and relation to outcome of hepatitis D.

The outcome of hepatitis D virus (HDV) superinfection varies among patients and in different geographical areas. To find out whether HDV genotype affects outcome, we used a simple genotyping method based on restriction-fragment length polymorphism with enzymes XhoI and SacII for cleavage of PCR products of the HDV genome. Of samples from 88 patients studied, the genotypes of 61 were confirmed by two methods--analysis with both enzymes or by combined restriction-enzyme and direct sequencing analyses--with consistent results. No genotype III HDV was detected among these patients. The majority of patients with acute HDV infection (35/41 [85%]) had genotype II HDV. Among the 41 patients with acute infection, four of six with genotype I had fulminant disease compared with two of 35 with genotype II. Among patients in chronic stage, cirrhosis or hepatocellular carcinoma were found in 12 of 18 with genotype I HDV and eight of 29 with genotype II. Thus genotype II was the predominant HDV genotype in this study in Taiwan. Genotype II HDV was less frequently associated with fulminant hepatitis at the acute stage or with an unfavourable long-term clinical outcome at the chronic stage than was genotype I.

Acute Disease↗
PubMed1995

Jun-B oncogene aberrations in cervical cancer cell lines.

We describe here structural and expression analysis of the jun-B oncogene in two cervical cancer cell lines. In the CC7T-a cell line, results from both Southern analysis and cDNA cloning studies revealed the existence of two structurally altered jun-B alleles besides the normal gene. One of the altered alleles was due to a type 16 human papillomavirus (HPV-16) integration event, whereas the other allele was a consequence of a chromosomal translocation involving chromosome 19 (jun-B) and an EST182 locus residing in chromosome 15. In the HeLa cell line, which contains integrated HPV-18, an apparent structural aberration, a 3-fold amplification and a 3-fold overexpression of the jun-B gene were observed. Our observations suggest that deregulation of the jun-B gene expression may have contributed to the transformation process in these two cervical cancer cell lines.

Alleles↗
PubMed1995

Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences.

Exogenous DNA microinjected into one-cell mouse zygotes either integrates into the host genome within a short time span, or is rapidly degraded. On integration, a transgene sequence is frequently reiterated. In this report, we describe the enzymatic amplification analysis of transgene junctions of 12 transgenic mice carrying different copy numbers of the same transgene with dissimilar ends. The transgene was composed of the regulatory sequence of the type 18 human papillomavirus linked to the TAg gene of the SV40 virus. Nucleotide sequences of 36 of these junctions were also determined. Deletions were found in 33 (91.7%) of the junctions analysed. At the crossover regions, 55.6% contained short overlapping sequences of one to six nucleotides. Insertions of 2-6 extraneous nucleotides were also found in 8.3% of the transgene junctions. Within a 10-nucleotide sequence on both sides of the transgene junctions, topoisomerase I (topo I) cleavage sites, runs of homogeneous purines or pyrimidiens, alternating purine-pyrimidine tracks and (A-T)-rich sequences were found frequently. Stringent control experiments were also performed to ascertain that the observations made were not artefacts resulting from the polymerase chain reaction. Our data therefore indicate that damage had occurred quite frequently and extensively in our transgene construct. Such transgene damage may also occur to various extents in mice carrying other transgenes. Primary structure of the nucleotide sequences of the injected DNA seems to influence the process of transgene reiteration and aberration.

Animals↗
PubMed1995

Inhibition of alpha-fetoprotein production in a hepatoma cell line by antisense oligonucleotide analogues.

alpha-Fetoprotein (AFP) is a fetal protein which is absent in adult serum. However, the AFP gene is expressed in some neoplastic cells. According to the literature, AFP may play a role in accelerating the growth of cancer cells. In this report, 15 meric antisense oligonucleotide analogues (phosphorothioates and methylphosphonates) and their chimeric forms, which were complementary to different regions of AFP mRNA, were synthesized, and their physical characteristics such as stability, melting temperature, and toxicity were compared. They were examined as to their inhibitor effects on the translation of AFP mRNA in a AFP-producing hepatoma cell line, HuH-7. We found that chimeric oligomers with methylphosphonate or phosphorothioate linkages at both the 5' and 3' ends were more effective than prototypic oligomers. Inhibition of 72% was achieved with a chimeric oligomer against the translational initiation region, at a concentration of 25 microM. No suppressive effect of the oligomers was observed on cell viability or albumin production, indicating the specificity of the inhibition.

Base Sequence↗
PubMed1995

Evidence of transmission of hepatitis D virus to spouses from sequence analysis of the viral genome.

