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D M Gilbert

Publications and source records attributed to D M Gilbert.

At least 37 records · Page 2Linked to original sources
PubMed1997

Origin-specific initiation of mammalian nuclear DNA replication in a Xenopus cell-free system.

The introduction of Chinese hamster ovary (CHO) cell nuclei into Xenopus egg extracts provides the only cell-free system that can efficiently initiate replication at a specific metazoan replication origin. With intact late-G1-phase nuclei as a substrate, the pattern of initiation sites for replication at the CHO dihydrofolate reductase (DHFR) locus is indistinguishable from that observed in cultured cells. By contrast, with early-G1-phase nuclei or with late-G1-phase nuclei that have damaged nuclear envelopes, these same extracts efficiently initiate replication at apparently random sites. Thus, at a distinct point during G1 phase [origin decision point (ODP)], nuclei experience a transition that is required for specific recognition of the DHFR origin by Xenopus egg cytosol. Described here are the basic requirements to achieve origin-specific initiation, which include: 1) a cell line that can be synchronized in G1 phase, 2) a method to prepare intact nuclei, 3) a technique to map origins with a few million cells, and 4) a small colony of Xenopus laevis. Immunodepletion of specific gene products allows one to test hypotheses about the requirements for origin recognition. Here we show that depletion of the Xenopus origin recognition complex subunit XORC2 from Xenopus egg extracts has no influence on the efficiency of replication or the pattern of initiation sites with either pre-ODP or post-ODP nuclei.

Animals↗
PubMed1997

The replication origin decision point is a mitogen-independent, 2-aminopurine-sensitive, G1-phase event that precedes restriction point control.

At a distinct point during G1 phase (the origin decision point [ODP]), Chinese hamster ovary (CHO) cell nuclei experience a transition (origin choice) that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. We have investigated the relationship between the ODP and progression of CHO cells through G1 phase. Selection of the DHFR origin at the ODP was rapidly inhibited by treatment of early G1-phase cells with the protein kinase inhibitor 2-aminopurine (2-AP). Inhibition of the ODP required administration of 2-AP at least 3 h prior to phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and the restriction point (R point). Cells deprived of either serum or isoleucine from metaphase throughout early G1 phase acquired the capacity to replicate in Xenopus egg extract (replication licensing) and subsequently passed through the ODP on the same schedule as cells cultured in complete growth medium. After growth arrest at the R point with hypophosphorylated Rb protein, serum- or isoleucine-deprived cells experienced a gradual loss of replication licensing. However, recognition of the DHFR origin by Xenopus egg cytosol remained stable in growth-arrested cells until the point at which all nuclei had lost the capacity to initiate replication. These results provide evidence that the ODP requires a mitogen-independent protein kinase that is activated after replication licensing and prior to R-point control.

2-Aminopurine↗
PubMed1996

A distinct G1 step required to specify the Chinese hamster DHFR replication origin.

Nuclei isolated from Chinese hamster ovary (CHO) cells at various times during the G1 phase of the cell cycle were stimulated to enter S phase by incubation in Xenopus egg cytosol. Replication of DNA initiated within the dihydrofolate reductase (DHFR) origin locus in nuclei isolated late in G1, but at random sites in nuclei isolated early in G1. A discrete transition point occurred 3 to 4 hours after metaphase. Neither replication licensing nor nuclear assembly was sufficient for origin recognition. Thus, a distinct cell cycle-regulated event in the nucleus restricts the initiation of replication to specific sites downstream of the DHFR gene.

Animals↗
PubMed1996

The role of colpocleisis with urethral lengthening in transsexual phalloplasty.

OBJECTIVE: Transsexual surgery is an unique area of rarely performed surgery. This study examines factors that have significance in the prevention of major morbidity in this unusual surgery. The role of the gynecologist in the psychologic, endocrine, and operative management is presented. STUDY DESIGN: Initial operations were complicated by fistulas at the urethra-to-phallus anastomosis site. After reviewing these complications, we modified our approach to include a two-stage procedure allowing for healing before microsurgery and sparing of the anterior vaginal wall during vaginal hysterectomy and colpocleisis. By sparing the anterior vaginal wall, we were able to better extend the urethra for later phallus attachment. RESULTS: Using the two-stage procedure at colpocleisis allowed a significant reduction in the fistula rate. (p = 0.0087) with the effective elimination-fistulas, the use of stiffeners during phalloplasty for better functional results is possible. CONCLUSION: Extending the urethra during colpocleisis allows for better healing and significantly decreased fistula formation. Proper blood supply for microvascular surgery and adequate tissue for the anastomosis site contribute to better results.

Female↗
PubMed1996

Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts.

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase-arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.

Animals↗
PubMed1995

Mimosine arrests DNA synthesis at replication forks by inhibiting deoxyribonucleotide metabolism.

