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)

Glucosidase I initiates the processing of asparagine (N-) linked glycoproteins by removing the distal alpha1,2-linked glucosyl residue of the tetradecasaccharide Glc(3)Man(9)GlcNAc(2). The gene encoding this enzyme was isolated and its structural organization and promoter activity determined. The major transcript for glucosidase I on northern blot appeared to be 3.1 kb; Southern blotting and DNA sequencing indicated the size of the gene to be 6.8 kb, comprising four exons separated by three introns. The first exon encodes the cytoplasmic tail and transmembrane domain; the fourth encodes the putative catalytic domain of the enzyme. Exon-intron junctions are flanked by consensus splice donor and acceptor sequences. Transcription initiation sites were mapped by primer extension, ribonuclease protection assay and RT-PCR analysis. Primer extension results showed multiple initiation sites at -150, -156, and -272 bp relative to the translation initiation codon ATG. Sequence analysis of 5' flanking region showed no canonical TATA box, a high GC content, Sp1 and ETF binding sites (typical of a housekeeping gene promoter). Also noteworthy, the promoter region contains several generic STAT factor binding sites, one nearly perfect, and two half GR binding elements. Other cis- acting elements recognized by transcription factors such as AP-2, NF-kappaB, estrogen receptor, and progesterone receptor (PR) were also present in the putative promoter region. To determine the promoter activity, a construct encompassing the region between -2114 to -5 bp of the putative promoter was ligated to the chloramphenicol acetyltransferase (CAT) reporter plasmid and transiently transfected into COS 7 cells. CAT assay results clearly show transcriptional activity of the promoter.
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PMID:Genomic organization and promoter activity of glucosidase I gene. 1040 45

The T1alpha gene is one of few markers for the type I cell phenotype in the adult mammalian lung. Type I cells form a large, thin epithelial layer that facilitates gas exchange and transport of fluids between the air spaces and capillaries. The T1alpha gene has a complex pattern of developmental expression in lung and brain; in vitro studies indicate that expression is regulated in part by thyroid transcription factor 1, forkhead proteins, and Sp1/Sp3 proteins. To explore the mechanisms that confine T1alpha expression in intact adult animals to alveolar type I and choroid plexus epithelial cells, we generated mice bearing a 1.3-kb T1alpha promoter-chloramphenicol acetyltransferase (CAT) gene. In situ hybridization and RNase protection assays show that the 1.3-kb promoter confers a pattern of CAT expression that largely matches the endogenous T1alpha in embryos and mid-term fetuses in lung and central nervous system. However, the 1.3-kb promoter lacks elements important for perinatal up-regulation of T1alpha in the lung and maintenance of that expression in the adult lung and brain. The final adult pattern of T1alpha expression may be directed by elements outside the 1.3-kb fragment, perhaps those 5' to the 1.3-kb fragment as we show herein, or in 3' and intronic regions. Dev Dyn 1999;215:319-331.
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PMID:1.3 kilobases of the lung type I cell T1alpha gene promoter mimics endogenous gene expression patterns during development but lacks sequences to enhance expression in perinatal and adult lung. 1041 21

A lambdaEMBL3 rat genomic library was screened to clone a phage that contained the promoter region of the kidney-type mitochondrial glutaminase gene. The resulting lambdaGA1 phage contained 13.7 kb of genomic DNA that was mapped by Southern blotting and restriction analysis. The 2.22 kb and 0.83 kb SacI fragments of lambdaGA1 were sequenced and the transcription initiation site was identified by RNase mapping. The reported sequence contains 2287 bp of the promoter, the entire exon 1 (542 bp), and 223 bp of the initial intron of the glutaminase gene. The initial exon contains 141 bp of 5'-nontranslated sequence and 401 bp of coding sequence that encodes the 72-amino acid mitochondrial targeting presequence and 61 amino acids from the N-terminus of the mature 66 kDa glutaminase subunit. Various segments of the GA promoter were cloned into a chloramphenicol acetyltransferase (CAT) expression vector. The resulting GA-CAT constructs were transfected into LLC-PK(1)-F(+) kidney cells to assess the promoter function of the isolated genomic DNA. The GA(-402)CAT construct produced a 10-fold greater CAT activity than the promoter-less pCAT vector. Analysis of various deletion constructs indicated that elements located between -402 and -63 bp must act in synergy with more proximal elements to create a functional promoter. The initial 402 bp segment lacks a TATA sequence but is GC-rich and contains two CCAAT boxes and two Sp1 sites.
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PMID:Isolation and characterization of the promoter region of the rat kidney-type glutaminase gene. 1126 68

Although the primary function of U1 snRNA is to define the 5' donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5' donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], beta-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3' cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.
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PMID:Reduction of target gene expression by a modified U1 snRNA. 1128 60

The human folate receptor (FR) is overexpressed in ovarian carcinoma. FR transcripts are heterogeneous due to the use of two promoters, P1 and P4, and alternative splicing of exon 3. RNase protection assay and RT-PCR revealed higher levels of the transcripts that include exon 3 in lines and specimens from ovarian carcinoma. A P1-chloramphenicol acetyltransferase (CAT) construct containing exon 3 demonstrated efficient reporter expression only in ovarian carcinoma. 5' and 3' deleted variants of the P1-CAT construct were analyzed by RT-PCR of the exogenous transcripts and reporter activity. A 5' splice site and 35 bp downstream intronic region of exon 3 appeared to regulate enhanced FR expression in ovarian carcinoma.
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PMID:Exon 3 of the alpha folate receptor gene contains a 5' splice site which confers enhanced ovarian carcinoma specific expression. 1147 43

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences.
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PMID:Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites. 1156 76

Human immunodeficiency virus type 1 (HIV-1) replication requires active nuclear export of unspliced and incompletely spliced HIV-1 RNA transcripts. This process is evolutionally made possible by expression of HIV-1 Rev, one of the three HIV-1 proteins encoded by completely spliced HIV-1 RNAs. Evidence has accumulated to suggest that Sam68 plays an important role in HIV-1 replication through HIV-1 Rev protein. In the present study, we further examined the structure-function relationship of Sam68 protein in relation to HIV-1 replication. We identified a Sam68 domain located between aa269 and aa321 to be involved in the HIV-inhibitory effects of Sam68 dominant negative mutants lacking the nuclear localization signal (NLS). Deletion of this domain abrogated inhibition of HIV-1 replication by these mutants. HIV-1 Rev protein appeared to mediate the HIV-inhibitory effects of these mutants and by this domain, as assessed by Rev-dependent chloramphenicol acetyltransferase reporter gene assay, in trans rev-defective HIV-1 complementation assay, and RNase protection assay. The HIV-inhibitory mutants containing this domain were further found to have diminished binding affinity to the wild-type Sam68 and to be associated with cytoplasmic retention of exclusively nuclear localized wild type Sam68. Taken together, these results further ascertain the important role of Sam68 in HIV-1 Rev function and viral replication, and suggest that the HIV-inhibitory effects of Sam68 dominant negative mutants directly result from their binding to endogenous Sam68 and their interference with nuclear localization of endogenous Sam68.
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PMID:Requirement of an additional Sam68 domain for inhibition of human immunodeficiency virus type 1 replication by Sam68 dominant negative mutants lacking the nuclear localization signal. 1623 70


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