Major Transcription Start Point Research Articles

The structure and evolution of the MaSMC4 gene of common vole Microtus arvalis (Arvicolidae, Rodentia)

In eukaryotes, the SMC (Structural Maintenance of Chromosomes) gene family is represented by at least six genes. Some of these genes have tissue-specific homologs. Eukaryotic SMC structural proteins are the members of biochemical complexes responsible for cohesion of sister chromatids, recombination, repair, regulation of gene expression, and formation of mitotic chromosomes. In the present study, the structure of the SMC4 sub-family gene was examined in common vole Microtus arvalis. Comparative analysis of rodent (M. arvalis, Mus musculus. and Rattus norvegicus), human, and Xenopus SMC4 orthologous genes was carried out, and the main patterns of their organization and regulation were established. The SMC4 genes contain 24 exons; open reading frame starts at exon 2. The SMC4 5' regions contain the CpG islands, extending in the region of exon-intron I and exon 2. The SMC4 genes are characterized by the presence of multiple transcription startpoints. The region of the major transcription startpoint contains the INR CCA,1TTTT element. The SMC4 5' region is characterized by the presence of putative binding site for basal transcription factor Sp and factor E2F, typical of the genes induced in the G I/S phase of the cell cycle. The divergence level of the SMC4 coding region was examined. The mean Ka/Ks ratio for the SMC4 genes examined was 0. 123. The region of exon 2 was found to be the most variable (Ka/Ks = 0.715), while the most conservative was the region coding for the C-globular domain, which contained the DA box (Ka/Ks = 0.024).

Activation of Zoosporogenesis-Specific Genes in Phytophthora infestans Involves a 7-Nucleotide Promoter Motif and Cold-Induced Membrane Rigidity

Infections of plants by the oomycete Phytophthora infestans typically result from zoospores, which develop from sporangia at cold temperatures. To help understand the relevant cold-induced signaling pathway, factors regulating the transcription of the zoosporogenesis-specific NIF (nuclear LIM-interactor-interacting factor) gene family were examined. Sequences required for inducing PinifC3 were identified by analyzing truncated and mutated promoters using the beta-glucuronidase reporter in stable transformants. A 7-nucleotide (nt) sequence located 139 bases upstream of the major transcription start point (GGACGAG) proved essential for the induction of PinifC3 when sporangia were shifted from ambient to cold temperatures. The motif, named the cold box, also conferred cold inducibility to a promoter normally activated only during sexual development. An identical motif was detected in the two other zoosporogenesis-specific NIF genes from P. infestans and three Phytophthora sojae orthologues, and a closely related sequence was found in Phytophthora ramorum orthologues. The 7-nt motif was also found in the promoters of other zoosporogenesis-induced genes. The presence of a cold box-interacting protein in nuclear extracts of P. infestans sporangia was demonstrated using electrophoretic mobility shift assays. Furthermore, zoospore release and cold box-regulated transcription were stimulated by the membrane rigidizer dimethyl sulfoxide and inhibited by the membrane fluidizer benzyl alcohol. The data therefore delineate a pathway in which sporangia perceive cold temperatures through membrane rigidity, which activates signals that drive both zoosporogenesis and cold-box-mediated transcription.

Functional characterization of the promoter of the human glucose transporter 10 gene

The human SLC2A10 gene encodes the high-affinity glucose transporter 10 (GLUT10) and is widely expressed in adult tissues, including organs which play major roles in glucose homeostasis. Its function and genomic location in a region linked to Type 2 diabetes susceptibility are consistent with a potential role in Type 2 diabetes. Analysis of the CpG-rich promoter revealed the presence of two major transcription start points with differential use in tissues and cell lines. Mapping of transcriptionally active regions in the 5′ flanking sequence identified a region, located between nucleotides −70 and −14 (relative to the major transcription start point) as the SLC2A10 basal promoter. This sequence harbors consensus binding sites for Sp, AP2α, and other transcription factors. A juxtaposed Sp/AP2α motif located between −25 and −11 is critical for core promoter function. In cells expressing Sp and AP2 factors, the two motifs are required for maximal activation of the basal promoter. In cells lacking AP2α, transcription is dependent on the integrity of the Sp site. Using electrophoresis mobility shift assays, we demonstrate that Sp1 and Sp3 bind to the GC-box in site 5 forming specific complexes. In addition, a silencer region is present upstream of −696 which down-regulates SLC2A10 promoter activity independently of its distance to the transcript start site.

