Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.
Mol Carcinog 1991
PMID:Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17 beta-estradiol and 2-aminofluorene in V79 Chinese hamster cells. 179 87

A stable transformation procedure has been developed for Phytophthora infestans, an oomycete fungus that causes the late blight diseases of potato and tomato. This is the first description of reliable methods for transformation in an oomycete pathogen. Drug-resistant transformants were obtained by using vectors that contained bacterial genes for resistance to hygromycin B or G418 fused to promoters and terminators from the Hsp70 and Ham34 genes of the oomycete, Bremia lactucae. Using polyethylene glycol and CaCl2, vector DNA was introduced into protoplasts as a complex with cationic liposomes or with carrier DNA only. Transformants were obtained at similar frequencies with each combination of promoter and selectable marker and were confirmed by DNA and RNA hybridization and phosphotransferase assays. Transformation occurred through the integration of single or tandemly repeated copies of the plasmids into genomic DNA, conferring mitotically stable drug-resistant phenotypes. The sizes of the marker gene mRNAs in each transformant and the results of transcript mapping studies were consistent with the function of the B. lactucae regulatory sequences in P. infestans. A hygromycin-resistant transformant was tested and found to maintain pathogenicity, indicating that the gene transfer procedure will be useful for the molecular analysis of genes relevant to disease.
Mol Plant Microbe Interact
PMID:Transformation of the oomycete pathogen, Phytophthora infestans. 180 4

Two human liver UDP-glucuronosyltransferase cDNA clones (HLUGP1 and HLUG25) were individually inserted into the eukaryotic expression vector pKCRH2. Each recombinant plasmid was cotransfected with a SFVneo vector, thereby allowing establishment of several V79 cell lines retaining the exogenous UDP-glucuronosyltransferase cDNA after selection with G418 (Geneticin). Southern blot analysis suggested that the cDNAs were integrated into the host cell genome. Northern blot and immunoblot analyses indicated that the cDNAs were correctly transcribed and translated for the production of functional enzymes. The established recombinant V79 cell lines stably expressed the UDP-glucuronosyltransferase activities towards 1-naphthol (HLUGP1) and hyodeoxycholic acid (HLUG25) at levels 10-20-fold higher than with transient expression, and in the range found in human liver. These high levels of expression of UDP-glucuronosyltransferase activity allowed the determination of apparent kinetic constants and substrate specificities of glucuronidation in the genetically engineered cell lines. HLUG25 cDNA encoded an isoform with restricted specificity towards the 6-OH group of the bile acid hyodeoxycholic acid. The other steroids, bile acids, endobiotics, and xenobiotics tested as substrates were glucuronidated in various samples of human liver microsomes, but not by this isoenzyme. This study, allowing the expression of individual UDP-glucuronosyltransferases in heterologous cells with no endogenous transferases, offered a unique solution for the characterization of UDP-glucuronosyltransferase functional heterogeneity.
Mol Pharmacol 1991 Feb
PMID:Stable expression of two human UDP-glucuronosyltransferase cDNAs in V79 cell cultures. 184 92

We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations.
Mol Cell Biol 1991 May
PMID:Cotransformation and gene targeting in mouse embryonic stem cells. 185 Jan 4

We have shown that the Leishmania major transfection vector pR-NEO (or derivatives thereof) can be introduced and stably maintained in four species complexes of pathogenic Leishmania (L. tropica, L. mexicana, L. donovani, L. braziliensis), and the genera Endotrypanum and Crithidia; transfection of Trypanosoma cruzi or Trypanosoma brucei was not successful. Quantitative plating assays showed that the transfection efficiencies were high in L. major and Leishmania amazonensis (5x10(-5)/cell) and about 10-fold less for Leishmania panamaensis and Crithidia. Leishmania donovani transfected with pR-NEO retained the ability to infect hamsters, and amastigotes recovered after 2 months yielded G418-resistant promastigotes which retained high levels of extrachromosomal pR-NEO DNA. In promastigotes, the transfected DNA existed as extrachromosomal circles, and expressed the predicted 2.4-kb hybrid NEO/DHFR-TS mRNA bearing the trans-spliced miniexon. Large quantitative differences were observed only in Crithidia: relative to transfected Leishmania species, the copy number of pR-NEO was elevated 20-fold, while the levels of the NEO/DHRFR-TS mRNA or Escherichia coli beta-galactosidase (synthesized from the expression vector pX-beta GAL) were reduced 80 and more than 1000-fold, respectively. Thus, genetic signals derived from L. major DNA that mediate RNA expression or stability are recognized by the heterologous Leishmania species but less efficiently by Crithidia. These studies suggest that pR-NEO derived vectors may be applied to the study of genes expressed throughout the life cycle in a wide range of pathogenic trypanosomatids.
Mol Biochem Parasitol 1991 May
PMID:Stable DNA transfection of a wide range of trypanosomatids. 190 80