To study sexual transmission of hepatitis D virus (HDV), 52 spouses of 56 index patients were observed and HDV genomes from antibody to HDV (anti-HDV)-positive couples were sequenced. Of the spouses, 11 (21%) were serum HBsAg positive, 3 (27%) of whom were also anti-HDV positive. The HDV sequences between spouses were found to be nearly identical (98% to 98.8%) in the region analyzed (nt 911 to nt 1260). Only one couple showed an identity > 90% with the genotype I HDV strains. The HDV sequences of the remaining two couples showed > 95% identity with each other and > 91% homology with genotype II, but they shared only a 73.1% to 73.7% homology with those of the first couple. The regions corresponding to the autocatalytic cleavage sites, the junction between the middle and the carboxyl terminal one-third domains, and the middle domain of the open reading frame for delta antigen on the antigenomic HDV RNA were more conserved with < 19% divergence among the three couples. Interestingly, there was a 56% divergence in the region corresponding to the carboxyl end of the open reading frame for the large delta antigen on the antigenomic HDV RNA. In summary, this study provides a direct nucleotide evidence of a common source of HDV infection in each couple. Despite divergence in the viral nucleotide sequence, both genotypes I and II were found in Taiwan and were transmitted from patients with a history of prostitute contact to spouses through sexual contact.

Base Sequence↗
PubMed1994

Analysis of deletion of the integrated human papillomavirus 16 sequence in cervical cancer: a rapid multiplex polymerase chain reaction approach.

A protocol for a rapid physical mapping of the integrated type 16 human papillomavirus (HPV16) sequences in biopsied and paraffin-embedded archival cervical cancer samples is described. The procedure involves the use of an anchor primer and a mixture of indicator primers in a multiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleotide (nt) 6102-941, containing the entire regulatory region and the E6 and E7 open reading frames (ORFs). A general deletion domain of 1,465 bp in the integrated viral genome has been defined between nt 1417-2881, covering most of the E1 ORF at the 3'-half and 60 bp at the 5' terminus of the E2 ORF. This common deleted sequence contains an ATPase active domain speculated to be associated with a DNA helicase function essential for the viral replication, and it also falls within the actively spliced E1-E2 segment of the primary RNA transcripts. Detection of the loss of the 3'-half of the E1 ORF would be an ideal marker for PCR-based rapid determination of HPV integration in cervical cancer cells.

Chromosome Mapping↗
PubMed1994

Prenatal diagnosis of beta-thalassemic mutations in Chinese by multiple restriction fragment-single strand conformation polymorphism analysis.

A modified approach of polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) has been developed for prenatal diagnosis of beta-thalassemic mutations. In this method, a single PCR product (1,350-bp) is first generated, which is then digested by restriction enzymes (BbsI, BamHI, DraI) to generate multiple shorter restriction fragments for electrophoretic analysis in an SSCP gel. The method is thus termed multiple restriction fragment (MRF)-SSCP. Two cases of chorionic samples and the blood samples of their parents were studied. The previously described amplification-created restriction site (ACRS) method was used to confirm the SSCP results obtained. Two polymorphic sites of the beta-globin gene which would influence the MRF-SSCP patterns are described, including a previously undescribed polymorphic site located at codon 3 (CAT or CAC) of the beta-globin gene.

Base Sequence↗
PubMed1993

Absence of mutation in the p53 and the retinoblastoma susceptibility genes in primary cervical carcinomas.

Previous biochemical and structural studies using established cell lines have led to a hypothesis that the cellular p53 and the retinoblastoma susceptibility (Rb) proteins may play a crucial role in the pathogenesis process of cervical carcinoma. To further test this hypothesis, we have analyzed the structure of the p53 and Rb genes in 38 primary cervical carcinoma samples with known HPV status using the polymerase chain reaction-based single-strand conformation polymorphism (SSCP) approach. All 11 exons as well as 500 bp of the 5'-noncoding region of the p53 gene and exons 13-23 of the Rb gene were analyzed. None of these exons in all the samples analyzed showed band shift in the SSCP gel indicative of the presence of mutation. In the same experiments, however, all known exon mutations as well as previously unreported mutations in the intron sequences of the p53 and Rb genes in the HPV-free C33-A and HT-3 cell lines were detected. The absence of mutation in the p53 gene in all five HPV-negative samples was confirmed by direct sequence analysis. It appears that p53 and Rb mutations are a very rare event in cervical cancer and their occurrence is apparently not strictly correlated with HPV status.

Base Sequence↗
PubMed1993

Effect of an extract from Phyllanthus amarus on hepatitis B surface antigen gene expression in human hepatoma cells.

It has been suggested that Phyllanthus amarus may be helpful in the treatment of hepatitis B virus infection. We studied the effect of an aqueous extract of P. amarus on the cultured hepatoma cell line HepA2. This cell line had been transfected with tandemly arranged HBV DNA and continued to synthesize and secrete both HBsAg and HBeAg. Extract of P. amarus reversibly inhibited cellular proliferation and suppressed HBsAg production but not HBeAg production in HepA2 cells. We also found that P. amarus suppressed HBsAg gene expression at mRNA level in a time-dependent manner, and selectively abolished the HBsAg gene promoter driven CAT activity. Our results demonstrate that P. amarus contains some active components which can suppress the HBsAg gene expression in human hepatoma cells. Such suppression may contribute the antiviral activity of P. amarus in vivo.

