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)

The 5'-flanking region of protein kinase C (PKC) gamma gene was identified from a rat liver genomic library in a bacteriophage lambda Charon 4A. A 3.6-kilobase (kb) genomic fragment containing the 5'-flanking region, first exon, and first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 mapping and primer extension, was located 243 base pairs upstream from the translational initiation site. Promoter activity of a DNA segment spanning the 5'-flanking region was demonstrated by both in vitro transcription using HeLa cell nuclear extracts and chloramphenicol acetyltransferase assay by transfection of 293 cells with a PKC gamma-CAT fusion construct. Chloramphenicol acetyltransferase assay revealed that a fragment of about 0.16 kb from the transcriptional initiation site was sufficient for promoter activity in these cells, and the construct containing up to 1.6 kb from the cap site was expressed at a similar level. This promoter-active fragment contains several regions similar to defined transcriptional elements in other mammalian promoters, such as those for stimulatory protein 1 (Sp1), activator proteins 1 and 2 (AP1, AP2), c-myc, cAMP regulatory element-binding protein (CREB), and enhancer core (EnhC). Investigation of the genomic structure of PKC gamma gene may lead to the identification of cis-elements controlling tissue-specific and developmental stage-specific expression of PKC gamma.
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PMID:Characterization of the 5'-flanking region of the rat protein kinase C gamma gene. 224 72

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.
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PMID:Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32. 254 59

We have identified a positive modulator within the c-myc first exon downstream of the gene's transcription initiation sites, P1 and P2. We introduced myc-CAT (chloramphenicol acetyltransferase) hybrid genes into three cell lines (BJAB, COS and HeLa) and measured their expression by either CAT enzymatic activity, S1 nuclease protection or by a nuclear 'run-on' transcription assay. Removal of 46 bp from the 3' end of the first exon results in a decrease of myc-CAT expression and P2 activity. A 438-bp exon 1 segment, lacking the normal myc promoters, efficiently drives the expression of SV40 early promoters. We find that this first exon segment efficiently functions as a positive modulator only in its sense orientation, 3' of a nearby promoter. The positive effects of the myc first exon and the SV40 enhancer are complementary.
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PMID:The first exon of the c-myc proto-oncogene contains a novel positive control element. 303 Jul 32

Recombinant plasmids of the human c-myc promoter-leader region and the bacterial chloramphenicol acetyltransferase (cat) gene were constructed. After transfection into different rodent and human cells, the 862-base-pair (bp) PvuII fragment carrying both c-myc promoters and 350 bp of the untranslated leader conferred 1/15 to 1/30 of the CAT activity mediated by the simian virus 40 promoter. The presence of additional sequences upstream of the PvuII fragment had an overall negative effect on c-myc promoter activity detectable by titration analysis with small amounts of transfected plasmid DNA. The analysis of numerous deletion constructs in the c-myc promoter-leader region as well as S1 mapping experiments demonstrated that the high CAT activity depended largely on the presence of the second promoter. By cotransfection of c-myc-cat constructs with plasmids carrying different parts of the c-myc promoter locus, targets for positively acting cellular factors were identified. Two positive regulatory elements were mapped within the 862-bp PvuII fragment. One was localized within the 248-bp PvuII-SmaI fragment -101 to -349 bp upstream of the first cap site and the other within the 142-pb XhoI-NaeI fragment of the first exon, comprising positions -95 to +47 relative to the second cap site. We conclude that the dual promotor of the human c-myc gene represents a strong eucaryotic promotor regulated by cooperation of positively and negatively acting cellular transcription factors.
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PMID:Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene. 303 16

The involvement of c-myc in the genesis of animal neoplasia is now well documented for several systems. In order to define the precise role played by the myc gene in tumorigenesis, a better understanding of the normal regulation of myc expression is necessary. We have begun a study of the cis-acting regulatory sequences within the 5' flanking domain of the human c-myc gene. Regions important for myc promoter function have been identified by linkage to the coding sequences of the bacterial chloramphenicol acetyltransferase (cat) gene. Promoter deletion studies and in vivo competition assays for c-myc/cat recombinant plasmids have allowed the identification of a proximal 'core' promoter region capable of directing high levels of CAT activity. Further upstream a negative regulatory element (NRE2) has been identified which is capable of repressing cat gene expression and which functions by interaction with a transacting factor(s). Preliminary data suggests detection of NRE2 is dependent on both the type and amount of carrier DNA used in transient CAT assays. Initial experiments further indicate the involvement of at least two other distal regulatory domains, a negative regulatory domain (NRE1) and a putative enhancer-type region (E). In vitro footprint analysis has allowed the identification of DNA binding proteins which interact with NRE2 and the 'core' promoter. NRE2 contains binding sites for transcription factors Sp1 and CTF. The 'core' promoter domain appears to be highly complex and possesses several Sp1 binding sites.
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PMID:Transcriptional regulation of the human c-myc gene. 307 67

The translational efficiency of chloramphenicol acetyltransferase (CAT) mRNA containing a 5' noncoding sequence derived from exon 1 of the murine c-myc gene (360CAT) has been examined at different stages of Xenopus egg development. In contrast to its reduced translation in the Xenopus oocyte, 360CAT mRNA is translated as efficiently as CAT mRNA when injected into either mature Xenopus eggs or Xenopus embryos. No significant alteration of 360CAT mRNA stability was observed up to 10 h post-fertilization in Xenopus embryos as compared to that of CAT mRNA. The increase in 360CAT mRNA translational efficiency in Xenopus embryos was not observed with CAT mRNAs possessing other inhibitory 5' noncoding sequences. The increase in 360CAT mRNA translational efficiency is attributed to a trans-acting factor synthesized or activated following Xenopus oocyte maturation. The possible significance of the 5' noncoding region of c-myc mRNA in developmental expression of c-myc is discussed.
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PMID:Developmental regulation of translation by the 5' noncoding region of murine c-myc mRNA in Xenopus laevis. 307 57

