Biomedical subjects

B Thyagarajan

Publications and source records attributed to B Thyagarajan.

15 recordsLinked to original sources

PubMed2005

New approaches for genotyping paraffin wax embedded breast tissue from patients with cancer: the Iowa women's health study.

BACKGROUND: The use of paraffin wax embedded tissue samples as a source of DNA for genotype analysis has been limited because of difficulties in DNA extraction and single nucleotide polymorphism (SNP) analysis. AIMS: To test the feasibility of applying the combination of a commonly used DNA isolation procedure, PureGene, and a high throughput SNP analysis method, the polymerase chain reaction (PCR)-INVADER assay, to genotype several types of paraffin wax embedded breast tissues. METHODS: Twenty formalin fixed, paraffin wax blocks were obtained from five participants in the Iowa women's health study. Each participant provided several types of tissue including normal lymph node, normal nipple/areola tissue, inflammatory/fibrotic breast tissue, or normal breast tissue, and tumour tissue. RESULTS: Good quality DNA (260/280 ratio >1.6) was obtained from all tissues. Normal lymph nodes yielded the largest amount of DNA (97.1 mug). DNA obtained from the samples was tested for a germline C1183T polymorphism in the MnSOD gene by three methods-PCR-RFLP (restriction fragment length polymorphism), INVADER assay, and PCR-INVADER assay. Of the 20 samples, PCR-RFLP genotyped 16, the PCR-INVADER assay 18, and the INVADER assay two. This methodology was then used to analyse five additional genotypes and confirmed the general applicability of the method. CONCLUSIONS: This study demonstrated the feasibility of (1) using several paraffin wax embedded breast tissues as a source of DNA for germline genetic analysis, with lymph nodes providing the highest yield, and (2) using the combination of a common extraction method with a high throughput SNP analysis method, the PCR-INVADER assay.

Breast Neoplasms↗

PubMed2001

Directed evolution of a recombinase for improved genomic integration at a native human sequence.

We previously established that a unidirectional site-specific recombinase, the phage phiC31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.

Base Sequence↗

PubMed2001

Expression of Trp3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling: evidence for a role of Trp3 as a subunit of capacitative Ca2+ entry channels.

The role of Trp3 in cellular regulation of Ca(2+) entry by NO was studied in human embryonic kidney (HEK) 293 cells. In vector-transfected HEK293 cells (controls), thapsigargin (TG)-induced (capacitative Ca(2+) entry (CCE)-mediated) intracellular Ca(2+) signals and Mn(2+) entry were markedly suppressed by the NO donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (3 microm) or by authentic NO (100 microm). In cells overexpressing Trp3 (T3-9), TG-induced intracellular Ca(2+) signals exhibited an amplitude similar to that of controls but lacked sensitivity to inhibition by NO. Consistently, NO inhibited TG-induced Mn(2+) entry in controls but not in T3-9 cells. Moreover, CCE-mediated Mn(2+) entry into T3-9 cells exhibited a striking sensitivity to inhibition by extracellular Ca(2+), which was not detectable in controls. Suppression of mitochondrial Ca(2+) handling with the uncouplers carbonyl cyanide m-chlorophenyl hydrazone (300 nm) or antimycin A(1) (-AA(1)) mimicked the inhibitory effect of NO on CCE in controls but barely affected CCE in T3-9 cells. T3-9 cells exhibited enhanced carbachol-stimulated Ca(2+) entry and clearly detectable cation currents through Trp3 cation channels. NO as well as carbonyl cyanide m-chlorophenyl hydrazone slightly promoted carbachol-induced Ca(2+) entry into T3-9 cells. Simultaneous measurement of cytoplasmic Ca(2+) and membrane currents revealed that Trp3 cation currents are inhibited during Ca(2+) entry-induced elevation of cytoplasmic Ca(2+), and that this negative feedback regulation is blunted by NO. Our results demonstrate that overexpression of Trp3 generates phospholipase C-regulated cation channels, which exhibit regulatory properties different from those of endogenous CCE channels. Moreover, we show for the first time that Trp3 expression determines biophysical properties as well as regulation of CCE channels by NO and mitochondrial Ca(2+) handling. Thus, we propose Trp3 as a subunit of CCE channels.

Anthranilate Synthase↗

PubMed2001

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase.

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

3T3 Cells↗

PubMed2000

A phage integrase directs efficient site-specific integration in human cells.

