PubMed Health⌕ Search

Biomedical subjects

H E Ruley

Publications and source records attributed to H E Ruley.

At least 37 records · Page 2Linked to original sources
PubMed1994

Generation of Chinese hamster ovary cell glycosylation mutants by retroviral insertional mutagenesis. Integration into a discrete locus generates mutants expressing high levels of N-glycolylneuraminic acid.

Retroviral insertional mutagenesis can both generate somatic cell mutants and pinpoint the genomic locus associated with a mutant phenotype. In the present study, this approach was applied to Chinese hamster ovary cells (CHO) made susceptible to Moloney murine leukemia virus (MoMuLV) infection by stable expression of an ecotropic retrovirus receptor. These CHO cells were infected with a replication incompetent MoMuLV construct with a promoterless hygromycin phosphotransferase (hygro) gene inserted into the U3 region of the long terminal repeat and a second selectable marker, neomycin phosphotransferase (neo), expressed from an internal promoter. CHO clones containing integrated proviruses were selected with hygromycin or G418, and the subset of these with reduced cell surface Neu5Ac were then selected with wheat germ agglutinin (WGA). The majority of the resulting clones had a phenotype not previously described for WGA-resistant CHO mutants arising spontaneously or from chemical mutagenesis: Neu5Ac was almost completely replaced by Neu5Gc. We have provisionally termed these clones SAP mutants, for sialic acid phenotype. Southern analysis of HindIII digested DNA from four SAP mutants revealed that the MoMuLV provirus is present in a 10.4-kilobase (kb) fragment. Probing with a flanking CHO sequence resulted in equivalent hybridization to a 4.6-kb fragment and the 10.4-kb provirus-containing fragment in all four cases, while uninfected parental cells and non-SAP glycosylation mutants generated in the same retrovirus insertional mutagenesis experiments yielded only the 4.6-kb fragment. Sequencing of the 3'-flanking DNA revealed that each of the four SAP mutants had a unique provirus integration site falling within a 796 bp region of the CHO genome. The frequency with which SAP mutants arise suggests that this may be a preferred site for retrovirus integration.

Animals↗
PubMed1994

A murine homolog of the yeast RNA1 gene is required for postimplantation development.

A gene has been characterized that is required for postimplantation mouse development. The gene, designated fug1, was disrupted in embryonic stem cells by the U3Neo gene trap retrovirus, and the disrupted allele was introduced into the germ line. Homozygous mutant embryos arrest at the egg cylinder stage at about embryonic day 6 and are mostly resorbed by day 8.5. The appearance of the proamniotic cavity is delayed, and epiblast cells that surround the cavity are disorganized. fug1 transcripts are undetectable at E6 but are induced throughout the embryo after E6.5. The gene is expressed at low levels in all adult tissues examined, maps to chromosome 15, and is conserved among mammals. The cDNA sequence encodes a protein of 589 amino acids, the first 400 of which are 38% identical to the Saccaromyces cerevisiae RNA1 gene. Regions of greatest similarity include a long acidic domain and 11 leucine-rich motifs, thought to mediate high affinity protein-protein interactions. These similarities suggest that Fug1 may be required for developmental changes in RNA processing or chromatin structure prior to gastrulation.

Amino Acid Sequence↗
PubMed1994

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.

Animals↗
PubMed1993

p53-dependent apoptosis modulates the cytotoxicity of anticancer agents.

Although the primary cellular targets of many anticancer agents have been identified, less is known about the processes leading to the selective cell death of cancer cells or the molecular basis of drug resistance. p53-deficient mouse embryonic fibroblasts were used to examine systematically the requirement for p53 in cellular sensitivity and resistance to a diverse group of anticancer agents. These results demonstrate that an oncogene, specifically the adenovirus E1A gene, can sensitize fibroblasts to apoptosis induced by ionizing radiation, 5-fluorouracil, etoposide, and adriamycin. Furthermore, the p53 tumor suppressor is required for efficient execution of the death program. These data reinforce the notion that the cytotoxic action of many anticancer agents involves processes subsequent to the interaction between drug and cellular target and indicate that divergent stimuli can activate a common cell death program. Consequently, the involvement of p53 in the apoptotic response suggests a mechanism whereby tumor cells can acquire cross-resistance to anticancer agents.

