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D Y Kwoh

Publications and source records attributed to D Y Kwoh.

At least 19 recordsLinked to original sources
PubMed2000

Identification of three new members of the phospholipid scramblase gene family.

Phospholipid (PL) scramblase is a 35 kDa protein that is thought to mediate Ca2+-induced bidirectional transbilayer movement of plasma membrane phospholipids in activated, injured, or apoptotic cells. We recently reported the molecular cloning of a PL scramblase of human (HuPLSCR1) and mouse origin, respectively. In the present study, the gene for HuPLSCR1 was cloned from a human genomic library. The gene size is 29.7 kb and includes nine exons. Analysis of the 5' flanking genomic sequence with luciferase reporter constructs located the promoter to a region spanning from -95 to +60 of the first (untranslated) exon. Furthermore, we report the molecular cloning of three additional novel cDNAs encoding proteins with high homology to HuPLSCR1. The predicted open reading frames encode proteins with 59% (HuPLSCR2; 224 aa), 47% (HuPLSCR3; 295 aa) and 46% (HuPLSCR4; 329 aa) identity, respectively, to HuPLSCR1. All members of the PLSCR gene family conserve those residues contained in the segment of the PLSCR1 polypeptide that was previously shown to bind Ca2+. With the exception of HuPLSCR2, these proteins also each contain multiple PXXP motifs and a PPXY motif located near the N-terminus, implying the potential for interaction with SH3 or WW domain-containing proteins, respectively. HuPLSCR1, 2, and 4 were found to be closely clustered on chromosome 3 (3q23), whereas HuPLSCR3 is located on chromosome 17. Northern blots revealed that the expression of HuPLSCR2 is restricted to testis, whereas HuPLSCR1, 3 and 4 are expressed in most of the 16 tissues examined. Notable exceptions were HuPLSCR4, which was not detected in peripheral blood lymphocytes, and HuPLSCR1 and HuPLSCR3, which were not detected in brain.

Amino Acid Sequence↗
PubMed1999

Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver.

We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly-l-lysine and plasmid DNA. We have characterized poly-l-lysines of different chain lengths for DNA condensation and strength of DNA binding. Poly-l-lysine chains >20 residues bound DNA efficiently in physiological saline, while shorter chains did not. Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly-l-lysine/DNA polyplexes appeared as toroids 25-50 nm in diameter or rods 40-80 nm long; conjugation of asialoorosomucoid to the polylysine component increased the size of resulting polyplexes to 50-90 nm. In water, poly-l-lysine and asialoorosomucoid-PLL polyplexes have effective diameters of 46 and 87.6 nm, respectively. Polyplexes containing only poly-l-lysine and DNA aggregated in physiological saline at all charge ratios and aggregated at neutral charge ratios in water. Attachment of asialoorosomucoid lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly-l-lysine sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces. For efficient in vivo gene delivery, polyplexes will need to be sterically stabilized to prevent aggregation and interaction with serum components.

Animals↗
PubMed1997

Non-viral gene delivery: vehicle and delivery characterization.

The development of non-viral gene therapy has been hampered by an inability to reproducibly manufacture and characterize delivery system components and final formulations. Formation of interpolyelectrolyte complexes as the basis of various gene delivery methods has been approached as the first step towards development of synthetic viruses. We have found that preparation of interpolyelectrolyte complexes from disperse reagents gives a more homogeneous gene delivery vehicle than other methods. Methods which increase homogeneity also result in higher transfection efficiency in vivo. Expression levels of human growth hormone and other reporter proteins in mice confirm the potential of parenteral non-viral gene delivery for some therapeutic applications. Serum is demonstrated to inhibit transfection efficiency in vivo. Our results suggest that further development of methods to manufacture homogeneous disperse non-viral delivery vehicles with stealth characteristics may enhance both the potency and reproducibility of gene transfer in vivo.

Animals↗
PubMed1993

Rapid comparison of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris using polymerase chain reaction amplification.