Interleukin (IL)-15 and IL-2 Reciprocally Regulate Expression of the Chemokine Receptor CX3CR1 through Selective NFAT1- and NFAT2-dependent Mechanisms

We have recently reported that interleukin (IL)-15 and IL-2, which signal through IL-2Rbetagamma, oppositely regulate expression of the proinflammatory chemokine receptor CX3CR1. Here we delineate molecular mechanisms responsible for this paradox. By using a luciferase reporter plasmid, we identified a 433-bp region spanning the major transcriptional start point of human CX3CR1 that, when expressed in human peripheral blood mononuclear cells (PBMCs), possessed strong constitutive promoter activity. IL-2 and IL-15 treatment increased and abolished this activity, respectively, mimicking their effects on endogenous CX3CR1. IL-2 and IL-15 have been reported to also have opposite effects on the immunoregulatory transcription factor NFAT (nuclear factor of activated T cells), and the 433-bp region contains a kappaB-like NFAT site. The effects of IL-15 and IL-2 on both CX3CR1 reporter activity and endogenous CX3CR1 transcription in PBMCs were abolished by the NFAT inhibitors cyclosporin A and VIVIT. Moreover, mutation of the kappaB-like NFAT sequence markedly attenuated IL-2 and IL-15 modulation of CX3CR1 promoter-reporter activity in PBMCs. Furthermore, chromatin immunoprecipitation revealed that IL-15 promoted specific recruitment of NFAT1 but not NFAT2 to the CX3CR1 promoter, whereas IL-2 had the converse effect. This appears to be relevant in vivo because mouse CX3CR1 mRNA was expressed in both PBMCs and splenocytes from NFAT1-/- mice injected with recombinant IL-15 but was undetectable in cells from IL-15-injected NFAT1+/+ BALB/c mice; as predicted, IL-2 up-regulated cx3cr1 in both mouse strains to a similar extent. Thus, by pharmacologic, genetic, and biochemical criteria in vitro and in vivo, our results suggest that IL-15 and IL-2 oppositely regulate CX3CR1 gene expression by differentially recruiting NFAT1 and NFAT2 to a kappaB-like NFAT site within the CX3CR1 promoter. We propose that expression of CX3CR1 and possibly other immunoregulatory genes may be determined in part by the balance of NFAT1 and NFAT2 activity in leukocytes.

AmfR, an essential gene for aerial mycelium formation, is a member of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) acts as a chemical signalling molecule that triggers morphological differentiation and secondary metabolism. A transcriptional activator, AdpA, in the A-factor regulatory cascade switches on a number of genes required for both processes, thus forming an AdpA regulon. amfR encoding a regulatory protein similar to response regulators of bacterial two-component regulatory systems and essential for aerial mycelium formation was found to be a member of the AdpA regulon. AdpA bound two sites at nucleotide positions approximately -200 (site 1) and -60 (site 2), with respect to the major transcriptional start point of amfR, and accelerated the transcription of amfR by assisting RNA polymerase in forming an open complex at an appropriate region including the transcriptional start point. Site 2 contributed more to the transcriptional activation of amfR by AdpA than site 1, although AdpA showed a much lower affinity to site 2 than to site 1. The amfR transcription enhanced by AdpA subsequently ceased at day 2 when aerial hyphae began to be formed in the wild-type strain, whereas in an adsA null mutant amfR was continuously transcribed even until day 3. This implied that amfR was repressed growth dependently by a gene product under the control of sigma-AdsA. Transcription of the promoter upstream of amfT depended on amfR, which is consistent with the idea that AmfR serves as an activator for amfTSBA in the amf operon. The observations that the amfR gene contains a TTA codon, a potential target for bldA-mediated regulation, and a conserved Asp-54 residue, which might be phosphorylated by a sensor kinase, suggest that the amf operon is under transcriptional, translational and post-translational control systems.

