PubMed Health⌕ Search

Biomedical subjects

L Matsumoto

Publications and source records attributed to L Matsumoto.

13 recordsLinked to original sources
PubMed2001

MDR1 bicistronic vectors: analysis of selection stringency, amplified gene expression, and vector stability in cell lines.

The human multidrug resistance-1 gene (MDR1) is a dominant selectable and amplifiable marker in mammalian tissue culture cells. MDR1 is also being investigated as a gene therapy tool, both to protect normal cells against chemotherapy-related toxicity and to serve as an in vivo selectable marker for the overexpression of non-selectable therapeutic genes. The success of these strategies will depend on whether MDR1 expression can be sustained at levels high enough to confer a survival advantage on target cells. However, the MDR1 selection system is quite stringent, requiring high gene expression for transduced cells to survive in the presence of drug. The current report is a detailed molecular analysis of MDR1 selection stringency compared with the common neo selectable marker. A bicistronic vector encoding MDR1 and neo genes linked through an internal ribosome entry site was transferred into NIH 3T3 mouse fibroblasts and K562 human leukemia cells; cells were then exposed to colchicine (to select for MDR1 expression) or to G418 (to select for neo expression). Surviving populations and individual clones of cells were analyzed for expression levels of MDR1 and neo gene products; resistance to colchicine, paclitaxel, and G418; level and integrity of bicistronic mRNA; and structural integrity, integration number, and copy number of vector DNA. These studies provide direct evidence that colchicine selection is more stringent than G418 selection; that increased selection pressure with colchicine leads to increased gene expression; that increased gene expression can be accommodated primarily by gene amplification, even within an individual transduced clone and starting from a single-copy proviral integration event; and that the clonal diversity of a transduced population of cells is influenced significantly by the stringency of selection. Taken together, these results have important implications for the potential utility of MDR1 as a selectable marker and as a gene therapy tool in hematopoietic cells.

3T3 Cells↗
PubMed1999

Biological effects of a bifunctional DNA cross-linker. II. Generation of micronuclei and attached micronuclear-like structures.

Madin-Darby bovine kidney (MDBK) cells were treated with the bifunctional DNA cross-linker, L-7, to examine the generation of micronuclei and other nuclear abnormalities. The preceding paper demonstrates that L-7 treatment induces the formation of triradial and quadriradial chromosomes in MDBK cells. These chromosomes are believed to result from interduplex DNA cross-links formed between G-C rich centromeric satellite DNA regions on non-sister chromatids. Treatment produces a majority of centromere-positive micronuclei. In addition, many daughter cells remain attached by chromatin bridges which are sometimes beaded with micronuclei. Up to 15% of cell nuclei become lobular and fused with numerous micronuclear-like structures attached to their membranes. These attached structures are classified as attached micronuclear-like structures (AMNLS). Fluorescence in situ hybridization (FISH) using a centromeric satellite sequence was performed on treated cells. Hybridization reveals that intercellular bridges are composed of centromeric sequences and initiate at centromeric foci in daughter cells. Furthermore, the majority of junctions between AMNLS and nuclei contain an enhancement of centromeric signal. The frequency of AMNLS appears dependent on the concentration of L-7 and the duration of treatment. Similar results were found for the generation of cross-linked chromosome products in the previous paper. We suggest that AMNLS result from the abnormal mitotic segregation of cross-linked chromosome products.

Animals↗
PubMed1999

Biological effects of a bifunctional DNA crosslinker. I. Generation of triradial and quadriradial chromosomes.

Interduplex crosslinks by a bifunctional anthramycin DNA crosslinker produced triradial and quadriradial chromosomes. The crosslinker alkylates guanine at N-2. Bovine chromosomes contain GC-rich density satellite DNAs at the centromeric heterochromatin and is the basis for the formation of triradial and quadriradial chromosomes at the centromeres. The in situ crosslinking of interphase chromosomes indicates that the distance between centromeres is 17.5 A. We conclude that the nuclear matrix associated DNA in the centromeric heterochromatin of interphase chromosomes are positioned close enough for crosslinking to occur. We propose a model for the generation of triradial and quadriradial chromosomes based upon the number of interduplex crosslinks between two chromosomes.

Animals↗
PubMed1996

Bicistronic and two-gene retroviral vectors for using MDR1 as a selectable marker and a therapeutic gene.

