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)

Translocations involving chromosome band 11q23, found in acute lymphoid and myeloid leukemias, disrupt the MLL gene. This gene encodes a putative transcription factor with homology to the zinc fingers and other domains of the Drosophila trithorax gene product and to the "AT-hook" motif of high mobility group proteins. To map potential transcriptional activation or repression domains of the MLL protein, yeast GAL4 DNA-binding domain and MLL hybrid protein-expressing plasmids were cotransfected with chloramphenicol acetyltransferase reporter plasmids in a transient transfection system. We found that MLL contains a strong activation domain and a repression domain. The former, located telomeric (3') to the breakpoint region, activated transcription 18-fold to > 200-fold, depending on the promoter and cell line used for transfection. A repression domain that repressed transcription 4-fold was located centromeric (5') to the breakpoint region of MLL. The MLL AT-hook domain protein was expressed in bacteria and was utilized in a gel mobility shift assay to assess DNA-binding activity. The MLL AT-hook domain could bind cruciform DNA, recognizing structure rather than sequence of the target DNA. In translocations involving MLL, loss of an activation domain with retention of a repression domain and a DNA-binding domain on the der(11) chromosome could alter the expression of downstream target genes, suggesting a potential mechanism of action for MLL in leukemia.
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PMID:11q23 translocations split the "AT-hook" cruciform DNA-binding region and the transcriptional repression domain from the activation domain of the mixed-lineage leukemia (MLL) gene. 793

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.
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PMID:Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants. 805 69

The U1 and ACH-2 cell lines are subclones of human monocytic and T-lymphoid cells, respectively, persistently infected with human immunodeficiency virus type 1. These cell lines harbor the viral genome but produce only very low levels of viral progeny, which can be increased by stimulation with agents such as phorbol ester and cytokines. As such, they provide an in vitro model for human immunodeficiency virus type 1 latency. In order to examine the basis for their latent state, we have analyzed the activity of endogenous Tat protein in these cells and investigated the effect on viral replication of the addition of exogenous Tat protein. We find that U1 cells seem to have levels of Tat protein that are suboptimal for long terminal repeat (LTR) transcription, because transcription from a transfected LTR-chloramphenicol acetyltransferase plasmid can be enhanced by cotransfection of a Tat expression plasmid. Furthermore, viral replication can be stimulated in this cell line by incubation with purified Tat protein. In contrast, ACH-2 cells are not limited for LTR-chloramphenicol acetyltransferase transcription by endogenous levels of Tat, and virus production is not increased by the addition of exogenous Tat protein. By semiquantitative PCR analysis of viral RNA, we have demonstrated that Tat protein caused an increase in human immunodeficiency virus RNA expression in U1 cells but had no effect in ACH-2 cells. This suggests that a different mechanism underlies the latent state in U1 and ACH-2 cells.
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PMID:Analysis of Tat function in human immunodeficiency virus type 1-infected low-level-expression cell lines U1 and ACH-2. 810 61

Expression of the gene encoding the cytolytic granule protein perforin is restricted to cytotoxic lymphocytes. To undertake a functional analysis of the immediate 5'-promoter region of the mouse perforin gene, we transiently transfected mouse perforin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs into cytotoxic T, T lymphoid, B-lymphoid, and nonlymphoid cell lines. The transcriptional activity of the perforin promoter was restricted to cytotoxic lymphocytes. The perforin promoter was controlled by several positive (in perforin-positive cells) and negative (in perforin-negative cells) cis-acting regions, spread over at least 1.1 kilobases. The most specific expression of the CAT reporter gene in the interleukin-2-dependent cytotoxic T cell line CTLL-R8 was obtained with the mouse perforin promoter encompassing positions -1104 to +1 in relation to the RNA cap site. This construct expressed 65- to 70-fold higher CAT activity than the promoterless CAT construct in perforin-expressing cells but only 1- to 5-fold higher CAT activity than the promoterless construct in nonlymphoid cells. On the basis of these data, we used this most specifically active mouse perforin promoter, -1104 to +1, to express in CTLL-R8, a chimeric human receptor comprising the extracellular domains of human Fc gamma RI and the transmembrane and intracellular domains of TCR zeta. Selection in G418-containing medium produced CTLL-R8 transfectant clones that (1) expressed high levels of human Fc gamma RI mRNA; (2) expressed cell surface Fc gamma RI as demonstrated by immunoprecipitation and their ability to bind the Fc portion of human and mouse monoclonal antibodies (mAbs) in an isotype-specific manner, and (3) bound RBC expressing mucin-1 (Muc-1) peptide in the presence of a chimeric mouse-human anti-Muc-1 mAb. Activation of CTLL-R8 transfectants upon engagement of the human Fc gamma RI was evidenced by their ability to lyse tumor target cells in an mAb isotype-dependent manner. The successful expression of a functional chimeric gene in CTLL-R8 suggests that the mouse perforin promoter represents a novel reagent for expressing exogenous genes in cytotoxic T lymphocytes.
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PMID:Use of the 5'-flanking region of the mouse perforin gene to express human Fc gamma receptor I in cytotoxic T lymphocytes. 814 22