Isocitrate lyase of the yeast Kluyveromyces lactis is subject to glucose repression but not to catabolite inactivation.

KlICL1, encoding the isocitrate lyase of Kluyveromyces lactis, was isolated by complementation of the Saccharomyces cerevisiae icl1 deletion mutant. Sequence analysis revealed an open reading frame of 1626 nucleotides encoding a protein with 542 amino acids. The deduced protein shows extensive homologies to isocitrate lyases from various organisms, with an overall identity of 69% to the enzyme from S. cerevisiae. The KlICL1 gene has two major transcription start-points, located at -113 bp and -95 bp relative to the ATG translation start codon. The gene is expressed on ethanol medium only in respiratory-competent cells. Transcription is repressed by glucose. Mutants carrying a Klcat8 deletion lack the ability to derepress KlICL1 transcription. A Klicl1 deletion mutant does not grow on ethanol medium and lacks any isocitrate lyase activity. A strain lacking the gene KlFBP1, which encodes the gluconeogenic enzyme fructose 1,6-bisphosphatase, lacks the ability to grow on non-fermentable carbon sources. This implies that K. lactis does not contain additional isoenzymes catalyzing either of the reactions. Enzyme assays revealed that neither KlIcl1p nor KlFbp1p are subject to catabolite inactivation. However, the respective enzymes from S. cerevisiae are efficiently inactivated when expressed in K. lactis. Thus, despite the extensive sequence similarities of the enzymes involved, non-fermentative carbohydrate metabolism in the two yeasts displays distinct regulatory properties.

Functional Analysis of the Rat N-Methyl-D-aspartate Receptor 2A Promoter: MULTIPLE TRANSCRIPTION START POINTS, POSITIVE REGULATION BY Sp FACTORS, AND TRANSLATIONAL REGULATION

N-Methyl-d-aspartate (NMDA) receptor subunit 2A (NR2A) is an important modulatory component of the NMDA subtype of glutamate receptors. To investigate the transcription mechanism of the NR2A gene, we cloned the 5'-flanking sequence from a rat genomic library. RNA mapping with rat brain RNA revealed two sets of major and several minor transcription start points in a single exon of 1140 bp. Reporter gene and mutation studies indicated that core promoter activity resided in exon 1, whereas the 5'-flanking sequence up to 1.5 kb showed no significant impact on promoter activity. Fragments containing minor transcription start points were able to drive a reporter gene in transfected cells and produce nascent RNAs in an in vitro transcription system. All fragments tested showed more promoter activity in dissociated neurons of the rat embryonic cerebrocortex and cell lines expressing NR2A mRNA than that in glial cultures and non-neuronal cells. Within exon 1 there are three GC-box elements that displayed distinct binding affinity to both Sp1- and Sp4-like factors. Overexpression of Sp1 or Sp4, but not Sp3, significantly increased the activity of the promoter containing these elements. Inclusion of exon 2 and 3 sequences, which contain five short open-reading frames, attenuated promoter-driven reporter activity more than 3-fold but attenuated the level of reporter mRNA less than 1.4-fold. Our results suggest that the core promoter of the rat NR2A gene requires exon 1, that Sp factors positively regulate this core promoter, and that a post-transcriptional mechanism may negatively regulate expression of the gene.

Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa.

The genes for nitrous oxide (N(2)O) reduction, nosRZDFYL, are clustered on the chromosome of Pseudomonas aeruginosa. Promoter assays using transcriptional fusions to lacZ revealed that the structural gene for nitrous oxide reductase, nosZ, is transcribed with the upstream nosR gene. The nosR gene product is not required for the activity of the nosR promoter. A sequence similar to the consensus FNR-binding motif was found 41.5 bp upstream from the major transcriptional start point of nosR. Mutation of the motif significantly reduced the promoter activity. DNR, an FNR-related transcriptional regulator required for the expression of denitrification genes in P. aeruginosa, is necessary for the transcription of nosR, indicating that the motif is recognized by DNR. Nitrite (NO-2), nitric oxide (NO) and NO-generating reagents induced nosR promoter activity, but N(2)O did not. The NO-2-induced nosR promoter activity was reduced by mutation of the NO-2 reductase gene. However, a low concentration of NO-2 induced the promoter activity in a NO reductase mutant. These results indicate that NO is the inducer molecule for transcription of the nos genes.

