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M Gottesman

Publications and source records attributed to M Gottesman.

At least 19 recordsLinked to original sources
PubMed1992

Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells.

Gene therapy in humans requires the transplantation of genetically modified cells, and it is important to select only those cells capable of expressing high levels of protein from the transferred gene. Expression of the human multiple drug resistance (MDR) gene confers resistance to a variety of compounds in vitro and in vivo. To determine the feasibility of conferring recipient erythroid cells with the MDR phenotype, we have transduced mouse erythroleukemia cells (MELC) with the MDR gene in a retroviral vector. We show here that MELC clones resistant to exposure to colchicine (an MDR-responsive agent) can be isolated, and demonstrate high levels of MDR RNA and protein expression. Increasing doses of colchicine increase the level of MDR RNA and protein expression significantly. These results indicate that it is possible to transfer and express the human MDR phenotype in mouse erythroid cells by retrovirally mediated gene transfer, and that drug selection can be used to enrich or purify populations of cells containing and expressing this gene.

3T3 Cells↗
PubMed1992

Transfer and expression of the human multiple drug resistance gene into live mice.

The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1.

Experiments were performed to determine if retroviral-mediated transfer of the human multidrug resistance 1 gene (MDR1) into murine bone marrow cells would confer drug resistance to the cells and whether the MDR1 gene could be used as a dominant selectable marker in vivo. When mice transplanted with bone marrow cells containing a transferred MDR1 gene were treated with the cytotoxic drug taxol, a substantial enrichment for transduced bone marrow cells was observed. This demonstration of positive selection establishes the ability to amplify clones of transduced hematopoietic cells in vivo and suggests possible applications in human therapy.

Alkaloids↗
PubMed1991

Molecular manipulations of the multidrug transporter: a new role for transgenic mice.

Multidrug resistance in human cancer is associated with overexpression of the MDR1 gene which encodes a 170,000 molecular weight membrane glycoprotein that transports cytotoxic drugs out of cancer cells. The MDR1 gene is normally expressed in intestine, kidney, liver, and adrenal glands, and in tumors derived from these tissues, but it is not expressed in normal bone marrow. Transgenic mice that express the MDR1 gene in their bone marrow have been developed, and because of this expression these mice are resistant to the bone marrow-suppressive effects of daunomycin, doxorubicin, taxol, and several other anticancer drugs. These mice can be used in several different ways to develop new types of drugs to treat human cancer.--Pastan, I.; Willingham, M. C.; Gottesman, M. Molecular manipulations of the multidrug transporter: a new role for transgenic mice.

Animals↗
PubMed1991

HU and integration host factor function as auxiliary proteins in cleavage of phage lambda cohesive ends by terminase.

HU and integration host factor (IHF) are small, basic heterodimeric DNA-binding proteins which participate in transcription initiation, DNA replication, and recombination. We constructed isogenic Escherichia coli strains in which HU, IHF, or both proteins were absent. Bacteriophage lambda did not grow in hosts lacking both HU and IHF. Phage DNA replication and late gene transcription were normal in the double mutants, but packaging of lambda DNA was defective. Mature phage DNA molecules were absent, indicating that terminase was unable to linearize lambda DNA. Phage variants carrying a small substitution near cos or the ohm1 mutation in the terminase gene, Nul, formed plaques on HU- IHF- strains. We propose that HU or IHF is required to establish the higher-order DNA-protein structure at cos that is the substrate for lambda terminase.

Bacterial Proteins↗
PubMed1990

Integration host factor stimulates the phage lambda pL promoter.

Escherichia coli integration host factor (IHF) is a small dimeric protein that binds to a specific DNA consensus sequence and produces DNA bending. Transcription from the bacteriophage lambda pL promoter is stimulated three- to fourfold by IHF both in vivo and in vitro. IHF binds with high-affinity to two tandem sites located just upstream from the pL promoter and enhances the formation of RNA polymerase-promoter closed complexes. The rate of isomerization to open complex is not influenced by IHF. IHF may stimulate recognition of pL by one or more of several mechanisms: (1) by bending DNA; (2) by making protein-protein contacts with RNA polymerase; or (3) by occluding a competing promoter upstream from pL.