Mimosine has been reported to specifically prevent initiation of DNA replication in the chromosomes of mammalian nuclei. To test this hypothesis, the effects of mimosine were examined in several DNA replication systems and compared with the effects of aphidicolin, a specific inhibitor of replicative DNA polymerases. Our results demonstrated that mimosine inhibits DNA synthesis in mitochondrial, nuclear, and simian virus 40 (SV40) genomes to a similar extent. Furthermore, mimosine and aphidicolin were indistinguishable in their ability to arrest SV40 replication forks and mammalian nuclear chromosomal replication forks. In contrast to aphidicolin, mimosine did not inhibit DNA replication in lysates of mammalian cells supplied with exogenous deoxyribonucleotide triphosphate precursors for DNA synthesis. Mimosine also had no effect on initiation or elongation of DNA replication in Xenopus eggs or egg extracts containing high levels of deoxyribonucleotide triphosphates. In parallel with its inhibitory effect on DNA synthesis in mammalian cells, mimosine altered deoxyribonucleotide triphosphate pools in a manner similar to that reported for another DNA replication inhibitor that affects deoxyribonucleotide metabolism, hydroxyurea. Taken together, these results show that mimosine inhibits DNA synthesis at the level of elongation of nascent chains by altering deoxyribonucleotide metabolism.

Animals↗
PubMed1995

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.

Animals↗
PubMed1993

Site-specific initiation of DNA replication in metazoan chromosomes and the role of nuclear organization.

We have asked whether or not Xenopus eggs or egg extracts, which have previously been shown to replicate essentially any DNA molecule, will preferentially utilize a known mammalian OBR. Our results reveal that Xenopus egg extracts can preferentially initiate DNA replication at sites chosen in vivo by the hamster cell, provided that the DNA substrate is presented to the extract in the form of a nucleus rather than bare DNA. Thus, site-specific initiation of DNA replication in metazoan cell chromosomes appears to be determined by nuclear organization as well as DNA sequence. We have also considered whether or not BPV, which was previously reported to regulate its copy number through negative as well as positive cis-acting sequences, provides a suitable paradigm for cellular origins. The BPV genome was found to contain cis-acting sequences that can suppress DNA replication driven by a lytic virus such as PyV. However, this suppression did not require any BPV protein, did not limit PyV origin activity to one initiation event per S phase, and did not affect BPV origin activity. These results, together with data from other laboratories, strongly suggest that BPV is simply a slow-replicating version of SV40 and PyV and therefore is not an appropriate model to explain how initiation of cellular DNA replication is limited to once per cell cycle.

Animals↗
PubMed1993

Estradiol-inducible squelching and cell growth arrest by a chimeric VP16-estrogen receptor expressed in Saccharomyces cerevisiae: suppression by an allele of PDR1.

We have constructed and characterized a flexible system for analyzing the phenomenon of squelching and estrogen receptor function in the yeast Saccharomyces cerevisiae. The A/B region of the human estrogen receptor was replaced with the transcriptional activating domain of VP16 and expressed in yeast cells from high-copy-number plasmids. Addition of hormone resulted in an immediate inhibition of expression (squelching) of a chromosomally integrated GAL1:lacZ reporter gene and the eventual arrest of cell growth (toxicity). In order to determine whether a relationship exists between toxicity and squelching, mutations were made in this chimeric receptor (VEO) and their effects on transcriptional activation, squelching, and toxicity were compared. A direct correlation was found between mutations in VEO that reduced VP16 transactivation ability in yeast cells and those that reduced both squelching and toxicity. Surprisingly, mutations in the DNA binding domain (DBD) of VEO dramatically reduced squelching and completely relieved toxicity, suggesting a role for the DBD in squelching and strengthening the correlation between squelching and toxicity. To demonstrate the utility of this system for carrying out genetic selection, a plasmid-based yeast genomic bank was screened for genes that can relieve the toxicity of VEO by means of an elevated copy number, resulting in the repeated cloning of an allele of the PDR1 (pleiotropic drug resistance) gene. We present evidence that mutations in PDR1 can modulate the intracellular availability of estradiol by the same mechanism that leads to multiple drug resistance in yeast cells. Taken together, our results provide evidence that cell growth arrest occurs when squelching exceeds a certain threshold and that strong squelching requires both a DBD and a transcriptional activating domain. Furthermore, we show that growth arrest can provide a useful phenotype for carrying out the genetic analysis of both squelching and estrogen receptor function in yeast cells.

Cell Division↗
PubMed1992

Ligand dependence of estrogen receptor induced changes in chromatin structure.

