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 rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Bandshifting and DNase-I footprinting experiments using this region of the RII beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.
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PMID:Identification and characterization of the GC-rich and cyclic adenosine 3',5'-monophosphate (cAMP)-inducible promoter of the type II beta cAMP-dependent protein kinase regulatory subunit gene. 131 46

The 5'-flanking region of the human lactoferrin gene was isolated from a human placental genomic library. This genomic clone contains a 16-kilobase pair (kbp) insert and produces seven fragments when digested with the SacI restriction enzyme. We sequenced one of the fragments that comprises 1294 bp of the 5'-flanking sequence, 79 bp of the first exon, and 690 bp of the first intron. A major transcription start site was mapped by primer extension. The region immediately upstream from the transcription initiation site following the first exon is abundant in G and C nucleotides. In the promoter and 5'-flanking region within a 300-bp stretch (-465 to -165) of the DNA, we found a noncanonical TATA box (ATAAA), CAAT-like sequence (CAAC) and sequences homologous to the consensus SP1 binding site, Pu.1/Sp.1 binding element (PU box), two half-palindromic estrogen response elements (EREs; GGTCA), an imperfect ERE (GGTCAAGGCGATC), and a sequence resembling the chicken ovalbumin upstream promoter transcription factor (COUP-TF) binding site (GTCTCACAGGTCA). The COUP-TF binding site and the imperfect ERE shared five nucleotides (GGTCA). With the exception of the two half-palindromic EREs, the elements with very well matched sequences were also found in the corresponding positions in the mouse lactoferrin gene. The synthetic oligonucleotide, including the 26 bp of COUP/ERE sequence, was cloned before the SV40 promoter in a chloramphenicol acetyltransferase reporter construct. These chimeric plasmids were transiently transfected into human endometrium carcinoma RL95-2 cells to assess hormone responsiveness. We found that the COUP/ERE element acted as an enhancer in response to estrogen stimulation. In vitro DNase I footprinting analysis showed binding of the estrogen receptor on the imperfect ERE. In contrast to the mouse lactoferrin COUP/ERE element, COUP-TF does not interact with this element, as demonstrated by band shift assay and site-directed mutagenesis. Therefore, the molecular mechanisms of the estrogen action that govern the lactoferrin gene expression differ between mouse and human.
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PMID:Differential molecular mechanism of the estrogen action that regulates lactoferrin gene in human and mouse. 148 Jan 83

Sequencing studies have indicated that the unique component of the human herpesvirus 6 (HHV-6) genome and the unique long segment of the human cytomegalovirus genome are genetically colinear. Of particular interest is the identification of a region of local CpG dinucleotide suppression in the genome of HHV-6, a feature conserved in the genomes of human cytomegalovirus, murine cytomegalovirus, and simian cytomegalovirus, and a characteristic of the major immediate-early loci of these viruses. Adjacent to this region in HHV-6 are approximately 30 copies of a 103- to 108-bp sequence element, which contains consensus binding sites for the transcription factors AP2 and NF kappa B, in addition to a single KpnI recognition site. Together, these KpnI repeat units may compose an immediate-early enhancer, analogous to those found in the cytomegaloviruses. We present the sequence of this region of HHV-6 and demonstrate that a transactivating function is encoded by this region. We have used polymerase chain reaction to synthesize fragments containing open reading frames and 5' sequences with or without the upstream KpnI repeat units. Effector plasmids containing these HHV-6 coding and 5' sequences were able to effect activation of heterologous promoter-chloramphenicol acetyltransferase (CAT) constructs, including adenovirus E3-CAT and E4-CAT, human T-cell lymphotropic virus type I long terminal repeat (LTR)-CAT, and human immunodeficiency virus LTR-CAT, in cotransfection experiments in Vero cells and peripheral blood lymphocytes. Furthermore, we have identified the major open reading frame (RF4; 2.3 kb) as being essential for activation, and we have shown that the NF kappa B, SP1, and TATA box motifs in the human immunodeficiency virus LTR are all required for full induction of the promoter by the HHV-6-encoded transactivator.
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PMID:Identification of a transactivating function mapping to the putative immediate-early locus of human herpesvirus 6. 165 46