In transformed NIH 3T3 cells, murine gamma interferon reduces the expression of the long terminal repeat-controlled oncogenes v-mos, c-myc, and v-Ha-ras by a direct effect on the activity of retroviral promoters, as revealed by analyses of RNA half-life and transcriptional activity of retroviral genes as well as by analyses of chloramphenicol acetyltransferase activity in cells transformed with the cat gene under the control of long terminal repeats.
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PMID:Gamma interferon regulates long terminal repeat-controlled oncogene expression in transformed mouse fibroblasts at the level of mRNA transcription. 312 65

We have used a competition assay to identify the targets of trans-acting elements that modulate the expression of the human c-myc gene (designated MYC in human gene nomenclature). For this purpose, a c-myc hybrid indicator gene was formed by joining the c-myc promoter region, first noncoding exon, and intron to the bacterial gene for chloramphenicol acetyltransferase (CAT). The test assay consisted of cotransfecting the indicator gene with competing fragments of DNA derived from suspected control regions of the c-myc gene. Such experiments test the hypothesis that control regions are often targets for the binding of trans-acting regulatory factors that can be diverted to competing fragments of DNA. A negatively acting element will be diverted from the indicator gene, allowing the gene's enhanced expression, whereas a positively acting element will behave oppositely. Control indicator genes driven by non-myc promoters assess the specificity of the effect. Using this approach, we find three c-myc regions that are capable of enhancing the expression of the indicator gene in competition assays (i.e., putative sites of negative modulation). In addition, we find sequences near the c-myc promoters that suppress expression in competition assays (i.e., putative binding sites of positively acting factors). These results, with appropriate controls, suggest the existence of target sites near the c-myc gene that specifically modulate its expression both positively and negatively. Their locations fit well with regions damaged or lost in many Burkitt lymphoma and murine plasmacytoma translocations.
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PMID:Trans-acting elements modulate expression of the human c-myc gene in Burkitt lymphoma cells. 346 6

Previous work demonstrated that a herpes simplex virus type 1 (HSV-1) immediate-early function up-regulates beta interferon but not chloramphenicol acetyltransferase reporter genes driven by the strong simian virus 40 (SV40) or cytomegalovirus promoter-enhancer regions in both transient assays and stable cell lines. The different 3' mRNA stabilization and RNA-processing signals from these two reporter genes appeared to be primarily responsible for this phenomenon. We now report that the HSV-1 ICP27 itself is sufficient to stimulate both steady-state accumulation and increased half-life of beta interferon reporter gene mRNA. Furthermore, the ability to respond directly to cotransfected ICP27 can be transferred to chloramphenicol acetyltransferase reporter genes by replacement of their SV40-derived splicing and poly(A) signals with the 3' AU-rich and poly(A) RNA-processing signals from the normally highly labile beta interferon and c-myc mRNA species. ICP27 expressed in bacteria bound specifically to in vitro-generated RNA from both the beta interferon and c-myc intronless AU-rich 3' RNA-processing regions, but not to the SV40-derived early-region splice signal and poly(A) sequences. By site-specific mutagenesis, we also show that individual ICP27 C-terminal amino acid residues that are positionally conserved in ICP27 homologs in other herpesviruses (D-357, E-358, H-479, C-400, C-483, and C-488) are critical for trans-regulatory activity. Importantly, several of these positions match mutations that are known to be essential for the role of ICP27 in the early-to-late switch during the virus lytic cycle. Therefore, our findings support the notion that HSV ICP27 modulates gene expression posttranscriptionally in part by targeting RNA.
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PMID:Herpes simplex virus trans-regulatory protein ICP27 stabilizes and binds to 3' ends of labile mRNA. 747 40

Proliferation of LNCaP 104-S cells, a clonal subline of the human prostate cancer cell line, was very slow in androgen-depleted medium but increased 10-13-fold in the presence of 0.1 nM of a synthetic androgen, R1881. This induction of proliferation was diminished at higher concentrations of R1881, indicating the biphasic nature of the androgen effect. After 20-30 passages in androgen-depleted medium, these cells progressed to 104-I cells, which exhibited much lower proliferative sensitivity to 0.1 nM R1881. After another 20-30 passages, LNCaP 104-I cells gave rise to 104-R cells, which proliferated rapidly without additional androgen. Proliferation of 104-R cells was induced 2-fold by 0.01 nM R1881 but was repressed by 0.1 nM R1881 and above. Thus, androgen induction and repression of proliferation could be seen at lower concentrations of androgen as the cells progressed. During the transition of 104-S cells to 104-R cells, the androgen receptor mRNA level increased 2.5-fold whereas the androgen receptor protein level increased 15-fold in the absence of androgen. Androgen receptor transcriptional activity, measured by androgen induction of prostate-specific antigen mRNA and chloramphenicol acetyltransferase activity in transfected cells, increased up to 20-fold during the progression. LNCaP cells, therefore, appear to be able to adapt to reduced androgen availability by increasing their sensitivity to androgen, raising questions concerning the therapeutic strategies used against prostate cancer. Androgen induction of c-myc expression in 104-R cells occurred at a 10-fold lower concentration (0.01 nM) than in 104-S cells (0.1 nM). In all stages, cell proliferation and c-myc expression were repressed by androgen at a high concentration (20 nM), but the repression of cell proliferation was blocked by retroviral overexpression of c-myc.
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PMID:Increased androgen receptor activity and altered c-myc expression in prostate cancer cells after long-term androgen deprivation. 751 Oct 45


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