The nucleotide sequence of the mdr1 gene encoding a putative drug efflux pump (P-glycoprotein) is homologous to a class of bacterial membrane-associated transport proteins. These bacterial proteins are part of a multicomponent system that includes soluble periplasmic proteins that bind substrates, channeling them through the membrane in an energy-dependent manner. We have investigated the possibility that a similar multicomponent transport system exists in a multidrug-resistant human MCF-7 breast cancer cell line that was initially selected for resistance to doxorubicin (AdrR MCF-7). AdrR MCF-7 cells overexpress both the mdr1 gene and the pi class isozyme of glutathione S-transferase (GST-pi) (EC 2.5.1.18). The latter is one of several isozymes known to have a ligand-binding function in addition to drug-metabolizing capabilities. Although we have recently shown that transfection of a functional GST-pi expression vector is insufficient to confer resistance to doxorubicin in cells that lack P-glycoprotein expression [Mol. Pharmacol. 36:22-28 (1989)], we examined the possibility that GST-pi interacts with P-glycoprotein to alter multidrug resistance. To do this, we have cloned cDNAs encoding these proteins from AdrR MCF-7 cells, constructed expression vectors containing these two genes, and transfected these vectors sequentially into drug-sensitive MCF-7 cells. The human mdr1 cDNA isolated from AdrR MCF-7 is a variant gene whose sequence differs from that isolated previously from vinblastine-resistant KB cells [Cell 53:519-529 (1989)], resulting in an amino acid substitution of alanine to serine at position 893 (mdr1/893ala). Transfection of eukaryotic expression vectors containing the mdr1 gene isolated from AdrR MCF-7 cells produced a multidrug-resistant phenotype in recipient cells, with a cross-resistance pattern similar to that in the AdrR MCF-7 cells. To determine whether GST-pi expression could augment resistance provided by mdr1, two clones transfected with mdr1, one with high levels (153% of mdr1 RNA in AdR MCF-7 cells) and one with low levels (10% of mdr1 RNA in AdrR MCF-7 cells), were subsequently cotransfected with a GST-pi expression vector and pSVNeo and selected for resistance to G418. Six of these clones contained levels of GST-pi that were 8- to 18-fold greater than GST levels found in mdr1-expressing clones transfected with nonspecific DNA. We found no difference in the degree of resistance to doxorubicin, actinomycin D, and vinblastine between the clones expressing mdr1 only and the clones expressing both mdr1 and GST-pi.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1990 Jun
PMID:Multidrug resistance in cells transfected with human genes encoding a variant P-glycoprotein and glutathione S-transferase-pi. 197 72

We report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.
Mol Gen Genet 1991 May
PMID:Stable transformation of the moss Physcomitrella patens. 203 4