The mouse Ha-ras oncogene is activated by point mutation and overexpressed in developing papillomas during two-stage skin carcinogenesis in SENCAR mice. One of our research aims is to characterize the factors regulating Ha-ras gene expression at the transcriptional level in SENCAR mouse epidermis. Towards this goal, we sequenced 1400 bp of the 5' upstream region of the mouse Ha-ras gene so as to characterize various cis-regulatory elements present in the gene. We identified seven sites with the proper consensus sequence for binding the SP1 transcription factor and three potential binding sites for the CTF-1 factor. In addition, we located a 13-base sequence with 92% homology to the consensus sequence for an estrogen response element and two hexamers with consensus sequences identical to the core sequence of the glucocorticoid response element. A series of transient gene expression vectors was constructed in which various regions of the mouse Ha-ras 5' upstream region were fused to the chloramphenicol acetyltransferase (CAT) gene. These expression plasmids were transfected into newborn and adult primary SENCAR epidermal cells, the epidermal cell population that presumably contains the stem cells involved in two-stage skin tumorigenesis. Transient gene expression assays carried out after 48-72 h indicated that a 2.3-kb Ha-ras 5' fragment produced CAT activity comparable to that produced by pSV2CAT and pdolCMVCAT, both of which are plasmids with strong viral promoters and enhancers driving CAT gene expression. Maintenance of transfected keratinocytes under both nondifferentiating (0.05 mM calcium) and differentiating (1.2 mM calcium) culture conditions demonstrated that the mouse Ha-ras upstream region was relatively unresponsive to changes in calcium concentration in transient expression assays carried out in either newborn or adult keratinocytes. Our results demonstrated the power of the cloned mouse Ha-ras promoter and upstream region in driving transient gene expression after transfection into primary keratinocytes.
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PMID:Transient expression of the cloned mouse c-Ha-ras 5' upstream region in transfected primary SENCAR mouse keratinocytes demonstrates its power as a promoter element. 191 Apr 81

Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes. 212 73

Insulin-like growth factor I (IGF-I) and insulin regulate expression of the endogenous delta 1-crystallin gene in embryonic lens cells that express receptors for both peptides. To further analyze the transcriptional component of this hormonal effect, transient transfections of lens cells were prepared with DNA constructs containing deletions of the delta 1-crystallin promoter and the chloramphenicol acetyltransferase reporter gene. A 77-nucleotide DNA segment of the delta 1-crystallin promoter from nucleotide positions-120 to -43 confers sensitivity to insulin and IGF-I. The hormonal effect is dose-dependent, and maximal stimulation of promoter activity (2- to 2.5-fold induction) is obtained with 10(-8) M IGF-I and 10(-7) M insulin. Mobility-shift DNA-binding analysis shows specific binding of nuclear protein(s) to the delta 1-crystallin promoter DNA between positions -120 and +23, which appears to be regulated by IGF-I. An SP1-binding motif is involved in this DNA-protein interaction. The bivalent IgG fraction of an anti-insulin receptor antiserum (B-10), known to mimic insulin action in other systems, stimulates promoter activity to the same extent as insulin.
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PMID:Transcriptional stimulation of the delta 1-crystallin gene by insulin-like growth factor I and insulin requires DNA cis elements in chicken. 218 66

A group of five cDNA clones, representing the gadd genes, were recently isolated from Chinese hamster ovary (CHO) cells as genes induced upon growth arrest and after DNA damage (Fornace, A. J., Jr., Nebert, D. W., Hollander, M. C., Luethy, J. D., Papathanasiou, M., Fargnoli, J., and Holbrook, N. J. (1989) Mol. Cell. Biol. 9, 4196-4203). We have isolated and characterized one of these genes, gadd153. The gene spans five kilobases and contains four exons. The 5'-flanking region of the gene, within 420 base pairs of the transcription initiation site, contains a number of cis elements associated with transcriptional regulation in other genes. These include a Hogness box, ATAAAA, an inverted GCCAAT box; seven SP1 transcription factor binding sites, and an AP-1 site. This region is rich in G + C content (greater than 70%) and contains an unusually long stretch of alternating CpG residues. The 800-base pair region immediately upstream of the transcription start site can drive expression of the bacterial chloramphenicol acetyltransferase (CAT) gene, but only in its endogenous orientation, in three different cell lines: HeLa, CHO, and Jurkat. The gadd153 promoter is strongly activated by methyl methanesulfonate, hydrogen peroxide, and UV irradiation, but not by growth arrest signals. This suggests that separate and very different regulatory pathways are involved in the induction of the gadd153 gene by growth cessation and DNA damage.
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PMID:Isolation and characterization of the hamster gadd153 gene. Activation of promoter activity by agents that damage DNA. 239 62