Transcriptional Analysis of the DNA Polymerase Gene of Shrimp White Spot Syndrome Virus

The white spot syndrome virus DNA polymerase (DNA pol) gene (WSSV dnapol) has already been tentatively identified based on the presence of highly conserved motifs, but it shows low overall homology with other DNA pols and is also much larger (2351 amino acid residues vs 913-1244 aa). In the present study we perform a transcriptional analysis of the WSSV dnapol gene using the total RNA isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with a WSSV dnapol-specific riboprobe found a major transcript of 7.5 kb. 5′-RACE revealed that the major transcription start point is located 27 nucleotides downstream of the TATA box, at the nucleotide residue A within a CAGT motif, one of the initiator (Inr) motifs of arthropods. In a temporal expression analysis using differential RT-PCR, WSSV dnapol transcripts were detected at low levels at 2–4 h.p.i., increased at 6 h.p.i., and remained fairly constant thereafter. This is similar to the previously reported transcription patterns for genes encoding the key enzyme of nucleotide metabolism, ribonucleotide reductase. Phylogenetic analysis showed that the DNA pols from three different WSSV isolates form an extremely tight cluster. In addition, similar to an earlier phylogenetic analysis of WSSV protein kinase, the phylogenetic tree of viral DNA pols further supports the suggestion that WSSV is a distinct virus (likely at the family level) that does not belong to any of the virus families that are currently recognized.

Identification and characterization of a novel promoter of the mouse mu opioid receptor gene ( Oprm) that generates eight splice variants

Our previous studies isolated and characterized eight splicing variants containing exon 11 as the first coding exon. The location of exon 11 about 10 kb upstream from exon 1 dual promoters implied another promoter to drive expression of the exon 11 variants. I now identify the second promoter in the 5′-flanking region of exon 11. One major transcriptional start point was determined. Sequence analysis indicated that the 5′-flanking region of the exon 11 contained a putative TATA box, several CAAT boxes and a number of cis-acting elements. Functional analysis suggested that exon 11 promoter activity was most evident in neuronal-like cells. A basal core region containing the TATA box, a negative region and a positive region were identified. Electrophoretic mobility shift assays with the nuclear extracts from NIE-115 cells revealed several protein complexes that likely contained TATA box-associated factors, NF-1-like and cMyc-Max-like proteins, respectively. It also showed that a TATA-binding protein specifically bound to the TATA box fragment. Mutation analysis suggested that the TATA box in the basal core region played a fundamental role in the exon 11 promoter, whereas the NF-1 site acted as a positive element.

Genomic organization of the mouse peroxisome proliferator-activated receptor β/δ gene: alternative promoter usage and splicing yield transcripts exhibiting differential translational efficiency

Peroxisome proliferator-activated receptor (PPAR) beta/delta is ubiquitously expressed, but the level of expression differs markedly between different cell types. In order to determine the molecular mechanisms governing PPARbeta/delta gene expression, we have isolated and characterized the mouse gene encoding PPARbeta/delta. The gene spans approx. 41 kb and comprises 11 exons of which the six exons located in the 3'-end of the gene are included in all transcripts. Primer-extension and 5'-rapid amplification of cDNA ends experiments revealed the presence of multiple transcription start points and splice variants, originating from the use of at least four different promoters. One of these transcription start points was found to be used predominantly in all tissues examined. Initiation from this major transcription start point gives rise to a transcript with a 548 nt 5'-untranslated leader containing eight upstream AUG codons. We show that the presence of the 548 nt leader resulted in a low translational efficiency of the corresponding PPARbeta/delta mRNA and propose, based on structural features of the 5'-untranslated region, that translational initiation may be mediated via an internal ribosome entry site-dependent mechanism.

Identification of a novel first exon of prolactin receptor gene expressed in the rat brain.