We describe a series of two-gene and bicistronic retroviral vectors that use the human MDR1 gene as a selectable marker for the overexpression of a second heterologous gene in transduced cells. The vectors use Harvey murine sarcoma virus sequences for viral expression and packaging functions and include sites for cloning foreign genes of interest under the control of either an internal promoter (two-gene vectors) or an internal ribosome entry site (bicistronic vectors). To characterize these vectors, we used neo as a reporter gene for foreign gene expression and as an independently selectable marker for comparison with MDR1. Each of the vector constructions supported high-titer retrovirus production and transduction of mouse and human cell lines. Using MDR1-neo virus supernatants in parallel titering assays, we found that titers based on colchicine resistance were 10- to 20-fold lower than titers based on G418 resistance, suggesting that MDR1 is a more stringent selectable marker than neo in NIH 3T3 and KB-3-1 cell lines. Whereas neo gene expression with the two-gene vectors was subject to host-specific limitations on internal promoter activity, the bicistronic vectors were highly active in three cell lines tested. In K562 cells, using the bicistronic vector, selection with colchicine led to at least 20-fold higher expression of the MDR1 gene product than did selection with G418, suggesting that the stringent MDR1 selection system is very efficient for obtaining overexpression of foreign genes. Retroviral vectors carrying MDR1 as a selectable marker plus a second, heterologous gene of interest could have widespread utility for in vitro and in vivo applications of gene transfer technology, including gene therapy.

3T3 Cells↗
PubMed1995

Transduction of NIH 3T3 cells with a retrovirus carrying both human MDR1 and glutathione S-transferase pi produces broad-range multidrug resistance.

In the experiments, we examined the ability of a retroviral vector, pHaMASV, to encode two potential chemoprotective genes on separate transcription units. We previously described the pHaMSV vector, which includes the human MDR1 gene as a selectable marker and chemoprotective gene, plus an internal SV40 promoter for expressing a second heterologous gene along with MDR1 [M. E. Metz, D. M. Best, and S. E. Kane. Virology, 208: 634-643, 1995]. To test the ability of this vector to deliver two therapeutic genes simultaneously, the cDNA for human glutathione S-transferase pi (GST pi, the most abundant member of the glutathione S-transferase family in human tumor cells) was inserted into pHaMASV, and this plasmid was transfected into ecotropic packaging cells. The resulting pHaMASV.GST pi ecotropic retrovirus, which was produced at a titer of 2 x 10(6) colony-forming units/ml, was used to transduce NIH 3T3 cells. After initial selection in 60 ng/ml colchicine, a population of transduced cells was exposed to stepwise increasing colchicine concentrations to select for amplified expression of MDR1. As MDR1 expression increased, the expression of GST pi increased in concert, as demonstrated by Northern analysis, Western analysis, and measurement of glutathione S-transferase activity. Transduced cells growing in 1280 ng/ml colchicine had about 3-fold higher total glutathione S-transferase activity than nontransduced cells and 2.5-fold higher activity than transduced cells growing in 60 ng/ml colchicine. Northern hybridizations demonstrated a 3-5-fold increase in both the full-length retroviral message encoding MDR1 and the subgenomic mRNA encoding GST pi after amplification of resistance from 60 to 1280 ng/ml colchicine. The cytotoxic effects of several xenobiotics were evaluated in NIH 3T3 cells transfected with MDR1 (3T3.MDR) or transduced with the MDR1-GST pi retrovirus (3T3.GST640 or 3T3.GST1280) to evaluate the ability of our vector to produce a spectrum of drug resistances specific for the genes expressed. 3T3.MDR and 3T3.GST1280 cells expressing equivalent levels of MDR1 had identical levels of resistance to doxorubicin or colchicine. These results suggest that GST pi expression did not contribute to doxorubicin resistance in this model system. However, 3T3.GST640 cells were about 4-fold resistant to ethacrynic acid and 1-chloro-2,4-dinitrobenzene compared to cells expressing MDR1 alone, consistent with the ability of GST pi to conjugate both of these cytotoxins. Increases in drug resistance paralleled increases in gene-specific mRNA and recombinant protein levels in all cases.4+ chemotherapy.

3T3 Cells↗
PubMed1995

Nonrandom arrangement of bovine satellite I DNA within the interphase nucleus of Madin-Darby bovine kidney cells.

The distribution of bovine satellite DNA was examined in Madin-Darby bovine kidney cells for each hour of interphase. Cells were grown on coverslips and probed using biotinylated cloned fragments of bovine satellite I DNA. Hybridization was detected using a streptavidin-alkaline phosphatase conjugate. Cells were projected onto a grid of fixed dimensions and the distribution of hybridization signal was recorded. A chi 2 analysis of fit compared this distribution of signals to a random distribution generated from the poisson distribution. Nuclear localization of bovine satellite I DNA was found to be nonrandom throughout interphase except at the G1/S border and S = 6 h. Moreover, distinct patterns of hybridization were also observed at specific times during interphase.

Animals↗
PubMed1989

Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in lambda excisive recombination.