To determine whether the human estrogen receptor requires ligand to bind to its cognate estrogen receptor element (ERE) in vivo, we have examined the structure of chromatin at a chromosomally integrated ERE-URA3 reporter gene in yeast, and the influence of ligand bound and ligand free estrogen receptors on that structure. Using indirect end-labelling to map DNaseI and micrococcal nuclease sensitive sites, we found that receptor induced alterations in chromatin structure were completely dependent upon the presence of estradiol. These same alterations in chromatin structure were induced by a truncated estrogen receptor with both TAF-1 and TAF-2 transactivation functions deleted, suggesting that DNA binding per se disrupts chromatin structure. These results support models in which the estrogen receptor requires ligand to bind to the ERE in vivo.

Chromatin↗
PubMed1992

Mouse genomic DNA sequences homologous to sea urchin TU elements are genetically stable polydispersed repeats useful for analysis of multiple RFLPs.

Segments of the murine genome that hybridize to the inverted repeat regions of the transposable TU elements of sea urchins include tandem repeats of a sequence (CTCC) that encodes the recognition site for the restriction enzyme Mnl1, as do the analogous polypurine/polypyrimidine (pPu/pPy) stretches in humans. The Mnl1-sensitive repeats, which exist as a microsatellite sequence 200-300 bp in length, lack the terminal dyad symmetry characteristic of the TU elements and are structurally and functionally distinct from these elements. DNA fragments containing these repeat units that are isolated from different generations of isogenic (or congenic) mice or from different tissues of genetically identical individuals are indistinguishable by RFLP analysis; however, they show restriction fragment length polymorphism in different strains. This polymorphism appears to reflect DNA sequence changes occurring at sites flanking the repeats rather than variability in the number of repeats. Their genetic stability and occurrence in a wide variety of animal species make the Mnl1 repeats useful in studying genetic variation that has occurred over an evolutionary time scale of greater duration than can be examined conveniently by VNTR analysis.

Animals↗
PubMed1992

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.

Animals↗
PubMed1990

Replication timing of DNA sequences associated with human centromeres and telomeres.

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.

Animals↗
PubMed1990

Position effects on the timing of replication of chromosomally integrated simian virus 40 molecules in Chinese hamster cells.

Simian virus 40 (SV40) DNA molecules chromosomally integrated at different sites in three Chinese hamster lung fibroblast lines replicated during the middle portion of S phase but not precisely at the same time in all three cell lines. The time of replication was unrelated to the presence of T antigen or to its relative activity in promoting SV40 replication. SV40 sequences and chromosomal DNA sequences adjacent to the SV40 insert in one cell line expressing a temperature-sensitive T antigen showed a T-antigen-independent difference in replication timing from the homologous, allelic locus not linked to SV40. Our results indicate that the timing of replication of these integrated SV40 molecules is dependent upon the site of integration and is not determined by the level of T antigen replication-promoting activity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Case report: ABO discrepancy due to vancomycin complicating a transfusion reaction investigation.

A 3-year-old patient with acute myelogenous leukemia developed fever and chills during transfusion of packed red cells. A preliminary workup suggested that a group AB donor unit had been issued to a Group A patient. However, a discrepancy between the ABO group of the original donor unit segment (A) and blood taken from the IV tubing (AB) and the patient's pre- and post-transfusion samples (A and AB, respectively) suggested mother reason for the weak reactivity of some samples with anti-8. The patient's chart revealed that vancomycin, reported to be a cause of non-immune agglutination of red cells, had been injected into the IV tubing one hour prior to transfusion. Further testing confirmed that the patient's febrile response to transfusion was consistent with a nonhemolytic transfusion reaction and was unrelated to the drug-induced, pseudo ABO problem.

Journal Article↗
PubMed1988

Phallic reinnervation via the pudendal nerve.

Total phallic reconstruction presents the genitourinary reconstructive surgeon with one of the most difficult surgical challenges. The development of microsurgical techniques and free tissue transfers have advanced phallic reconstruction by reducing the number of surgical procedures and by allowing more selectivity in choosing the best innervated donor tissue. During the last 5 years 16 patients underwent total phallic reconstruction using free tissue transfers from distant donor sites. The pudendal nerve was coapted routinely to the major sensory nerves of the donor free flap. The most accurate objective baseline parameters of penile sensibility are pressure and vibratory thresholds, and electrically evoked potentials. We examined 30 normal subjects and 7 patients at least 1 year postoperatively for penile (phallic) sensibility. A pressure aesthesiometer, a biothesiometer and electrodiagnostic studies were used for testing. The 7 postoperative patients (in all of whom the pudendal nerve was incorporated into the reconstruction) had an encouraging return of tactile and erogenous sensibility compared to normal subjects. This is a promising advance in phallic reconstruction.

Adolescent↗
PubMed1988

Transsexual surgery in the genetic female.

The increasing acceptance of the transsexual patient by society as well as a willingness of insurance companies to compensate for associated surgical procedures will result in an increase in patients presenting for sex reassignment. Data from gender identity centers suggest that the multidisciplinary approach leads to the best therapeutic results. Significant advances in phalloplasty techniques are yielding genitalia with more normal function and appearance.

Breast↗