Animals↗
PubMed1993

Hypermethylation of the human papillomavirus transgenome in transgenic mice.

We have previously reported an observation that the regulatory sequence of the human papillomavirus (HPV) type 18 genome elicits only basal and unregulated transcriptional activities in transgenic mice [Choo et al., Virology (1992) 188:378-383]. We have now extended the study to other transgenic mice carrying an entire or a subgenomic fragment of HPV-16 and -18. Extremely low levels of HPV transcripts were detected in various organs of the mice only by reverse transcriptase-based polymerase chain reaction. On analysis of the methylation status of the HPV transgenome sequences, hypermethylation was clearly observed, therefore suggesting a possible explanation for low level HPV transgenome transcriptional activity in HPV transgenic mice.

Animals↗
PubMed1993

Post-hybridization recovery of membrane filter-bound DNA for enzymatic DNA amplification.

We describe here a simple and rapid method for enzymatic DNA amplification using DNA template recovered from membrane filters previously used in hybridization analysis. This is done by first solubilizing membrane pieces carrying DNA of interest in dimethyl sulfoxide, followed by isopropanol precipitation and polymerase chain reaction amplification. The source of membrane-bound DNA successfully tested includes plasmid and human leukocyte DNA and DNA immobilized on bacterial colony filters and plaque lifts. The sensitivity of the procedure is such that DNA recovered from 0.5 microgram of filter-bound total human DNA was enough for PCR amplification of a 0.3-kb fragment. Our protocol will be useful for recycling of scarce DNA samples for cloning and sequencing purposes.

Biotechnology↗
PubMed1992

Detection of mutations in the p53 gene in human head and neck carcinomas by single strand conformation polymorphism analysis.

Using the polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis, we have examined the highly conserved regions of the p53 gene in 58 biopsy samples of head and neck tumors. Mutations were found in 13/58 (23%) tumor specimens, but not in 6 normal tissues. Ten of 13 mutations were due to single base changes and the remaining 3 were 1- or 8-base deletion mutants. These mutations were clustered in exons 5 and 7 and resulted in amino acid changes. Our results seem to indicate that mutations in the p53 gene contribute to a significant number of cases of the head and neck tumors including 20% of nasopharyngeal carcinoma biopsies. The relationship of Epstein-Barr virus or human papillomavirus and p53 gene mutations in this group of cancers was also analyzed and discussed.

Base Sequence↗
PubMed1992

Mutational analysis by a combined application of the multiple restriction fragment-single strand conformation polymorphism and the direct linear amplification DNA sequencing protocols.

We describe here an improved procedure for polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) for rapid mutational detection. To circumvent the restriction of having to analyze relatively short PCR fragments, restriction endonucleases were used to cleave a longer PCR product and the mixture of fragments was analyzed directly in SSCP gel electrophoresis. This multiple restriction fragment (MRF)-SSCP protocol was demonstrated by the detection of a 4-bp deletion in codons 41-42 and a point mutation in the IVS-2 sequence of the human beta-globin gene. The MRF-SSCP or the standard SSCP protocol was then combined with the linear amplification DNA sequencing (LADS) procedure for direct analysis of the PCR products without further purification for an exact characterization of the mutations detected. In the LADS analysis, homo- or heterozygosity of a mutation was easily distinguished by the appearance of a single- or double-lane band in the sequencing gel. The choice of isotope used and different labeling methods were compared and were found, in some cases, to produce SSCP patterns of different complexities. The combined MRF-SSCP/LADS protocol permits rapid mutational analysis of a large number of clinical samples using only very small amounts of materials and can easily be adopted for nonisotopic clinical applications.

Base Sequence↗
PubMed1992

Unregulated and basal transcriptional activities of the regulatory sequence of the type 18 human papillomavirus genome in transgenic mice.

Type 18 human papillomavirus (HPV18) is a genital virus closely associated with cervical carcinoma. To analyze the transcriptional activities of the long control region (LCR) of the HPV18 genome, we have produced 12 transgenic mice harboring the HPV18/LCR sequence fused to a promoterless SV40 T-antigen (TAg) gene. The mice were small in body size, generally very weak, and none lived longer than 110 days. Three mice with the longest life span (58-110 days) developed hyperplastic thymus and/or lymph node and were further analyzed. In these mice, Northern hybridization failed to detect TAg transcripts in any of the 25 organs studied. However, spliced TAg RNA was detected by polymerase chain reaction in the hyperplastic thymus and lymph node and in the normal submaxillary gland, stomach, large intestine, urinary bladder, and the cerebrum, indicating the presence of very low cellular levels of TAg RNA in these organs. When immunostaining was performed on the hyperplastic thymus, TAg protein was detected only in the ductal epithelial cells. Our results appear to indicate that the HPV18/LCR sequence was able to express only unregulated and basal levels of transcriptional activity in transgenic mice. Such a mode of transcription has become a major hindrance in the use of transgenic mouse system for the studies of the biology of the human papillomavirus.

