Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat.

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.     

Mapping of the late promoter of simian virus 40.

Mapping of the simian virus 40 (SV40) late promoter was carried out in the absence of the viral early protein, large tumor (T) antigen, and replication of the viral DNA template. SV40 late control region DNA fragments, containing specific deletions, were cloned directly upstream from the coding region of the herpes simplex virus-1 (HSV-1) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (tk). The promoter activities of the fragments were determined by measuring the tk transformation frequencies of the chimeric tk constructs in mouse L TK- APRT- (adenine phosphoribosyltransferase-negative) and human 143B TK- cell lines. The following results were obtained. (i) The SV40 control region functions with equal efficiencies in the early and late promoter orientations. (ii) A major late control element has been localized within the G+C-rich 21-base-pair (bp) repeat. Thus, in conjunction with our earlier results, the 21-bp repeat is a bidirectional promoter element functioning as a major component of both the early and late promoters and is an element that enhances the replication efficiency of SV40 DNA. (iii) Minor late promoters have been localized within the minimal replication origin and the 72-bp repeat. (iv) The minimal replication origin is not per se a constituent of the major late promoter; however, both the minimal replication origin and the 21-bp repeat are required for obtaining high levels of late gene expression observed at late times after infection by SV40. (v) The 72-bp repeat exerts a 4- to 5-fold enhancement of late promoter expression.     

Neuron-specific expression in vivo by defined transcription regulatory elements of the GnRH gene

The GnRH-expressing neurons are the ultimate regulator of reproductive function. GnRH gene expression is limited to this small population of neurons in the hypothalamus. Transfections using 3 kb of the rat or mouse 5'-regulatory region provide specific gene expression in the hypothalamic cell line GT1-7. The combination of two elements, a 300-bp enhancer and a 173-bp promoter, recapitulates specificity in GT1-7 cells. It was not known whether these elements could specifically target gene expression throughout development in the whole animal. We demonstrate that the 3-kb rat GnRH regulatory region provides a higher degree of specificity than the equivalent mouse sequence in a mouse hypothalamic cell line. Moreover, combination of the enhancer and the promoter of the rat gene targets expression to GnRH neurons in transgenic mice in a developmentally appropriate manner. Transgene expression is regulated by activin A, a known activator of GnRH gene expression. In contrast, the enhancer on a heterologous promoter produces inappropriate expression in vivo. We conclude that the enhancer and promoter regions of the rat GnRH gene are necessary for targeted expression to hypothalamic neurons and are sufficient to confer regulated, cell type-specific expression to a reporter gene in vivo.     

Genomic structure and chromosomal localization of the human deoxycytidine kinase gene.

Deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have isolated genomic clones encompassing its entire coding and 5' flanking regions and delineated all the exon/intron boundaries. The gene extends over more than 34 kilobases on chromosome 4 and the coding region is composed of 7 exons ranging in size from 90 to 1544 base pairs (bp). The 5' flanking region is highly G+C-rich and contains four regions that are potential Sp1 binding sites. A 697-bp fragment encompassing 386 bp of 5' upstream region, the 250-bp first exon, and 61 bp of the first intron was demonstrated to promote chloramphenicol acetyltransferase activity in a T-lymphoblast cell line and to have > 6-fold greater activity in a Jurkat T-lymphoblast than in a Raji B-lymphoblast cell line. Our data suggest that these 5' sequences may contain elements that are important for the tissue-specific differences in deoxycytidine kinase expression.     

Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons.     

S-phase-specific regulation by deletion mutants of the human thymidine kinase promoter.

The levels of thymidine kinase (TK; EC 2.7.1.21) mRNA were determined in nine established cell lines derived from TK-ts13, a temperature-sensitive mutant cell line that arrests in late G1 phase of the cell cycle at the restrictive temperature. The derivative cell lines carried either a cDNA or a minigene of human TK under the control of TK promoters of different lengths. A tenth cell line carried a human TK cDNA under the control of a simian virus 40 promoter. Two different assays were used to determine the S-phase-specific regulation of human TK mRNA levels in quiescent cells stimulated to proliferate. Results from these two assays indicated that (i) the first two introns of the human TK gene had no effect on the S-phase-specific regulation of TK mRNA levels, although the presence of introns increased the amount of TK mRNA; (ii) similar amounts of TK mRNA were present in cells containing constructs with an 83-base-pair (bp) promoter as with other TK promoters comprising up to approximately 4000 bp of 5' flanking sequence; (iii) a 456-bp promoter was fully S-phase-regulated, whereas the 83-bp promoter was only partially regulated; (iv) a 63-bp promoter was much less regulated than an 83-bp promoter; and (v) the crucial element in the 20-bp fragment comprising bp -83 to -64 has been localized, by site-directed mutagenesis, to the CCAAT element at -70.     