The integrase from the Streptomyces phage phiC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the phiC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

Bacteriophages↗

PubMed2000

Mammalian genomes contain active recombinase recognition sites.

Recombinases derived from microorganisms mediate efficient site-specific recombination. For example, the Cre recombinase from bacteriophage P1 efficiently carries out recombination at its loxP target sites. While this enzyme can function in mammalian cells, the 34bp loxP site is expected to be absent from mammalian genomes. We have discovered that sequences from the human and mouse genomes surprisingly divergent from loxP can support Cre-mediated recombination at up to 100% of the efficiency of the native loxP site in bacterial assays. Transient assays in human cells demonstrate that such pseudo-lox sites also support Cre-mediated integration and excision in the human cell environment. Pseudo sites for Cre and other recombinases may be useful for site-specific insertion of exogenous genes into mammalian genomes during gene therapy and other genetic engineering processes.

Animals↗

PubMed1999

Epstein-Barr virus plasmid model system for analyzing recombination in human cells.

Homologous recombination stimulated by a double-strand break at a desired target site offers a method to achieve site-specific integration useful for gene therapy and other genetic engineering. To test parameters needed for this strategy, we developed an Epstein-Barr virus shuttle vector model system as a genetic tool. This extrachromosomal plasmid assay system has several advantages over a chromosomal assay. The system detects all classes of recombination events without selection and allows rapid analysis of the frequency and nature of recombination events. We found that a double-strand break at the target site stimulated a large increase in recombination frequency. The resulting recombinants included one-sided insertion events, as well as two-sided or gene conversion events. A circular donor substrate was more effective in recombination than linearized donor DNA.

Base Sequence↗

PubMed1998

Nucleolin promotes homologous DNA pairing in vitro.

We purified to near homogeneity a previously identified 100 kDa mammalian homologous DNA pairing protein. The purified 100 kDa protein also catalyzed high levels of cell-free homologous DNA recombination activity. This ATP-dependent activity was capable of forming conservative recombinant products between two circular, double-stranded DNA molecules. We were unable to detect any DNA polymerase, DNA ligase, or 5' or 3' exonuclease activity associated with this purified material. The purified 100 kDa protein bound silver nitrate as well as a monoclonal antibody specific for nucleolin. A recombinant protein comprised of the Escherichia coli maltos-ebinding protein fused to the carboxyl-terminal two-thirds of human nucleolin possessed homologous DNA pairing activity. These data indicate that the 100 kDa homologous DNA pairing protein is nucleolin. The observation that nucleolin can carry out homologous DNA strand pairing in vitro raises the prospect that it may function similarly in vivo.

Base Pairing↗

PubMed1998

Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts.

Physiological and pathological Ca2+ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+ into mitochondria. Mobilization of Ca2+ stores with bradykinin (BK) increased the cytosolic Ca2+ concentration ([Ca2+]c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca2(+)-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca2+ concentration ([Ca2+]mt) of 2.0 +/- 0.2 microM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca2+]mt increase, although it did not affect the [Ca2+]c transient. Basal [Ca2+]c and [Ca2+]mt in control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 +/- 1% in normal cells to 19 +/- 2% in the PDH-deficient fibroblasts. BK-elicited [Ca2+]mt transients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca2+. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca2+.

Aequorin↗

PubMed1997

Elevated homologous recombination activity in fanconi anemia fibroblasts.

It is widely believed that Fanconi anemia cells possess a reduced ability to repair inter-strand DNA cross-links. While the mechanism through which inter-strand DNA cross-links are removed from mammalian chromosomes is unknown, these lesions are repaired via homologous recombination in lower eukaryotes and bacteria. Based on the hypothesis that a similar mechanism of DNA repair functions in mammalian somatic cells, we measured homologous recombination activity in diploid fibroblasts from healthy donors, and Fanconi anemia patients. Somewhat surprisingly, homologous recombination levels in nuclear protein extracts prepared from Fanconi anemia cells were nearly 100-fold higher than in extracts prepared from control cells. We observed a similar increase in the activity of a 100-kDa homologous DNA pairing protein in extracts from Fanconi anemia cells. Transfection studies confirmed that plasmid homologous recombination levels in intact Fanconi anemia cells were substantially elevated, compared with control cells. These results suggest that inappropriately elevated levels of homologous recombination activity may contribute to the genomic instability and cancer predisposition that characterize Fanconi anemia.

Cell Nucleus↗

PubMed1996

Mammalian mitochondria possess homologous DNA recombination activity.