Polymerase chain reaction amplification was used to compare different regions of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris, to examine homology within the species. Six 30-base polymerase chain reaction primers and three probes were synthesized on the basis of the published nucleic acid sequence of the cytochrome c3 gene from D. vulgaris, NCIMB 8303. Amplifications were performed on genomic DNA isolated from NCIMB 8303 as well as eight other strains. Six strains, NCIMB 8302, 8305, 8306, 8311, 11779, and DSM 2119, showed amplification products of equal size and quantity to those of strain 8303. Two other strains, NCIMB 8456 and DSM 1744, either showed reduced levels or no detectable amplification products. These results were confirmed by hybridization of amplified DNA to radiolabeled probes specific for each product. DNA sequencing of a 145-bp polymerase chain reaction fragment from strains NCIMB 8302, 8303, 11779, and DSM 2119 revealed complete sequence homology between these strains, whereas slight differences were seen with strain NCIMB 8456. Amino acid sequencing of the first 20 residues of cytochrome c3 purified from strains NCIMB 8456 and 8303 also showed differences in the two proteins. In contrast to the results obtained with strain NCIMB 8456, limited homology was observed between the first 20 amino acid residues of cytochrome c3 from strain DSM 1744 and strain NCIMB 8303.

Amino Acid Sequence↗
PubMed1990

Methodologies for in vitro nucleic acid amplification and their applications.

The capability to detect the genetic elements (DNA or RNA) of a particular pathogen as a means of identifying the infectious agent has been the traditional function of nucleic acid hybridization assays. The low copy number of genetic material from several types of viral pathogens has fostered the development of in vitro nucleic acid amplification methods as a means to increase the copy number of the characteristic genetic elements of pathogenic agents. The polymerase chain reaction (PCR) and a transcription-based amplification system (TAS) are two amplification methods that have been developed to serve this function. Both methods have been employed to study both genetic and infectious disease problems. This review discusses the characteristics of these amplification methods and describes some of their applications, especially in the study of HIV-1.

DNA↗
PubMed1990

Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.

Base Sequence↗
PubMed1990

Detection of human immunodeficiency virus type 1 in AIDS patients using amplification-mediated hybridization analyses: reproducibility and quantitative limitations.

Eighty-six peripheral blood mononuclear cell (PBMC) samples from 30 patients with AIDS were analyzed using a transcription-based amplification system (TAS) and the polymerase chain reaction (PCR). Human immunodeficiency virus type 1 (HIV-1) sequences were detected by amplification-mediated hybridization in 98% of the samples, 52% of which were positive for p24 antigen by ELISA. Neither TAS (93%) nor PCR (95%) detected HIV-1 sequences in all 86 samples. The hybridization-detection methods (slot blot, bead-based sandwich, and solution) used to detect the HIV-1-specific TAS products had a clear influence on the efficiency of detecting and quantitating the levels of HIV-1 present in these samples. The reproducibility of amplification of constant amounts of HIV-1 RNA and beta-globin DNA by TAS and PCR was studied over 3 months. The results indicated that variations of 10- and 5-fold in the HIV-1 sequence levels could be detected between samples by TAS and PCR, respectively. Within the range of sensitivities for each assay used, the administration of zidovudine did not appear to reduce the amount of HIV-1 nucleic acid sequences as observed in PBMC obtained serially from six AIDS patients.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Target amplification systems in nucleic acid-based diagnostic approaches.

Currently, PCR is the standard method for target amplification because it is the oldest and most developed procedure. However, several new alternative approaches for target amplification have recently been developed. Although these new methods are at a relatively early stage of development, each has some advantages over PCR, such as greater amplification per cycle (TAS, 3SR, Q beta), isothermal reaction (3SR), or coupled amplification-mutation detection (LAR/LAS). As a result, each may eventually gain widespread use after further development.

DNA↗
PubMed1990

Unique features of the self-sustained sequence replication (3SR) reaction in the in vitro amplification of nucleic acids.

The development of a transcription-based amplification system and its application to a retrospective analysis of HIV-1-infected clinical samples demonstrated the specificity and sensitivity of this in vitro amplification procedure. The TAS protocol has been modified to mimic the retroviral strategy of replication, resulting in a self-sustained sequence replication (3SR) amplification reaction which operates under isothermal conditions (37 degrees C). The ability to specifically amplify only RNA sequences in the presence of DNA genomic copies containing the same sequence and the rapid kinetics of the 3SR reaction distinguish it from the well-used PCR protocol.

