Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A plasmid containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of a mammalian cAMP-regulated promoter was entrapped in H-2Kk antibody-coated liposomes composed of dioleoyl phosphatidylethanolamine, cholesterol, and oleic acid (pH-sensitive immunoliposomes). The entrapped or free DNA was injected intraperitoneally into immunodeficient (nude) BALB/c mice bearing ascites tumor generated by H-2Kk-positive RDM-4 lymphoma cells. About 20% of the injected immunoliposomes were taken up by the target RDM-4 cells. Uptake was much less when liposomes without antibody were used. The presence of the targeting antibody on liposomes also significantly decreased the nonspecific uptake of liposomes by the spleen. Significant CAT enzyme activity was detected in RDM-4 cells from mice treated with DNA entrapped in the pH-sensitive immunoliposomes. Furthermore, CAT expression in RDM-4 cells was under the control of cAMP, as only the cells from mice injected with 8-bromo-cAMP and 3-isobutyl-1-methylxanthine showed CAT activity. CAT activity in liver and spleen was much lower (by factors of 12 and 5, respectively) than in the RDM-4 cells, and the activities in these reticuloendothelial organs were not regulated by cAMP. CAT activity in RDM-4 cells from mice injected with DNA entrapped in pH-insensitive immunoliposomes (containing phosphatidylcholine in place of phosphatidylethanolamine) was approximately one-fourth that in RDM-4 cells from mice injected with pH-sensitive immunoliposomes, indicating the superior delivery efficiency of the pH-sensitive liposomes. These results are discussed in terms of the DNA-carrier potential of immunoliposomes in therapy of cancer and genetic diseases.
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PMID:pH-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse. 244 13

Studies of recombinants between murine leukemia viruses (MuLVs) that cause thymic or erythroid leukemias have shown that enhancer sequences in the long-terminal repeats (LTRs) can determine the target tissues for pathogenesis. It has been inferred that the enhancers may specifically target viral expression into the cells that then become neoplastic. However, the neoplasms in those studies formed after latencies and contained ultimate viruses (called MCFs) that differed from the injected viruses in their enhancer sequences and envelope (env) genes. Transcriptional activities of LTRs from these proximal and ultimate viruses have not been thoroughly analyzed in different hematopoietic lineages. We present evidence that the enhancer of Friend spleen focus-forming virus (SFFV), an ultimate erythroleukemogenic retrovirus, contains an unstable 42-nucleotide direct repeat. Other ultimate erythroleukemogenic MuLVs (Friend MCFs) contain an enhancer nearly identical to that of SFFV both in its sequence and in its specific instability. The instability occurs in sequences that contain inverted repeats and we propose that it occurs by a simple reverse transcriptase hop mechanism. We constructed plasmids that contain the two forms of the SFFV LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, and we compared these in transient transfection assays with LTR-CAT plasmids constructed from Friend and Moloney MuLVs. The assays employed erythroleukemia cells, thymic lymphoma cells, and fibroblasts. The tropisms of expression correlated only weakly with tissue specificities of pathogenesis and each LTR was active in all cells. The SFFV 42-nucleotide duplication reduced expression in erythroid cells and increased expression in fibroblasts. We conclude that retroviral enhancers do not stringently direct gene expression into specific cell lineages, but on the contrary they are leaky and contain replicative instabilities that also may facilitate viral entrenchment throughout the host. These results have important implications for understanding murine retroviral evolution and the multi-step process of leukemogenesis.
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PMID:An enhancer sequence instability that diversifies the cell repertoire for expression of a murine leukemia virus. 283 56