A novel first exon, E1(4), whose sequence was distinct from those of the three known first exons, E1(1), E1(2), and E1(3), of the rat PRL receptor (PRL-R) gene was identified by cDNA cloning for the 5'-end region of PRL-R mRNA expressed in the rat brain. Sequence analysis revealed the presence of two different length E1(4) cDNAs. The longer cDNA contained the 243-bp E1(4) sequence, and the shorter cDNA lacked the 139-bp sequence at the 5'-end of the longer one. Neither E1(4) cDNA has a second exon sequence, indicating that the E1(4) first exon is extensively spliced to the third exon. E1(4)-containing PRL-R mRNAs were detected only in the brain by RT-PCR and ribonuclease protection assay. The longer E1(4) mRNA was expressed as the major PRL-R mRNA species in the brain and was greatly increased in pregnant (d 18) and lactating (d 5) rats. A genomic clone containing the E1(4) first exon together with its 5'- and 3'-flanking regions was isolated from a rat kidney genomic library. Ribonuclease protection assay revealed that the position corresponding to the 5'-end of the shorter E1(4) cDNA is the major transcription start point for the E1(4) exon. The 5'-flanking region of E1(4) contained a TATA box-like element 23 bp upstream of the major transcription start point. Other putative transcription factor-binding sites, such as CCAAT, Sp1, and glucocorticoid-responsive elements, were observed at further upstream regions. These results suggest that PRL-R gene expression in rat brain is controlled by the promoter for the E1(4) first exon.

Convergence of two global transcriptional regulators on nitrogen induction of the stress‐acclimation gene nblA in the cyanobacterium Synechococcus sp. PCC 7942

Cyanobacteria respond to environmental stress conditions by degrading their phycobilisomes, the light harvesting complexes for photosynthesis. The expression of nblA, a key gene in this process, is controlled by the response regulator NblR in Synechococcus sp. PCC 7942. Here we show that, under nitrogen stress, nblA is also regulated by NtcA, the global regulator for nitrogen control. NtcA activation of nblA was found to be nitrogen-specific and did not take place under sulphur stress. Transcripts from the two major transcription start points (tsp) for the nblA gene were induced in response to nitrogen and sulphur starvation. The most active one (tspII) required both NblR and NtcA to induce full nblA expression under nitrogen starvation. NblR and NtcA bound in vitro to a DNA fragment from the nblA promoter region, suggesting that, under nitrogen stress, both NblR and NtcA activate the main regulated promoter (PnblA-2) by direct DNA-binding. The structure of PnblA-2 differs from that of the canonical NtcA-activated promoter and it is therefore proposed to represent a novel type of NtcA-dependent promoter. We analysed expression patterns from ntcA and selected NtcA targets in NtcA(-), NblR(-) and wild-type strains, and discuss data suggesting further interrelations between phycobilisome degradation and nitrogen assimilation regulatory pathways.

Myc and YY1 mediate activation of the Surf-1 promoter in response to serum growth factors

The human Surf-1 and Surf-2 genes are divergently transcribed and share a single bi-directional promoter. The addition of serum growth factors to serum-starved cells activates transcription in the Surf-1 direction, but has no effect on transcription in the Surf-2 direction. Mutations that block the binding of YY1 to a site immediately downstream of the major Surf-1 transcription start point abolish this response to serum factors. Here we show that over-expression of mitogen-activated protein (MAP) kinase phosphatase MKP-1, an inhibitor of the MAP kinase cascade, also blocks the response the Surf-1 promoter to serum factors. YY1 has previously been shown to interact with several transcription factors including Myc. We show that although the Surf-1/Surf-2 promoter does not contain Myc binding sites (E-boxes), Myc over-expression, or the activation of a Myc–oestrogen receptor fusion protein, activates transcription in the Surf-1 direction and that this response to Myc requires a functional YY1 binding site. Our data suggest that the MAP kinase cascade is required for the stimulation of Surf-1 promoter activity and that the Myc-YY1 interaction mediates this response.

Isolation and characterization of the promoter region of the rat vasopressin V1b receptor gene.