DNA Replication↗
PubMed1989

Synthesis of 5'-oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes.

A hydrazone-based method for conjugating synthetic nucleic acids and reporter molecules for use as nonradioactive hybridization probes is presented. Oligonucleotides complementary to the hepatitis B virus were derivatized at their 5' ends with hydrazine or homobifunctional acyl hydrazides. These derivatives reacted facilely with aldehydes to give hydrazones, which were characterized by uv spectroscopy and HPLC. Coupling of aldehyde-modified alkaline phosphatase with carbohydrazide-oligonucleotide derivatives provided a mixture of two enzyme-nucleic acid conjugates in 80-85% yield. The conjugates had a 1:1 and a 2:1 oligonucleotide/enzyme ratio, respectively, and were separated by ion-exchange chromatography. Both conjugates were able to detect 7 amol of target DNA in 1 h, using a colorimetric assay. In contrast, oligonucleotide-horseradish peroxidase conjugates were 40-fold lower in sensitivity of detection.

Alkaline Phosphatase↗
PubMed1989

Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format.

The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 10(6)-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 10(6) uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.

Cell Line↗
PubMed1988

Introduction and expression of the bacterial PaeR7 restriction endonuclease gene in mouse cells containing the PaeR7 methylase.

To study the factors essential for a functional restriction system, the PaeR7 restriction-modification system has been introduced and expressed in murine cells. Transfer of this system was accomplished in two steps. First, cells containing sufficient PaeR7 methylase to completely methylate the mouse genome were constructed. In the second step, the mouse metallothionein promoter-regulated, endonuclease expression vector linked to the hygromycin B resistance selection marker was used to transfect the high methylase-expressing cells. Sixty percent of the clones isolated contained PaeR7 endonuclease enzymatic activity. Transfected cells expressing both methylase and endonuclease were incapable of blocking infection by DNA viruses, and possible explanations are discussed.

Adenoviridae↗
PubMed1987

Hybridization properties of immobilized nucleic acids.

The 5'-end attachment of oligonucleotides to dextran supports facilitates the study of the hybridization properties of an immobilized oligonucleotide system. The hybridization properties which were studied include: hybridization capacity and kinetics, hybridization-complex stability, and reagents influencing hybridization efficiency. Results of these experiments reveal that the hybridization efficiencies of support-bound oligonucleotides were 75-80% and 40-50% for single-stranded oligonucleotide targets and long double-stranded targets, respectively. These hybridization efficiencies are dependent upon prehybridizing the support-bound oligonucleotides with dextran sulfate. In addition, comparisons of the relative hybridization efficiencies of the support-bound oligonucleotide and nitrocellulose-based systems have been made which indicate a retention of 13-28% of target sequences on the filters and a detection efficiency of 8-20%.

Acrylic Resins↗
PubMed1986

Introduction and expression of the bacterial PaeR7 methylase gene in mammalian cells.

An approach is devised for studying the role of DNA methylation in eukaryotic gene expression. The approach is based on the expression of site-specific bacterial methylase genes in animal cells. A model system using the cloned PaeR7 (an isoschizomer of Xho I) methylase gene was constructed to test the feasibility of this approach. Expression plasmids for the PaeR7 methylase gene were introduced into mouse Ltk- cells by cotransfection with the cloned chicken thymidine kinase (tk) gene. Several of the cell strains derived from Tk+ colonies were found to express the PaeR7 gene as judged by four criteria: the cellular DNA of these strains showed increased resistance to cleavage by Xho I; these strains contained cellular proteins that comigrated with pure PaeR7 methylase protein, as visualized by immunoblotting; PaeR7 methylase activity was found in vitro in crude extracts of total cellular protein from these strains; and murine adenovirus genomes grown on cells expressing PaeR7 methylase showed resistance to cleavage to PaeR7 endonuclease. The potential applications of this approach for the study of cellular and viral gene regulation, DNA repair, and restriction modification are discussed.

Animals↗