Bacterial Proteins↗
PubMed1990

Symptomatic scapholunatotriquetral carpal coalition with fusion of the capitatometacarpal joint. Report of a case.

Carpal coalition is not an uncommon entity. It is most often asymptomatic and coincidentally noted on roentgenograms. The most common location is between the lunate and the triquetrum, although nearly every combination has been reported. Reported is a 33-year-old man with a symptomatic scapholunatotriquetral carpal coalition associated with capitatometacarpal coalition. This condition seems not to have been previously reported in the literature.

Adult↗
PubMed1989

Stimulation of IS1 excision by bacteriophage P1 ref function.

Lysogenization by a c1ts variant of coliphage P1, P1c1.100, markedly increased the frequency of reversion of a galT::IS1 mutation. The formation of Gal+ colonies presumably occurs by microhomologous recombination between the 9-base-pair repeats in galT (CGCCGCTAC) generated by the transposition of IS1. The responsible P1 gene, ref, has been cloned and sequenced. ref encodes a 22.8-kilodalton protein and is located near the P1 site-specific recombination function, cre. Expression of ref was repressed by P1 c+. The absence of a distinctive ribosome-binding site is consistent with a poor translation of ref from an expression vector in vivo. Placement of a ribosome-binding site before ref resulted in the extensive synthesis of the Ref protein. Ref stimulated precise excision in recB or himA cells, but not in recA mutants. Ref was active in lexA3 mutants, suggesting that the recombination activity of RecA was directly involved in the reaction. We have constructed a P1c1.100 ref::Tn10 mutant. The absence of Ref did not appear to restrict dramatically the ability of P1 to grow lytically or to form lysogens. Thus, the role of ref in the physiology of P1 remains to be determined.

Amino Acid Sequence↗
PubMed1988

Neurology, age, hospital costs, and DRGs.

The DRG payment scheme is causing hospitals to examine the financial consequences of treating various patient populations. The purpose of this study was to examine resource utilization for hospitalized neurology patients treated during a 2-year period at an academic medical center. All patients (N = 1,993) were stratified by payor (Medicare, Medicaid, Blue Cross, and other) and age (0-35 years, 35-65 years, and 65 years and above). Mean hospital cost per patient (exclusive of MD fees) for each payor generally rose with age. Patients 35 years of age and over consumed a disproportionately larger share of resources than younger patients. DRG payment under an all payor system would have produced a substantial deficit for this group of patients. The mean hospital length of stay, number of diagnoses per patient, and mortality, as well as percent of admission through the emergency department generally rose with age. This study demonstrated that neurology patients 35 years of age and older generated higher resource utilization than their younger counterparts and were underpaid by the current DRG reimbursement. In this study, DRG payment appeared to provide significant financial disincentives to treat older neurology patients. If our findings are widespread, the quality of neurologic care and the elderly's access to it could become limited in the future.

Aged↗
PubMed1987

Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation.

Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage lambda cII protein were isolated. The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of cII or the activation by cII of the lambda promoters pE and pI. The sck-1, sck-2, and sck-3 mutations modify transcription termination. The growth of lambda, but not of the N-independent lambda variant, lambda nin-5, is hindered by these mutations, which act either alone or in concert with the bacterial nusA1 mutation. In contrast to their effect on lambda growth, the three mutations reduce transcription termination in bacterial operons. The E. coli pyrE gene, which is normally regulated by attenuation, is expressed constitutively in the mutant strains. The sck mutations appear to prevent pyrE attenuation by slowing the rate of transcriptional elongation of the pyrE leader sequence. The sck-6 mutation, unlike the other sck mutations, neither increases pyrE expression nor inhibits the ability of lambda to suppress transcription termination. Instead, the sck-6 mutation blocks the growth of the lambda variants lambda nin-5 and lambda red-3.

Bacteriophage lambda↗
PubMed1986

DNA-mediated transfer of cAMP resistance in CHO cells.