Regulation of pituitary vasopressin V1b receptors plays a critical role in regulating pituitary adrenocorticotropic hormone (ACTH) secretion during adaptation to stress. The objective of this study was to isolate the promoter regulatory region of the V1b receptor gene to better understand the molecular mechanisms involved in V1b receptor regulation. Screening of a rat genomic library using probes directed to the coding region and to the 5'UTR of the rat V1b receptor resulted in the isolation of several clones containing the 5'upstream regions of the V1b receptor cDNA. Sequencing of an 11.2 Kb fragment revealed 8.2 Kb upsteam of the reported cDNA sequence, which contains a putative promoter regulatory region. The 3' end of the clone contained 1472 base pairs corresponding to the recognized cDNA sequence, followed by 1506 bp of unknown sequence located at the end of the sixth transmembrane domain, probably corresponding to an intron, characteristic of these family of receptors. An additional 161 bp intron was found in the 5'UTR, similar to that described in the rat oxytocin receptor gene. 5'RACE and RNase protection analysis mapped two major putative transcription start points at -830 and -861 bp from the starting methionine. Analysis of the putative promoter region showed no indication of a proximal TATA box, but the presence of a CACA box, a GAGA box, several AP-1 and AP-2 sites and a cluster of Sp1 sites upstream of the AP-2 sites. A luciferase construct containing a 2.1-kb of putative promoter, and part of the 5'UTR including the first intron, showed promoter activity when transfected into COS-7, CHO and PC12 cell lines but not in AtT-20 cells. A similar construct without the intron and distal 5'UTR sequence has no promoter activity in the same cell lines. In summary, the V1b receptor gene contains at least 3 exons and 2 introns. The 5'flanking sequence contains several potential sites for transcriptional regulation, and induced luciferace activity only in constructs containing intron 1, suggesting that the latter is important for receptor gene activation. The data provide bases for future analysis of the regulatory elements controlling V1b receptor transcription.

Genomic organization of the human rod photoreceptor cGMP-gated cation channel beta-subunit gene.

We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel beta-subunit is complex, comprising non-overlapping transcription units that give rise to at least six transcripts (Ardell, M.D., Aragon, I., Oliveira, L., Porche, G.E., Burke, E., Pittler, S.J., 1996. The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit. FEBS Lett. 389, 213-218). To further understand the transcriptional regulation of this extraordinarily complex locus, and to develop a screen for defects in the gene in patients with hereditary disease, we determined its genomic organization and DNA sequence. The CNGB1 locus consists of 33 exons, which span approximately 100kb of genomic DNA on chromosome 16. The beta-subunit comprises two domains, an N-terminal glutamic acid-rich segment (GARP), and a C-terminal channel-like portion. Two additional exons encoding a short GARP transcript and a truncated channel-like transcript have been identified. A major transcription start point was identified 79bp upstream of the initiator ATG. To begin analysis of the basis for the generation of multiple transcripts, and to identify promoters driving expression in retina, approximately 2.5kb of the upstream region were sequenced. Putative cis-elements, which can bind the retina-specific transcription factors Crx and Erx, were found immediately upstream of the transcription start point, and may be important for gene expression in this tissue. From our analysis, a model is reported to account for at least four of the retinal transcripts.

Structural organization of the human gamma-glutamyl hydrolase gene.

Gamma-glutamyl hydrolase (GH) plays an important role in the metabolism of folic acid and the pharmacology of antifolates such as methotrexate. We have previously cloned and characterized the human GH cDNA. In this report, the complete organization and structure of the human GH gene was determined. The human GH gene spans 24 kb in the human genome, with nine exons sized from 51 to 371 bp. All of exon-intron splice junctions follow the GT-AG rule. The sequence upstream of exon 1 consists of a promoter-like, GC-rich region and a number of putative cis active elements including Sp1, AP1, and MZF1 sites. A TATA sequence in the 5' region of human GH gene was not observed, similar to housekeeping genes known to be tissue-specific and differentially expressed. S1 nuclease protection analysis with human liver, prostate, brain, and mammary gland revealed a major transcription start point at nucleotide -125 relative to the ATG start codon and several minor transcription start points. Analysis of GH cDNA isolated from human liver indicated a nucleotide change, T-->C, in the leader sequence of GH, which suggested a polymorphism. Studies of cDNA from different human tissue sources provided evidence that there is a single spliced cDNA species in human.