Two groups of transgenic rainbow trout (Oncorhynchus mykiss, Walbaum) have been produced and compared. One group harbored the reporter gene of chloramphenicol acetyltransferase (CAT) associated with mouse immunoglobulin (Ig) promoter/enhancer (pUCL-CAT-E). The other group carried the same reporter gene under the control of the cytomegalovirus promoter/enhancer (pCMV-CAT). Slot blot analysis of DNA from blood cells and other tissues from pUCL-CAT-E fish showed variation of copy number between the major tissues but not between red and white blood cells. Southern blot analysis indicated that multiple copies organized in concatemers were incorporated into the genome. The pCMV-CAT fish had a pronounced expression of CAT in both white and red blood cells. In contrast, activity of CAT was found in the white blood cells of all pUCL-CAT-E fish but not in their red blood cells. Expression in white blood cells was found preferentially in sIg+ cells, indicating that B cells are the major expressors. High expression was also found in spleen and kidney, but the activity found in thymocytes was equal to the background level. Analysis of some major tissues showed high white blood cell expression associated with low tissue expression, except that liver (known to contain lymphoid tissue in fish) was higher. Thus the regulatory elements of the Ig gene from mouse induce a tissue-specific expression in fish.
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PMID:Lymphocyte expression in transgenic trout by mouse immunoglobulin promoter/enhancer. 820 21

To examine the effects of Mycobacterium tuberculosis on human immunodeficiency virus type 1 (HIV-1) expression, the monocytoid cell line U1 containing integrated provirus was incubated with the H37Ra strain of M. tuberculosis. This resulted in heightened expression of virus in supernatant that was partially inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha). Purified protein derivative (PPD) prepared from M. tuberculosis also could activate HIV expression, and this was less affected by anti-TNF antibody. PPD could activate the HIV promoter in both U937, the monocytoid cell line from which U1 was derived, and Jurkat, a CD4+ lymphoid line. Activation was abolished by mutations in the nuclear factor (NF)-kB binding domains. Jurkat cells transfected with a plasmid construct linking 8 NF-kB binding domains to the chloramphenicol acetyltransferase (CAT) gene showed increased activity of the reporter gene after activation with PPD. Transcriptional activation of HIV expression by mycobacteria and mycobacterial products may enhance propagation of HIV in monocytoid and lymphoid cells. This may result in accelerated HIV disease progression in persons coinfected with M. tuberculosis.
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PMID:Mycobacterium tuberculosis and its purified protein derivative activate expression of the human immunodeficiency virus. 820 50

The regulatory mechanisms which control latency and reactivation of the Epstein-Barr virus (EBV) are not fully understood. To determine whether DNA methylation is involved, we examined the BamHI-H divergent promoter, which also encompasses the origin for lytic replication (oriLyt) of EBV. The divergent promoter was highly methylated in the stringently latent HH514cl16 cell line and largely unmethylated in the semipermissive FF41-1 cell line. Expression vectors in which the divergent promoter directed transcription of the chloramphenicol acetyltransferase gene were made. Using in vitro methylation and transient transfections, we found an inverse correlation between the number of sites methylated and level of gene expression. Similar patterns of inhibition were observed when the methylated promoter was activated by BZLF1 or BRLF1 and in lymphoid or epithelial cells. The role of two CpG dinucleotides in the BRLF1 binding sites of the divergent promoter was determined by site-directed mutagenesis. The results indicated that site-specific methylation of these CpGs was not solely responsible for inhibition of expression by methylation. DNA methylation also reduced DNA replication mediated by oriLyt. These results suggest that hypermethylation of the divergent promoter and oriLyt may suppress transcription and lytic replication of EBV.
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PMID:Regulation of Epstein-Barr virus BamHI-H divergent promoter by DNA methylation. 821 55