The nucleotide sequences of long terminal repeats (LTRs) from several mouse mammary tumor virus (MMTV) proviruses acquired in mouse T-cell lymphomas were determined. All MMTV proviruses cloned from a C57BL/6 lymphoma contained an identical LTR deletion of 491 base pairs (approximately -655 to -165), whereas an MMTV provirus from a BALB/c T-cell lymphoma had a 430-base-pair deletion in the same U3 region. MMTV proviruses with LTR deletions were acquired in these tumors 10 times more frequently than proviruses with intact LTRs. Because the deletions removed a portion of the glucocorticoid response element or "regulated" enhancer, the transcriptional activity of the deleted MMTV LTRs was assessed in both transient expression and stable transfection experiments. Plasmids were constructed in which the deleted or full-length MMTV LTRs were placed upstream of the chloramphenicol acetyltransferase gene. Results from transfection experiments with these constructs showed that the basal expression of the deleted MMTV LTR in the absence of glucocorticoids was higher than that of the full-length Mtv-17 or C3H MMTV LTRs under the same conditions. Moreover, the C3H LTR with a similar deletion (-637 to -255) also promoted high basal levels of chloramphenicol acetyltransferase activity. These results, coupled with the observation in lymphomas of high basal levels of transcription from MMTV proviruses with deleted LTRs, suggested that these proviruses lack negative regulatory elements in their LTRs. Loss of the negative regulatory element may contribute to the selective propagation of proviruses with deleted LTRs.
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PMID:Mouse mammary tumor virus proviruses in T-cell lymphomas lack a negative regulatory element in the long terminal repeat. 284 76

The effects of rearrangement and insertion of sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. The alterations were made by recombinant DNA manipulations on a plasmid subclone containing an M-MuLV LTR. Promoter activity of altered LTRs was measured by fusion to the bacterial chloramphenicol acetyltransferase gene, followed by transient expression assay in NIH 3T3 cells. M-MuLV proviral organizations containing the altered LTRs were also generated, and infectious virus was recovered by transfection. Infectivity of the resulting virus was quantified by XC plaque assay, and pathogenicity was determined by inoculating neonatal NIH Swiss mice. Inversion of sequences in the U3 region containing the tandemly repeated enhancer sequences (-150 to -353 base pairs [bp]) reduced promoter activity approximately fivefold in the transient-expression assays. Infectious virus containing the inverted sequences (Mo- M-MuLV) showed a 20-fold reduction in relative infectivity compared with wild-type M-MuLV, but the virus still induced thymus-derived lymphoblastic lymphoma or leukemia in mice, with essentially the same kinetics as for wild-type M-MuLV. We previously derived an M-MuLV which carried inserted enhancer sequences from the F101 strain of polyomavirus (Mo + PyF101 M-MuLV) and showed that this virus is nonleukemogenic. In Mo + PyF101 M-MuLV, the PyF101 sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at -150 bp). A new LTR was generated in which the PyF101 sequences were inserted to the 5' side of the M-MuLV enhancers (at -353 bp, PyF101 + Mo M-MuLV). The PyF101 + Mo LTR exhibited promoter activity similar (40 to 50%) to that of wild-type M-MuLV, and infectious PyF101 + Mo M-MuLV had high infectivity on NIH 3T3 cells (50% of wild type). In contrast to the nonleukemogenic Mo + PyF101 M-MuLV, PyF101 + Mo M-MuLV induced leukemia with kinetics similar to that of wild-type M-MuLV. Thus, the position of the PyF101 sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyF101 + Mo M-MuLV induced a different spectrum of neoplastic disease. In comparison with wild-type M-MuLV, which induces a characteristic thymus-derived lymphoblastic lymphoma with extremely high frequency, PyF101 + Mo M-MuLV was capable of inducing both acute myeloid leukemia or thymus-derived lymphoblastic lymphoma, or both. Tumor DNA from both the PyF101 + Mo- and Mo- M-MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus.
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PMID:Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro. 374 27

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30,000 M(r) serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5'-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5'-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5'-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5'-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines. The transcriptional activity of the RNK-Met-1 5'-flanking region was strong, restricted to the RNK-16 LGL leukemia and controlled by several positive cis-acting regions spread over at least 3.3 kb. The longest and most active 5'-flanking region (-3341 to -33) was also used to drive specific expression of beta-galactosidase in RNK-16. These data are consistent with the NK cell-specific expression of RNK-Met-1 and suggest the potential utility of this gene promoter in the development of transgene models of NK cell biology in vivo.
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PMID:Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5'-flanking sequences. 760 1