Adenovirus E1A Proteins↗
PubMed1993

Enrichment of insertional mutants following retrovirus gene trap selection.

The present study has investigated the use of gene trap retroviruses as insertional mutagens. A gene trap vector (U3Hygro) was used to target single-copy thymidine kinase (tk) genes, present at different sites in the genome. Cell populations isolated by gene trap selection contained a higher proportion of insertional mutants as compared with nonselected cells containing randomly integrated viruses. The number of integration events required to observe loss of gene function was reduced from 8-40 x 10(6) to 2-10 x 10(4), an overall enrichment of 100- to 1000-fold. The feasibility of targeting normally diploid genes was also demonstrated in hypodiploid Chinese hamster ovary cells. The cellular gene encoding GlcNAc transferase I was disrupted in one wheat germ agglutinin resistant clone selected from a total of 5 x 10(4) gene trap events. The clone was nullizygous for GlcNAc transferase I, indicating that the allele opposite the provirus was lost as a result of preexisting hemizygosity or by loss of heterozygosity. Finally, the total number of genes in the genome that could activate the expression of retrovirus gene traps was estimated at between 2 x 10(4) and 10(5), suggesting that most expressed genes can be mutagenized by gene trap selection.

3T3 Cells↗
PubMed1993

Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis.

Oncogenic transformation by human adenoviruses requires early regions 1A and 1B (E1A and E1B) and provides a model of multistep carcinogenesis. This study shows that the metabolic stabilization of p53 observed in adenovirus 5 (Ad5)-transformed cells can occur in untransformed cells expressing E1A alone. Stabilized p53 was localized to the nucleus and was indistinguishable from wild-type p53 with respect to its interactions with hsc70, PAb420, Ad5 p55E1B, and SV40 large T antigen. Moreover, binding of Ad5 p55E1B or SV40 large T antigen had no additional effect on p53 levels or turnover. Higher levels of p53 were also induced in a variety of cell types within 40 hr after transferring E1A genes. E1A also caused cells to lose viability by a process resembling apoptosis. The apoptosis appeared to involve p53, because p53 levels reverted to normal in surviving cells that had lost E1A, and E1B protected cells from the toxic effects of E1A. These results suggest that (1) the involvement of p53 in tumor suppression and/or apoptosis can be regulated at the level of protein turnover, and (2) a major oncogenic role for E1B is to counter cellular responses to E1A (i.e., stabilization of p53 and associated apoptosis) that preclude transformation by E1A alone. This represents the first physiological setting in which high levels of endogenous p53 are induced in response to an oncogenic challenge, with the apparent consequence of suppressing transformation.

Adenovirus E1A Proteins↗
PubMed1992

Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes.

Murine embryonic stem (ES) cells were infected with a retrovirus promoter trap vector, and clones expressing lacZ fusion genes (LacZ+) were isolated by fluorescence-activated cell sorting (FACS). Of 12 fusion genes tested, 1 was repressed when ES cells were allowed to differentiate in vitro. Two of three lacZ fusion genes tested were passed into the germ line, indicating that FACS does not significantly affect stem cell totipotency. The pattern of lacZ expression observed in vivo was consistent with that seen in vitro. Both fusion genes were expressed in preimplantation blastulas. However, a fusion gene whose expression was unaffected by in vitro differentiation was ubiquitously expressed in day-10 embryos, while the other, which showed regulated expression in vitro, was restricted to cells located along the posterior neural fold, the optic chiasm, and within the fourth ventricle. These results demonstrate the utility of using promoter trap vectors in conjunction with fluorescence sorting to disrupt developmentally regulated genes in mice.

Animals↗
PubMed1992

Selective disruption of genes expressed in totipotent embryonal stem cells.

Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.

Alcohol Oxidoreductases↗
PubMed1991

Selective targeting of gene products with the megakaryocyte platelet factor 4 promoter.