The promoter region of human prepro-nociceptin gene and its regulation by cyclic AMP and steroid hormones.

The promoter and upstream regulatory region of the human prepro-nociceptin gene has been cloned from adaptor-ligated genomic DNA libraries by polymerase chain reaction. This 1.7 kb region contains several potential binding sites for transcription factors, among which are binding sites for TF-IID, cyclic AMP response element binding protein, glucocorticoid receptor and estrogen receptor. Multiple start points for the transcription of prepro-nociceptin are identified by an 'oligoribonucleotide-capping' method, but the major one is located at -558(G). Promoter activity assays using luciferase reporter gene constructions with the 1.7 kb fragment and a series of deletion mutations demonstrate that the core promoter is located in the region from -639 to -521 (a region surrounding the major transcription start point -558). A TATA-box motif displays weak promoter activity. An increase of cellular cyclic AMP levels by forskolin treatment up-regulates prepro-nociceptin transcription. Estrogen also up-regulates gene transcription whereas glucocorticoid down-regulates transcription, each through their corresponding receptor response elements. These regulatory effects can be blocked either by mutations of the potential cyclic AMP- or estrogen receptor response elements or by the application of antagonists for glucocorticoid and estrogen receptors. These findings provide a molecular basis for the regulatory mechanisms of human prepro-nociceptin gene expression.

An initiator element and a proximal cis-acting sequence are essential for transcriptional activation of the complement factor I (CFI) gene.

Human complement factor I (CFI) is a serine protease which regulates the complement system by inactivation of C3b and C4b in the presence of appropriate cofactors. In this study, we have analyzed the mechanism controlling the constitutive transcriptional activation of the CFI gene. Using deletion analysis and transient CAT expression assays, we have mapped the minimal promoter to the region located between -46 and +160 bp relative to the major transcription start point (tsp), and also shown that cis-acting elements both upstream and downstream of the tsp are important for promoter activity. A silencer element was also found between -71 and -46 bp. The sequence surrounding the tsp was related to the mouse terminal deoxynucleotidyltransferase initiator element (Inr) and point mutations in this sequence, from -3 to +4, drastically reduced CFI promoter activity. Mutations in a -9 to -5 bp CTGAA sequence immediately upstream of the tsp also reduced promoter activity. Gel mobility shift analysis demonstrated the binding of nuclear factors to a CTGAA repeat located at -9 to -5 and +101 to +105. Our results suggest that CFI promoter contains a functional Inr element that is essential for promoter activity, and the interactions of the CTGAA element located between -9 and +5 with nuclear factor(s) may be part of the machinery required for CFI Inr-dependent transcription.

The expression of the trpD, trpC and trpBA genes of Streptomyces coelicolor A3(2) is regulated by growth rate and growth phase but not by feedback repression.

Transformation of tryptophan auxotrophs of Streptomyces coelicolor A3(2) and subsequent analysis have allowed the identification of four tryptophan biosynthetic genes. Subcloning, complementation of trp strains, nucleotide sequencing of 5.1 kb and 1.95 kb of DNA and subsequent homology comparisons identified the trpC, trpB and trpA genes and trpD gene respectively. The arrangement of genes in the trpCBA cluster is unusual in that trpC is separated by a small open reading frame, trpX, from the potentially translationally coupled trpB and trpA genes. Sequence analysis of the trpD gene revealed the presence of a large mRNA loop structure directly upstream of the trpD-coding region. S1 nuclease mapping studies of trpCXBA have revealed two major potential transcription start points, one just upstream of the trpC gene and the other located upstream of the trpX gene. S1 nuclease mapping of the trpD region revealed four fragment end-points. Quantitative S1 nuclease protection assays and a promoterless catechol dioxygenase reporter gene have revealed that the expression of all these genes is growth phase dependent and growth rate dependent, expression being maximal during early exponential phase and dropping off sharply in late exponential phase. This growth phase-dependent and growth rate-dependent regulation is the first reported in streptomycete primary metabolism.