Transcriptional regulation of the interleukin-5 (IL-5) gene in T lymphocytes appears to be of central importance in the control of the eosinophilia characteristic of allergic responses and certain parasite infections. Previous studies of IL-5 gene regulation have been hampered by the lack of a transfection assay, which detects the antigen-responsive enhancer in the IL-5 promoter. Here we show that stable transfection of the Th2 clone D10.G4.1 and the T lymphoma EL4.23 with chloramphenicol acetyltransferase reporter gene constructs carrying the region to -3859 gives inducible expression with the known regulatory characteristics of the endogenous IL-5 gene. To facilitate detailed analysis of the promoter region, 3.9 kb of DNA sequence immediately up stream of the start of transcription was determined and the minimum upstream region required for inducible expression was further localized, by stable transfection studies in EL4.23 cells, to the region up to -1016. A CTF/NF1 site in the upstream enhancer at -940 to -928 was shown to be required for regulated inducible expression. Mutation of this sequence motif abolished inducibility and also prevented binding of the sequence to a nuclear protein(s). A TCATTT-containing element in the proximal promoter region was also demonstrated to be essential for inducible expression of the IL-5 gene, similar to the role of this conserved element in the transcriptional regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 genes.
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PMID:Localization of the inducible enhancer in the mouse interleukin-5 gene that is responsive to T-cell receptor stimulation. 771 77

Three double negative (BoCD4-, BoCD8-) bovine T cell lines, BTL-PC3, BLT2, and Pr2181, which have been established from bovine lymphosarcomas, were examined for expression and molecular function of bovine c-myb genes. BTL-PC3 expressed 4.0- and 3.6-kilobase c-myb transcripts, and BLT2 and Pr2181 expressed a 3.8-kilobase c-myb message. The c-Myb protein (75 kDa) was detected in Pr2181 but not as a clear band in BLT2, while BTL-PC3 exhibited a 65-kDa c-Myb band in immunoprecipitation tests with anti-Myb antiserum. Nucleotide sequences for c-myb cDNA clones from BTL-PC3 and BLT2 indicated that the predicted bovine wild-type c-Myb from BLT2 consists of 640 amino acids whereas that from BTL-PC3 consists of 555 amino acids lacking 85 internal amino acids. This deleted DNA region (255 base pairs consisting of 85 amino acids) corresponds to the human genomic exon 9 encoding a negative regulatory domain in the c-myb gene. Upon cotransfections with reporter plasmids containing myb binding sites, the internally deleted c-Myb exhibited a 3-fold higher transcriptional activity than the wild-type c-Myb in chloramphenicol acetyltransferase assays. These results indicate that internal DNA deletion in the c-myb gene is directly involved in the enhancement of transcriptional activation in bovine T lymphoma cells.
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PMID:A spontaneous internal deletion of the c-myb protooncogene enhances transcriptional activation in bovine T lymphoma cells. 792 19

Nuclear protein binding sites in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV) were identified by the method of DNase I footprinting. Using nuclear protein extracts from a feline T lymphoma cell line, several discrete footprints were generated upstream of the transcriptional initiation site (-50 to -150). The specificity of protein binding was examined by competition with oligonucleotides representing consensus DNA binding sites for known transcription factors. Binding to AP-1 (-124) and ATF (-58) motifs was observed, with cross-competition between these sites. A strong footprint signal was also detected over a tandemly repeated C/EBP motif (-94, -86) and an adjacent weaker footprint was found to be specific for an NF1 motif (-72/-63). The effect on FIV LTR promoter activity of progressively deleting these nuclear factor binding sites was examined by linking LTR deletion mutants to the chloramphenicol acetyltransferase (CAT) gene. Deletion of the AP-1 site caused a 10- to 25-fold loss of CAT activity whereas deletion past the ATF site reduced activity virtually to background levels. The effects of deleting the C/EBP and NF1 sites were less marked and varied according to cell type. Transactivation of the LTR was assayed using constructs linked to a CAT reporter gene. The full-length FIV LTR was not significantly trans-activated. However, the expression of a deleted LTR construct lacking the AP-4/AP-1 site but retaining C/EBP and ATF sites was partially restored by co-infection with FIV or by co-transfection with an infectious molecular clone of FIV (FIV-PPR). These results show that host transcription factors responsive to cellular activation have a major role in regulating FIV expression, and suggest that virus-coded trans-activators acting through U3 may play a role in some cellular environments.
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PMID:Cis- and trans-regulation of feline immunodeficiency virus: identification of functional binding sites in the long terminal repeat. 812 51