We have used the 1.1 kilobases of the 5' upstream region of the platelet factor four (PF4) gene coupled to the prokaryotic beta-galactosidase gene to generate two lines of transgenic mice that express this construct. Studies of blood, bone marrow, spleen, and thymus reveal that platelets are the only circulating blood cells and megakaryocytes are the only hematopoietic precursor cells that possess the prokaryotic enzyme. The lack of transgene expression in brain, heart, intestine, kidney, liver, lung, and skeletal muscle was established by in situ staining of tissue sections as well as kinetic assay of tissue homogenates. These data suggest that this domain of the PF4 promoter contains most, if not all, of the tissue-specific region of the gene. Unexpectedly, the adrenal gland exhibits approximately 2% of the levels of beta-galactosidase possessed by megakaryocytes and the distribution of the prokaryotic enzyme corresponds to the location of mineralocorticoid-secreting cells. This result implies that either the PF4 gene is transcribed at low levels in specialized adrenal cells or that these specialized endocrine cells possess trans-acting factors similar to those that control the megakaryocyte promoter. The selective high-level expression of transgenes linked to the PF4 promoter should allow us to augment or suppress the in vivo levels of critical components in megakaryocytes and platelets and subsequently ascertain the effects of these modifications.

Animals↗
PubMed1991

Retrovirus promoter-trap vector to induce lacZ gene fusions in mammalian cells.

A retrovirus promoter-trap vector (U3LacZ) has been developed in which Escherichia coli lacZ coding sequences were inserted into the 3' long terminal repeat (LTR) of an enhancerless Moloney murine leukemia virus. The U3LacZ virus contains the longest reported LTR (3.4 kbp); nevertheless, lacZ sequences did not interfere with the ability of the virus to transduce a neomycin resistance gene expressed from an internal promoter. Duplication of the LTR placed lacZ sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA. Approximately 0.4% of integrated proviruses expressed beta-galactosidase as judged by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, and individual clones expressing lacZ were isolated by fluorescence-activated cell sorting. In all clones examined, beta-galactosidase expression resulted from the fusion of lacZ sequences to transcriptional promoters located in the flanking cellular DNA. Furthermore, by differential sorting of neomycin-resistant cell populations, clones were isolated in which lacZ expression was induced and repressed in growth-arrested and log phase cells, respectively.

Animals↗
PubMed1991

Mechanisms of oncogene cooperation: activation and inactivation of a growth antagonist.

Gene transfer experiments have defined limitations with regard to the ability of individual oncogenes to transform cultured cells to a tumorigenic state. The stable transformation of REF52 cells by either the ras or sis oncogenes requires the continuous expression of a second collaborating oncogene, such as adenovirus-5 E1A or SV40 large T-antigen. Our studies suggest that the function of the nuclear collaborators is to antagonize dominant growth controls which limit the ability of REF52 cells to proliferate in response to mitogenic stimuli.

Adenovirus Early Proteins↗
PubMed1991

Massive accumulation of neutral lipids in cells conditionally transformed by an activated H-ras oncogene.

Phenotypic consequences of ras oncogene expression were studied in cells conditionally transformed by T24 H-ras and a temperature-sensitive SV40 large T antigen (tsA58). Previous studies have demonstrated that transformation of REF52 cells by ras and SV40 large T antigen requires continuous T antigen expression. Thus, tsA58/T24 H-ras transformants ceased growing when transferred to a restrictive temperature for T antigen expression. Inhibition of cell growth was accompanied by massive accumulations of cholesterol esters, triglycerides and a third lipid species, identified as glycerol ethers on the basis of mobility on TLC. Cholesterol esters were derived from serum lipoproteins, and appeared to accumulate because LDL receptor expression and activity did not decline in growth arrested cells. Triglycerides and glycerol ethers were products of cell metabolism. The process lacked features characteristic of adipocyte differentiation, but may suggest mechanisms important in diseases, such as atherosclerosis, that involve abnormal accumulations of neutral lipids. Accumulating lipid species may also include metabolites induced by ras that accumulate in growth-arrested cells.

Adipose Tissue↗
PubMed1990

Structure of the human smooth muscle alpha-actin gene. Analysis of a cDNA and 5' upstream region.