We have used a host cell reactivation system to study the effect of 8-methoxypsoralen (8-MOP) reaction on CAT (chloramphenicol acetyltransferase) and NEO (aminoglycoside phosphotransferase) expression in normal human cells, as well as two cell lines with possible DNA repair-processing defects. Plasmid DNA was treated with psoralen plus near-ultraviolet (NUV) irradiation. The reacted plasmids, pSV2cat and pSV2neo, were transfected into Fanconi anemia (FA), xeroderma pigmentosum (XP), and normal human fibroblast cells for transient or stable assay. The cells were assayed for CAT activity at various times after transfection or selected for G418 resistance. The extent of adduct formation required to inhibit expression was much less (difference of D37 greater than 2.5) in FA or XP cells compared to normal. We conclude that in FA and XP cells, the reactivation of CAT was much less than in normal cells. The possibility of differential DNA uptake and/or degradation in transient assay was ruled out by analysis of plasmid DNA recovered from transfected cells. The data of the two independent assays indicate that FA and XP cells are deficient in cross-linked DNA repair.
Somat Cell Mol Genet 1991 May
PMID:Reactivation of psoralen-reacted plasmid DNA in Fanconi anemia, xeroderma pigmentosum, and normal human fibroblast cells. 204 39

To study the relationship between oncogenesis by v-src and normal cellular signalling pathways, we determined the effects of v-src on 3T3-TNR9 cells, a Swiss 3T3 variant which does not respond mitogenically to tumor promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We found that src was unable to transform these variant cells, whether the oncogene was introduced by infection with a murine retrovirus vector or by transfection with plasmid DNA. 3T3-TNR9 cells were not inherently resistant to transformation, since infection with similar recombinant retroviruses containing either v-ras or v-abl did induce transformation. Further analysis of Swiss 3T3 and 3T3-TNR9 cell populations infected with the v-src-containing retrovirus revealed that although the amount of v-src DNA in each was approximately the same, the level of the v-src message and protein and the overall level of phosphotyrosine expressed in the infected variants was much less than in infected parental cells. Cotransfection experiments using separate v-src and neo plasmids revealed a decrease in the number of G418-resistant colonies when transfections of TNR9 cells occurred in the presence of the src-containing plasmid, suggesting a growth inhibitory effect of v-src on 3T3-TNR9 cells, as has also been found for TPA itself. Since v-src cannot transform this variant cell line, which does not respond mitogenically to the protein kinase C agonist TPA, we suggest that src makes use of the protein kinase C pathway as part of its signalling activities.
Mol Cell Biol 1990 Aug
PMID:A Swiss 3T3 variant cell line resistant to the effects of tumor promoters cannot be transformed by src. 211 20

Nuclear run-on experiments were used to verify the hypothesis that extinction of expression of Ig synthesis in L cell x myeloma hybrids occurs at the transcriptional level. Both the H chain enhancer and promoter have been shown to be the targets for extinction in myeloma x T cell hybrids. To examine the expression of genes containing the immunoglobulin heavy chain gene (IgH) enhancer in stably transfected non-B cells, we used a vector with two selectable markers, one of which (gpt providing resistance to mycophenolic acid) either lacks an enhancer or contains the IgH enhancer, the other (neo providing resistance to G418) contains an SV40 enhancer. Stable transfectants of both myeloma (J558L) and L cells selected using G418 were tested to determine if they are also mycophenolic acid resistant. When the IgH enhancer is positioned 3' to the gpt gene, transfected J558L are mycophenolic acid resistant whereas stably transfected L-cells are mycophenolic acid sensitive. However, when large numbers of L cell transfectants are exposed to mycophenolic acid for a prolonged period, resistant subclones emerge. When the 700-bp IgH enhancer fragment was used, the majority of the subclones examined had amplified the vector, between 3 and 38 copies; when a 400-bp subfragment was used no change in the integrated genes was seen. In both cases, in the mycophenolic acid resistant subclones, increased accumulation of gpt and neo mRNA is seen. However, the gpt specific transcripts are heterogeneous in size whereas the neo transcripts are of a discrete size. The heterogeneity of the gpt transcripts results at least in part from heterogeneous initiation. When HXM-resistant L cell subclones are fused to the gamm 2b, k myeloma 4T001, extinction of Ig production occurs; therefore these cells are still capable of negatively regulating Ig expression. These results are discussed from the standpoint of both cis and trans regulatory elements and factors in non-lymphoid cells.
Mol Immunol 1990 Aug
PMID:Expression of genes containing the IgH enhancer in non-lymphoid cells. 211 78


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