Mitochondrial protein extracts from normal and immortalized mammalian somatic cells catalyze homologous recombination of plasmid DNA substrates. Mitochondrial homologous recombination activity required exogenous adenosine triphosphate, although substantial activity remained when non-hydrolyzable analogs were used instead. There was no requirement for added nucleoside triphosphates, and the reaction was not inhibited by dideoxyadenosine triphosphate or aphidicolin. The majority of recombinant plasmid molecules result from a conservative process, indicating that nuclease-mediated strand-annealing is not responsible for the mitochondrial homologous recombination activity. Affinity-purified anti-recA antibodies inhibited the reaction, suggesting that activity is dependent on a mammalian mitochondrial homolog of the bacterial strand-transferase protein. The presence of homologous recombination activity within mammalian mitochondrial extracts suggests that this process is involved in mitochondrial DNA repair.

Animals↗

PubMed1996

Characterization of homologous DNA recombination activity in normal and immortal mammalian cells.

Homologous DNA recombination levels were measured in normal and spontaneously immortalized murine and human fibroblasts, and in a number of primate and murine established fibroblast cell lines. Immortal cell lines and tumor-derived clones homologously recombined extrachromosomal plasmid substrates at frequencies approximately 100-fold higher than did normal cells. To further explore the mechanism responsible for this phenotype, homologous recombination frequency was measured using nuclear extracts derived from normal and immortalized murine and human fibroblasts. Extracts prepared from immortal cells catalyzed high levels of homologous recombination, whereas very little recombination activity was detected in extracts prepared from normal fibroblasts. Similarly, only extracts derived from immortal cells contained strand-transferase activity as measured by the recently described pairing-on-membrane assay. Mixing experiments indicated that a recombination enhancing factor or factors present in immortal cells, rather than a recombination inhibitor in normal cells, was responsible for the enhanced homologous recombination activity observed using extracts derived from the former.

Animals↗

PubMed1996

Elevated levels of recombinational DNA repair in human somatic cells expressing the Saccharomyces cerevisiae RAD52 gene.

The Saccharomyces cerevisiae RAD52 gene was introduced into the human fibrosarcoma-derived cell line HT1080. Transfected cell lines that expressed the yeast transgene catalyzed inter-plasmid homologous DNA recombination at frequencies approx. 12-fold higher than did control cells. Additional experiments revealed that yeast RAD52 gene expression increased the level of resistance to the DNA damaging agents diepoxybutane, and methyl methanesulfonate, but did not alter sensitivity to ultraviolet radiation. These results indicate that the S. cerevisiae Rad52 protein can function in a human somatic cell background and provide support for the idea that a homologous recombination-based DNA repair process functions in mammalian somatic cells.

Anti-Bacterial Agents↗

PubMed1996

Elevated levels of homologous DNA recombination activity in the regenerating rat liver.

We have characterized homologous DNA recombination activity in nuclear protein extracts prepared from quiescent and regenerating rat livers. Activity measured in regenerating liver extracts was elevated approximately 35-fold above control, and its appearance closely mirrored the first wave of DNA synthesis, peaking 24 hours after a regenerative stimulus, and returning fairly rapidly to basal levels. We also identified a strand-transferase protein of approximately 100 kDa whose presence in these extracts correlates with homologous recombination activity. Recent evidence suggests that mammalian somatic cells possess a recombinational DNA repair mechanism analogous to that described in the yeast Saccharomyces cerevisiae. Our results indicate that this recombinational repair process may be regulated in vivo by, or play a role in, progression through the cell division cycle.

Animals↗

PubMed1995

The effect of target site transcription on gene targeting in human cells in vitro.

We evaluate the effect of target site transcription on gene targeting in cultured human fibrosarcoma cells. A number of cell lines that harbored a plasmid recombination substrate within their chromosomal DNA were created. Gene targeting frequency was then measured at these different loci in the presence and absence of an agent that stimulated target site transcription. We observed that gene targeting was significantly enhanced by RNA transcription. The magnitude of transcription-stimulated gene targeting varied from 3-fold to > 20-fold. No increase in gene targeting was observed, however, when transcription proceeded away from, rather than through, the recombination site. Transcription-stimulated gene targeting was also observed when single-stranded plasmid vectors complementary to either the coding or template strand were used as recombination substrates. Our results indicate that gene targeting, like other forms of DNA recombination, can be stimulated by target site transcription. The implications of our observations on current models of transcription-stimulated recombination are discussed.

Binding Sites↗