Synergistic action of upstream elements and a promoter-proximal CRE is required for neuroendocrine cell-specific expression and second-messenger regulation of the gene encoding the human secretory protein secretogranin II

Secretogranin II (SgII) is a secretory polypeptide stored in large dense core vesicles of neuroendocrine and neuronal cells. In order to characterize the molecular mechanisms underlying the tissue-specific expression of the SgII gene and its regulation by second-messenger pathways in endocrine and neuronal cells, we have cloned and characterized the human SgII gene. Sequence analysis revealed the existence of numerous putative cis-regulatory elements in the SgII gene promoter, including a consensus cyclic AMP-responsive element (CRE). Constructs containing different portions of the human SgII promoter fused to the luciferase reporter were transfected in AtT-20, SH-SY5Y, LLC-PK1 or COS-7 cells. Northern blot analysis showed that the endogenous SgII gene is more highly expressed in AtT-20 cells than in SH-SY5Y cells, and not expressed at all in LLC-PK1 cells. Treatment by forskolin or 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a 1.5- and 10-fold increase, respectively, in SgII mRNA levels in SH-SY5Y cells but not in AtT-20 cells. Transfection experiments revealed that 4 kb of the human SgII promoter is sufficient to impart cell-specific expression of the reporter gene in the four cell lines studied. Specifically, in AtT-20 cells, a positive element located between -1.38 and -4 kb, in addition to the CRE, is responsible for the high expression of the SgII gene. In SH-SY5Y cells, a negative element located between -0.66 and -1.4 kb represses the activating effect of the CRE leading to an overall lower activity of fusion genes in these cells compared to the activity in AtT-20 cells. Finally, the promoter activity was very low in LLC-PK1 and COS-7 cells. Forskolin and TPA stimulated the activity of a SgII-luciferase fusion gene in SH-SY5Y but not in AtT-20 cells. Disruption of the CRE abolished the stimulatory effect of forskolin and TPA. These data suggest that the basal activity of the human SgII gene relies on cell-specific trans-acting factors in addition to factors that bind to the CRE and show that the regulation of this gene by second messengers is cell-specific and requires an intact CRE.     

Structure of the chromosomal gene for murine interleukin 3.

We have isolated the mouse interleukin 3 (IL-3) gene from a mouse sperm DNA library based on homology with the mouse mast-cell growth-factor (MCGF) cDNA sequence. The nucleotide sequence determined for the gene and its flanking regions reveals that the IL-3 gene is composed of four introns and five exons. The nucleotide sequence of the exons agrees with that determined for the MCGF cDNA. A "TATA"-like sequence preceded by a G + C-rich region is found in the 5' flanking region. At the 3' region of the second intron are nine repeats of a closely related 14-base-pair (bp) sequence. These repeated sequences share extensive homology with a 20-bp repeated sequence found in the human genome, which was shown to have enhancer activity. Eight out of nine repeats form a 73-bp duplicated sequence and each 73-bp repeat contains sequences homologous to the core sequence suggested for enhancer elements. These sequences may play a role in the expression of the IL-3 gene in concanavalin A- or antigen-stimulated T lymphocytes. Stable transformants of L cells generated by co-transformation of the IL-3 gene with pSV2neo constitutively express MCGF activity indicating that the isolated gene is functional in vivo.     

Systematic polymorphism discovery after genome-wide identification of potential susceptibility loci in a hereditary rodent model of human hypertension

Genetic strategies such as linkage analysis and quantitative trait locus (QTL) mapping have identified a multitude of loci implicated in the pathogenesis of hypertension in the spontaneously hypertensive rat (SHR). While several candidate genetic regions have been identified in the SHR and its control, the Wistar-Kyoto rat (WKY), systematic follow-up of candidate identification with polymorphism discovery has not been widespread. In the current report, we develop a data-mining strategy to identify candidate genes for hypertension in the SHR, and then sequence each gene in the SHR and WKY strains. We integrate blood pressure QTL data, microarray data and data-mining methods. First, we determined the set of genes differentially expressed in SHR and WKY adrenal glands. Next, the chromosomal position of all differentially expressed genes was compared with peak marker position of all reported SHR blood pressure QTLs. We also identified the set of differentially expressed genes with the most extreme fold-change. Finally, the QTL positional candidates and the genes with extreme differential expression were proposed as candidate genes if they had biologically plausible roles in hypertensive pathology. We identified seven candidate genes that merit resequencing (catechol-O-methyltransferase [Comt], chromogranin A [Chga], dopamine beta-hydroxylase [Dbh], electron transferring flavoprotein dehydrogenase [Etfdh], endothelin receptor type B [Ednrb], neuropeptide Y [Npy] and phenylethanolamine-N-methyltransferase [Pnmt]), and then discovered polymorphism in four of these seven candidate genes. Chga is proposed as the strongest candidate for additional functional investigation. Our method for candidate gene identification is portable and can be applied to microarray data from any tissue, in any disease model with a QTL database.     

Cloning and characterization of the human lung endothelial-cell-specific molecule-1 promoter

Endothelial-cell-specific molecule-1 (ESM-1) is a cysteine-rich protein that is expressed primarily in endothelial cells of the lung, kidney and gut. In the present study, we have cloned and sequenced 3,888 bp of the 5' flanking region of the human ESM-1 gene. The full-length promoter directed high-level expression of the luciferase reporter gene in bovine lung microvascular endothelial cells and bovine aortic endothelial cells, but not in nonendothelial cell types. In 5' deletion analyses, a region spanning -81 to +58 was shown to contain information for endothelial-cell-specific expression. Mutational analysis in transient transfection assays uncovered an important role for an Ets-binding motif located between -77 and -74 and a cAMP-response-element (CRE)-like motif located between -68 and -62 in mediating high-level expression in endothelial cells. A second Ets site (-63 to -60) as well as a novel 6-bp palindromic sequence (-58 to -53) were found to inhibit expression. In DNase footprint analyses, both the Ets-binding motifs were protected specifically in endothelial cells, while the CRE-like element and palindrome were protected in endothelial and nonendothelial cells alike. Taken together, these results provide an important foundation for studying endothelial-cell-subtype-specific gene regulation.