Amino Acid Sequence↗
PubMed1992

Characterization of a monoclonal antibody (P40) against the 68 kD major allergen of Penicillium notatum.

A monoclonal antibody (MoAb P40) against the 68 kD major allergen of Penicillium notatum (P. notatum) was obtained by immunizing the mouse with a crude extract of P. notatum. Analysed by two-dimensional gel electrophoresis and immunoblotting, P40 reacted with two different isoforms of the 68 kD component of P. notatum with pIs of 5.4 and 5.5. In addition to P. notatum, P40 showed positive ELISA activity to Aspergillus fumigatus (A. fumigatus) but not to components of six other fungi including Alternaria porri, Cladosporium cladosporoides, Aureobasidium pullulans, Fusarium solani, Rhizopus arrhizus and Candida albicans. Analysed by ELISA, MoAb P40 also showed positive activity to two (P. frequentans and P. roseopurpureum) of the 10 other Penicillium species and two (A. terreus and A. flavus) of the four other Aspergillus species tested. SDS-PAGE and immunoblotting studies demonstrated P40 positive reactivity to components with MW of about 67 kD in all these Penicillium and Aspergillus species with positive ELISA activity to P40. Furthermore, immunoblotting activity of MoAb P40 to the 67 kD component of A. niger was also observed. The epitope of the 68 kD allergen of P. notatum recognized by MoAb P40 was resistant to treatment of periodate oxidation with concentration of NaIO4 up to 20 mM. This MoAb may thus be useful in the characterization and purification of the 68 kD allergen from crude extracts, and in the molecular cloning of allergen genes.

Allergens↗
PubMed1991

A common precursor for a putative hemorrhagic protein and rhodostomin, a platelet aggregation inhibitor of the venom of Calloselasma rhodostoma: molecular cloning and sequence analysis.

Rhodostomin is a platelet aggregation inhibitor secreted by the venom gland of Calloselasma rhodostoma. We report here the isolation of a 1.67-kilobase (kb) lambda gt11 cDNA clone using degenerate oligonucleotide probe based on a partial amino acid sequence of rhodostomin. The amino acid sequence deduced from an open reading frame of the cDNA indicates that (i) the 68-amino acid sequence of rhodostomin is located at the carboxyl terminus of the precursor polypeptide and (ii) a peroxisomal targeting sequence (ser.his.ala.) exists between the stop codon and the rhodostomin sequence of the precursor. Since the amino-terminal segment of the deduced sequence shows a high degree of identity with hemorrhagic proteins, which are zinc-metalloproteinases, found in the venom of some crotalid and viperid snakes, our results also predict the existence of at least one such hemorrhagic protein in the venom of Calloselasma rhodostoma. The derivation of a platelet aggregation inhibitor and a hemorrhagic protein from the same precursor protein is consistent with the fact that these proteins may be synergistic in function.

Amino Acid Sequence↗
PubMed1991

Transgenome transcription and replication in the liver and extrahepatic tissues of a human hepatitis B virus transgenic mouse.

We have produced a transgenic mouse (B32-1) carrying the complete genome of the human hepatitis B virus (HBV). High titers of the viral surface (HBsAg) and the e antigen (HBeAg) were detected in the serum of the mouse. In the liver and 12 of 16 extrahepatic tissues analyzed, Northern blot hybridization indicated the presence of the 2.1-kilobase (kb) and the 3.5-kb major HBV transcripts. A liver cDNA library was constructed from which the liver RNAs from four cDNA clones with splicing were found. Sequencing analysis showed that the splicing occurred between nucleotides 2451 and 487 of the viral genome, resulting in a truncated viral polymerase gene, as in human hepatocytes. Southern blot analysis of total DNA preparations of the tissues revealed the presence of episomal HBV genome, indicating replication of the viral transgenome in these tissues. However, replication was detected only in some but not all of the tissues that transcribed the 3.5-kb RNA. Partial double-stranded as well as full-length and subgenomic-length single-stranded HBV DNA species of discrete sizes were detected which may represent replication intermediates of preferred replication termination sites of the HBV transgenome. Since many molecular characteristics of mouse B32-1 were similar to those found in HBV-infected humans, HBV transgenic mice similar to B32-1 would be useful in further elucidation of other aspects of the replication and transcription mechanisms of HBV in the liver and extrahepatic tissues.

Animals↗