Chinese hamster ovary (CHO) strain 10215 carries a dominant mutation which confers resistant to cAMP by virtue of an altered catalytic subunit of the cAMP-dependent protein kinase (Evain et al., 1979). This mutation was transferred to wild-type CHO cells by DNA-mediated gene transfer. Based on the absence of cAMP growth inhibition, seven transformant colonies were isolated. One of these, 11586, was studied in detail. This transformant showed the same phenotype as the mutant, including resistance to the morphological changes and growth inhibitory effects of 1 mM 8-Br-cAMP, reduced total cAMP dependent protein kinase activity and lowered sensitivity of the kinase to cAMP activation. When the cAMP-dependent protein kinase was fractionated on a DEAE-cellulose column, the transformant was lacking in type II cAMP dependent protein activity, to the same degree as the mutant. The transformant and mutant, but not wild-type cells, also failed to phosphorylate a 52,000-dalton protein in a cAMP-dependent manner. These characteristics support the conclusion that the gene for the mutant cAMP-dependent protein kinase has been transferred. The ability to transfer this gene by DNA-mediated transfer suggests that this methodology may be useful for the molecular isolation of the gene encoding the catalytic subunit of cAMP-dependent protein kinase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Translation initiation of bacteriophage lambda gene cII requires integration host factor.

Escherichia coli integration host factor (IHF), a DNA-binding protein, positively regulates expression of the lambda cII gene. Purified IHF stimulates cII protein synthesis in vitro, suggesting a direct role for host factor in cII expression. Further evidence for a direct role for IHF was obtained with operon and gene fusions between cII and lacZ or cII and galE. Analysis of these fusions in vivo demonstrated that IHF is essential for the initiation of cII translation. Replacement of the entire cII coding sequence with lacZ yielded a gene fusion which was still IHF dependent. However, a cII-galE fusion carrying a hybrid ribosome binding region expressed galE in IHF mutants. These results indicate that sequences which make cII translation IHF dependent lie between the ribosome binding region and the initiating codon of cII. Failure to translate cII activates a transcription terminator located within cII and results in polar effects on downstream transcription. This polarity is suppressed by the lambda N antitermination function. When cloned into another context, the terminator is active in both wild-type and IHF mutant strains. The amino terminus of cII is located near an IHF binding site in a region with considerable dyad symmetry. The role of IHF in cII translation may be to prevent formation of an RNA-RNA duplex that sequesters the ribosome binding site of cII. The binding of IHF might influence RNA structure by altering the rate of the dissociation of RNA from the DNA template.

Bacterial Proteins↗
PubMed1985

Regulation of single and multicopy his operons in Escherichia coli.

We fused segments of the Escherichia coli his regulatory region to galK in single-copy and multicopy vectors. These fusions demonstrated that (i) derepression of his by histidine starvation is due exclusively to attenuation; (ii) the his promoter is metabolically regulated; and (iii) both regulatory systems operate when the his regulatory region is present on a multicopy plasmid. Thus, there is no evidence for titration of his regulatory elements. Deletions of the his anti-attenuator region, carried on multicopy plasmids, cause low-level galK expression. This expression is not stimulated by histidine starvation, but is growth rate dependent. We replaced the his attenuator with the efficient lambda terminator, to. In the context of the his regulatory region, however, lambda to only partially terminates transcription.

Bacteriophage lambda↗
PubMed1984

Control of bacteriophage lambda CII activity by bacteriophage and host functions.

We have studied the regulation of the lambda cII gene in vivo using cloned lambda fragments. Lambda N protein stimulated cII expression. Surprisingly, although very high cII protein levels were detected by gel electrophoresis, little cII protein activity, measured as stimulation of the lambda pI and pE promoters, was observed. The half-life of cII protein depended critically on its initial level. At low concentrations its half-life was as short as 1.5 min, whereas at high cII protein levels, it could be as long as 22 min. The Escherichia coli mutant ER437 directs lambda towards lysogeny; cII protein was more stable in this strain than in the wild type. On the other hand, although cyclic AMP is required for efficient lysogeny, it did not appear to influence the synthesis, stability, or activity of cII protein.

Bacteriophage lambda↗