The structures of a cDNA and the 5' upstream region of the human smooth muscle alpha-actin gene have been characterized. Transcriptional start sites and the non-coding first exon were mapped by primer extension analysis and by comparing cDNA and genomic sequences. The deduced human smooth muscle alpha-actin protein sequence is identical to the corresponding bovine protein sequence, and thus confirms that the previously determined human genomic sequence contained a mutation at codon 312. Human smooth muscle cells express only a single, 1.4-kilobase smooth muscle alpha-actin transcript. 5' Noncoding sequences that have the greatest similarity to the chicken gene are located in five noncontiguous segments, extending from approximately 250 base pairs upstream of the cap site through the first exon. Conserved sequences encompass a region required for expression and tissue-specific regulation of chicken smooth muscle alpha-actin and therefore are probably also important for expression of the human gene.

Actins↗
PubMed1990

Isolation of cellular promoters by using a retrovirus promoter trap.

A retrovirus vector has been used to isolate transcriptional promoters from mammalian cells. The virus contains a selectable gene encoding histidinol dehydrogenase (his) in the U3 region of the 3' long terminal repeat (LTR). When the virus is passaged, duplication of LTRs places his sequences just 30 nucleotides from the adjacent cellular DNA. As a result, selection for histidinol resistance generates cell clones in which his is expressed on transcripts initiating in the flanking cellular DNA. Upstream cellular sequences, cloned after amplification by polymerase chain reaction, hybridized to RNA from uninfected cells, indicating that the adjacent promoters were transcriptionally active prior to virus integration. Two cloned transcribed flanking sequences also contained highly active transcriptional promoters, as estimated by their ability to activate expression of a linked reporter gene. Thus, U3His vectors provide a rapid and efficient means to isolate promoters active in different cell types. Moreover, by selecting for cell clones containing proviruses integrated in expressed genes, the virus may make an effective insertional mutagen.

Base Sequence↗
PubMed1990

Retroviruses as genetic tools to isolate transcriptionally active chromosomal regions.

By exploiting the ability of retroviruses to move genes into random sites of mammalian genomes and by exploiting some features of their replication, retrovirus vectors have been developed that select for instances in which the virus integrates into expressed genes. Since integrated proviruses tag transcriptionally active sites, the vectors provide a means to identify and isolate promoters active in different cell types. Furthermore, the viruses may be useful as insertional mutagens, since they select for instances in which integration occurs into expressed sites. This reduces the number of integrants needed to screen for loss of gene function and may enable genes controlling phenotypes in mammalian cells to be isolated.

Chromosomes↗
PubMed1989

Identification of cellular promoters by using a retrovirus promoter trap.

A retrovirus vector has been developed that selects for instances in which the provirus integrates in close proximity to cellular promoters. Coding sequences for a selectable marker (histidinol dehydrogenase) were inserted into the 3' long terminal repeat (LTR) of an enhancerless Molony murine leukemia virus. Although 1.8 kilobase pairs in size, the elongated LTR did not appear to interfere with virus replication or integration. Thus, when the virus was passaged, the elongated LTRs efficiently duplicated, placing histidinol dehydrogenase-coding sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA. Selection for histidinol expression generated cell clones in which histidinol gene sequences in the 5' LTR were invariably expressed on transcripts initiating in nearby cellular sequences. The efficiency of transducing histidinol resistance was 2,500-fold lower than the efficiency of transducing neomycin resistance when the neomycin phosphotransferase gene was located within the retrovirus and expressed from an independent promoter. By tagging transcriptionally active sites, the vector provides a means to identify and isolate promoters active in different cell types. Furthermore, the virus may be useful as an insertional mutagen, since selection for cell populations containing proviruses only in expressed sites is expected to reduce the number of intergrants needed to screen for loss of gene function.

Alcohol Oxidoreductases↗
PubMed1988

Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene.

Established REF52 cells (rat embryo fibroblasts) completely resist stable transformation by ras oncogenes, and simian virus 40 large tumor (T) antigen collaborates with ras to convert REF52 cells to tumorigenic state. A temperature-sensitive simian virus 40 large T antigen (encoded by tsA58) allowed the T24 Ha-ras oncogene to transform REF52 cells in a temperature-dependent manner. Two thirds of the clones transformed with tsA58 and ras became arrested in G2 or late S phase when shifted to a nonpermissive temperature for T antigen stability. Thus, ras induced growth arrest rather than stable transformation in the absence of a functional collaborating oncogene. These results indicate that collaborating oncogenes can regulate cellular responses to ras and have implications regarding therapeutic strategies to control tumor cells expressing activated ras oncogenes.

Animals↗