The early events in site-specific excisive recombination were studied with phage lambda half-att sites that have no DNA to one side of the strand exchange region; they carry a single core-type integrase binding site and either P or P' arm flanking DNA. These half-attR and half-attL sites exhibit normal properties for the initial (covalent) top-strand transfer and form stable intermediates independent of later steps in the reaction. With these novel substrates we show that Xis specifically promotes the first strand exchange and that attL enhances Int cleavage at the top-strand site of attR. It is also shown that synapsis and initial strand transfers do not require DNA-DNA pairing but are mediated by protein-protein and protein-DNA interactions. These involve the two top-strand Int binding sites (required for the first strand exchange) and, in addition, one of the two bottom-strand sites (C') responsible for the second strand exchange.

Attachment Sites, Microbiological↗
PubMed1987

Site-specific recombination intermediates trapped with suicide substrates.

A family of novel substrates was designed to enable the efficient accumulation of intermediates in site-specific recombination. Strategically placed nicks allow these "suicide substrates" to initiate the reaction but prevent its completion or reversal. Consequently, it has been possible to determine that lambda site-specific recombination proceeds by a pair of sequential single-strand exchanges. These results rule out that class of models invoking a concerted cutting of all four DNA strands. The sequential strand exchanges are executed in a strictly prescribed order that is the same in both integrative and excisive recombination. This specified order appears to be governed by the arrangement of bound proteins distal to the sites of strand exchange. Furthermore, when provided with an appropriate 5' OH acceptor, the Integrase protein has the capacity to execute a single DNA strand transfer in a nonreciprocal reaction.

Bacteriophage lambda↗
PubMed1981

Two distinct intervening sequences in different ribosomal DNA repeat units of Sciara coprophila.

We have prepared a partial gene library of sheared DNA from the fungus fly, Sciara coprophila, by dA-T tailing and insertion into pBR322. Two ribosomal DNA clones which differ from the usual ribosomal DNA organization in this organism were studied in detail. Clone pBc 1L-1 has an intervening sequence of 1.4 kb, and clone pBc 6D-6 has an intervening sequence of 0.9 kb. These intervening sequences occur in about the same position in 28S rDNA, but do not appear to share sequence homology with one another. Previously we found that 90% of Sciara ribosomal DNA is homogenous and lacks an intervening sequence, and our present data explains the size heterogeneity found in most of the remaining 10%. We have found no evidence of size heterogeneity in the nontranscribed spacer.

Animals↗
PubMed1977

Complex forms and replicative intermediates of mitochondrial DNA in tissues from adult and senescent mice.

The occurrence and types of complex forms and replicative intermediates of mitochondrial DNA (mtDNA) were investigated in tissues from C57BL/6J mice aged 10-11 months or 29-30 months. Total mtDNA from brain, heart, kidney and liver was isolated in ethidium bromide-CsCl gradients and examined by electron microscopy after aqueous or formamide spreading. Contour length measurements indicated no difference in the monomer size of mtDNA according to either tissue or donor age. The frequencies of catenated mtDNA, ranging from 4 to 8%, varied significantly according to tissue but changed relatively little as a result of donor age. The main age-related effect observed in this study was a significant increase in the frequency of circular dimers, from about 0.05% in adult tissues to 0.3% in kidney, 0.5% in liver, 0.6% in heart and 1.9% in brain of senescent mice. The frequency of D-loop DNA varied from 30 to 60% and that of larger replicative intermediates from 1 to 10%, suggesting differences in the rate of mtDNA replication according to tissue. The frequencies and types of the various replicative intermediates were unaffected by donor age.

Adult↗
PubMed1976

Complex mitochondrial DNA in animal thyroids. A comparative study.

1. The frequency of circular dimers and catenanes was determined in thyroid mitochondrial DNA (mtDNA) from rabbits, mice, pigs, sheep and cattle. 2. The mtDNA from freshly removed thyroids was isolated by buoyant density centrifugation in ethidium bromide/CsCl gradients after DNAase treatment of the mitochondrial pellet. Typically, more than 90% of the recovered mtDNA was found in the lower band, indicating a low rate of nicking during isolation. A sample of the total mtDNA (upper and lower bands) was examined by electron microscopy after preparation by the aqueous protein film technique. 3. The frequency of circular dimers generally ranged from 0.1 to 0.3%. However, in an mtDNA sample from cow thyroid, the frequency of circular dimers was 0.6% (0.9% if circular dimers occuring in catenanes are included(, differing significantly from the frequency of these forms in bull thyroid, 0.1%. A small but significant variability also occurred in the frequency of catenanes ranging from 2 to 8% in the different groups; this variation is within the limits usually observed in normal tissues. 4. These observations indicate that thyroids, like other normal tissues examined so far, have a low content of circular dimers. A high frequency of these forms seems to be the trademark of some genetically and physiologically abnormal cells such as certain established cell lines, virus-transformed cells and malignant or otherwise pathological tissues.

Animals↗
PubMed1974

Mitochondrial DNA replication in sea urchin oocytes.

Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [(3)H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.

Animals↗