The human deoxycytidine kinase gene is a single copy gene and is comprised of seven exons that are spread over more than 34 kb of the genome. The 5'-flanking region is highly G/C rich and does not contain CAAT or TATA boxes. This region, when cloned into a recorder gene construct containing the chloramphenicol acetyltransferase gene, is capable of mediating CAT activity in human lymphoid cell lines and appears to have greater activity in human T, as compared to B, lymphoblast cell lines. The expression of the gene at the mRNA level does not appear to be cell-cycle regulated in that the levels of mRNA in human peripheral blood T lymphocytes remain constant as the cells progress from a resting to a proliferating state. Since this enzyme catalyzes the conversion of a number of chemotherapeutic agents to their corresponding monophosphate form and is thus essential for their activation, it will be important to define further the genetic elements which regulate the expression of this gene.
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PMID:Regulation of human deoxycytidine kinase expression. 835 17

The induction of the viral lytic cycle in latently Epstein-Barr virus (EBV)-infected B cells is initiated by activation of the BZLF1 gene, whose expression is sufficient to disrupt EBV latency, suggesting that BZLF1 acts as the switch to change from a latent to a lytic replicative cycle. In the present studies, a series of deletion plasmids encompassing positions bp -552 to +12 of the BZLF1 promoter were constructed and tested for their response to anti-immunoglobulin (anti-Ig), an inducer of the viral lytic cycle, upon transfection into EBV-negative and -positive lymphoid cells. The promoter consisted of three functionally distinct regions. Region I (bp -552 to -221) had a negative influence on promoter activity; its deletion made the promoter highly responsive to anti-Ig. Region II (bp -203 to -177) was important for conferring responsiveness to anti-Ig. The response to anti-Ig did not require the presence of the EBV genome or EBV gene products. This sequence also enhanced expression of the chloramphenicol acetyltransferase (cat) gene from the simian virus 40 promoter in response to anti-Ig, even when inserted downstream of the cat gene. Region III (-134 to -116) was a positive element that was transactivated by the BZLF1 gene product.
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PMID:Analysis of the BZLF1 promoter of Epstein-Barr virus: identification of an anti-immunoglobulin response sequence. 838 98

The human CD22 gene is expressed specifically in B lymphocytes and likely has an important function in cell-cell interactions. A nearly full length human CD22 cDNA clone was used to isolate genomic clones that span the CD22 gene. The CD22 gene is spread over 22 kb of DNA and is composed of 15 exons. The first exon contains the major transcriptional start sites. The translation initiation codon is located in exon 3, which also encodes a portion of the signal peptide. Exons 4 to 10 encode the seven Ig domains of CD22, exon 11 encodes the transmembrane domain, exons 12 to 15 encode the intracytoplasmic domain of CD22, and exon 15 also contains the 3' untranslated region. A minor form of CD22 mRNA likely results from splicing of exon 5 to exon 8, skipping exons 6 and 7. A 4.6-kb XbaI fragment of the CD22 gene was used to map the chromosomal location of CD22 by fluorescence in situ hybridization. The hybridization locus was identified by combining fluorescent images of the probe with the chromosomal banding pattern generated by an Alu probe. The results demonstrate that CD22 is located within the band region q13.1 of chromosome 19. Two closely clustered major transcription start sites and several minor start sites were mapped by primer extension. Similarly to many other lymphoid-specific genes, the CD22 promoter lacks an obvious TATA box. Approximately 4 kb of DNA 5' of the transcription start sites were sequenced and found to contain multiple Alu elements. Potential binding sites for the transcriptional factors NF-kappa B, AP-1, and Oct-2 are located within 300 bp 5' of the major transcription start sites. A 400-bp fragment (bp -339 through +71) of the CD22 promoter region was subcloned into a pGEM-chloramphenicol acetyltransferase vector and after transfection into B and T cells was found to be active in both B and T cells. Further studies of the CD22 gene should lead to a greater understanding of the expression of CD22 during B cell development and differentiation.
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PMID:Genomic structure and chromosomal mapping of the human CD22 gene. 849 2


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