Adult T cell leukemia/lymphoma (ATL) is derived from CD4+ T cells and has a poor prognosis because of its resistance to chemotherapy. To evaluate the effectiveness of gene therapy for ATL, the effect of ganciclovir on ATL cell lines transfected with the thymidine kinase gene of herpes simplex virus type 1 (HSV-TK) was analyzed. To transfer the HSV-TK gene to ATL cells, a human immunodeficiency virus (HIV) vector that has specific infectivity to CD4+ cells was used. HSV-TK was inserted into the long terminal repeats of HIV-1 and driven by the SL3 promoter HXBSL3TK. HXBSL3TK was co-transfected with HXBCAT as a reporter into MT2 or HUT102 cells by DEAE-dextran. The cells were incubated with ganciclovir, and chloramphenicol acetyltransferase (CAT) activity was analyzed. The CAT activity of the MT2 cells and HUT102 cells transfected with HXBSL3TK decreased dose-dependently with ganciclovir. HXBSL3TK was also co-transfected into COS cells with an HIV-1 packaging vector that has gag, pol, and env driven by a cytomegalovirus promoter. The supernatant was transferred to MT2 cells or Raji cells and incubated with ganciclovir. Ninety percent of the MT2 cells transduced by HXBSL3TK and incubated with ganciclovir were killed, but Raji cells were not killed. In addition, HXBTK that expresses the HSV-TK gene and Tat gene driven by the LTR of HIV-1 was constructed. HXBTK had a higher expression of the HSV-TK gene and higher sensitivity to ganciclovir than did HXBSL3TK.
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PMID:Gene therapy for adult T cell leukemia using human immunodeficiency virus vector carrying the thymidine kinase gene of herpes simplex virus type 1. 895 10

Glucocorticoids inhibit transcription of the murine cytoplasmic thymidine kinase gene (Tk-1). Glucocorticoid regulation of Tk-1 transcription can be demonstrated in cells that are arrested in late G1. This observation indicates that inhibition of Tk-1 expression is not dependent upon redistribution within the cell cycle but is due to glucocorticoid regulation of this gene. Transfection studies have been carried out using chimeric genes in which restriction fragments of the Tk-1 promoter were fused to chloramphenicol acetyltransferase or neomycin phosphotransferase. These chimeric reporters were assayed for stable expression and glucocorticoid regulation in P1798 lymphoma cells. A 140-bp fragment, extending from -143 to -3 bp with respect to the thymidine kinase translational start site, was capable of both basal and glucocorticoid-regulated transcription of reporter genes. The extent of inhibition by glucocorticoids was similar to that observed for the endogenous gene, and no increase in basal expression or the extent of inhibition was observed with constructs containing additional 5'-flanking DNA. The 140-bp Tk-1 core promoter fragment binds to transcription factors in extracts from P1798 cells. Control cell extracts contain factors that bind to and protect (from deoxyribonuclease I) a distal promoter element from -106 to -87 bp, relative to the translational start site. A second, proximal element was protected at -43 to -36 bp. The proximal element of the Tk-1 promoter resembles an RNA polymerase II initiator element. No other elements were protected. Glucocorticoids inhibit the amount or activity of the transcription factor that binds to this initiator-like element within the Tk-1 promoter. This element, when fused to upstream activation sequences from the herpes simplex virus thymidine kinase promoter, conveys glucocorticoid sensitivity in cis.
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PMID:Glucocorticoid regulation of a transcription factor that binds an initiator-like element in the murine thymidine kinase (Tk-1) promoter. 896 Dec 64


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