Herpes simplex virus type 1 (HSV-1) and some of its immediate-early genes stimulate expression of the human immunodeficiency virus (HIV) long terminal repeat (LTR) sequences and the replication of HIV itself. To demonstrate this, the HIV LTR was linked to the indicator gene chloramphenicol acetyltransferase (CAT) and transfected into Vero cells with or without the trans-activating gene (tat) of HIV. Infection of these cells with HSV-1 strain KOS or temperature-sensitive mutant tsB21 or tsE6 resulted in a large increase in CAT activity in the absence of tat and further augmentation in the presence of tat. This stimulation was seen at both their permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures, implying either that HSV-1 infection or immediate-early gene expression is all that is required. In cotransfection assays in Vero cells, cloned HSV-1 immediate-early genes ICP0 and ICP4 stimulated CAT activity in the presence of tat, while ICP27 had no effect. On the other hand, in SW480 cells, ICP4 and, to a lesser extent, ICP0 genes caused stimulation of CAT activity in the absence of tat. Deletion mutants within the HIV LTR showed that the target for HSV stimulation is distinct from the tat-responsive area and maps near the SP1 binding sites. In Hela cells, ICP0 or ICP4 stimulated the replication of a cotransfected clone of HIV, as shown by an increase in reverse transcriptase activity in the culture supernatant.
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PMID:Activation of the human immunodeficiency virus by herpes simplex virus type 1. 244 5

To understand the basis of osteonectin (SPARC) transcriptional regulation, we have isolated a bovine genomic clone (lambda Og15) encoding exon 1 and 15 kilobase pairs (kb) of flanking DNA. Direct RNA sequencing of the 5' end of the osteonectin message showed it contained a sequence identical to that of a 2.4-kb EcoRI-BamHI fragment located midway in the clone lambda Og15. The results indicate exon 1 is located 10 kb away from exon 2 in the bovine genome. The DNA sequence unit CCTG is repeated five times in exon 1 which is composed exclusively of untranslated sequence. Sequence analysis of the 5'-flanking DNA revealed the presence of many regulatory motifs including a "GC" box with four overlapping SP1 consensus sequences. Immediately downstream from the GC box is a 72-base pair purine-rich stretch composed primarily of direct repeats of the sequence motifs GGGGA and GGA (GAGA box). Digestion of the flanking DNA in vitro with S1 endonuclease showed a site for the enzyme at position -55 which is just 3' to the GAGA box. Chimeric chloramphenicol acetyltransferase constructs were prepared containing the S1-sensitive site and showed substantial transcriptional activity in UMR-106 and fetal and adult human bone cells which are known to be high producers of the protein. The results indicate a potential regulatory activity of the S1 site in osteonectin gene activation.
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PMID:Osteonectin promoter. DNA sequence analysis and S1 endonuclease site potentially associated with transcriptional control in bone cells. 253 44

Tyrosine hydroxylase (TH) is selectively expressed in catecholaminergic neurons and in chromaffin cells of the adrenal medulla. Constructs in which 5' flanking sequences of the rat TH gene directed expression of bacterial chloramphenicol acetyltransferase (CAT) were transfected into cell lines and assayed for transient expression of CAT. In most nonexpressing cell lines, CAT levels were less than 5% of that found in a TH-positive pheochromocytoma line (PC8b). In two lines described here, a rat anterior pituitary cell line (GH4) and a rat fibroblast line (Fr3T3), CAT expression reached 12 and 20%, respectively, of the PC8b level. Greater than 90% of the PC8b activity was lost when sequences between -212 and -187 (in relation to the transcriptional initiation site) were deleted. Further deletions that removed the cyclic AMP response element (CRE) (-45) and the TATA box at -29 reduced transcriptional activity to background in all three lines. These data suggest that 212 nucleotides of the 5' sequence are sufficient for pheochromocytoma expression and that information between -212 and -187, which includes an AP1 site (-206 to -200), is essential for full transcriptional activity. In addition, sites for other protein transcription factors (AP2, POU/Oct, SP1, and CRE) reside between -221 and -38 and are largely conserved between the human and rat gene.
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PMID:5' flanking DNA sequences direct cell-specific expression of rat tyrosine hydroxylase. 257 79


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