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PubMed1998

Sp1 and Sp3 regulate transcriptional activity of the facilitative glucose transporter isoform-3 gene in mammalian neuroblasts and trophoblasts.

The murine facilitative glucose transporter isoform 3 (Glut 3) is developmentally regulated and is predominantly expressed in neurons and trophoblasts. Employing the primer extension and RNase protection assays, the transcription start site (denoted as +1) of the murine Glut 3 gene was localized to 305 base pairs (bp) 5' to the ATG translation start codon. Transient transfection assays in N2A, H19-7 neuroblasts, and HRP.1 trophoblasts using sequential 5'-deletions of the murine Glut 3-luciferase fusion gene indicated that the -203 to +237 bp region with reference to the transcriptional start site contained promoter activity. Repressor function was limited to the -137 to -130 bp region within the transcriptional activation domain. The nuclear factors Sp1 and Sp3 bound this GC-rich region in N2A, H19-7, and HRP.1 cells. Dephosphorylation of Sp1 was essential for Glut 3 DNA binding. The related Sp3 protein also bound this same region of mouse Glut 3 in all three cell lines. Mutations of the Sp1-binding site employed in transient transfection and mobility shift assays confirmed the nature of the DNA-binding proteins, while supershift assays with anti-Sp1 and anti-Sp3 IgGs characterized the differences in the two DNA-binding proteins. Co-transfection of the Glut 3-luciferase fusion gene with or without mutations of the Sp1-binding site along with the Sp1 or Sp3 expression vectors in Drosophila SL2 cells confirmed a reciprocal effect, with Sp1 suppressing and Sp3 activating Glut 3 gene transcription.

Animals↗

PubMed2000

Genomic organization of the dog dystroglycan gene DAG1 locus on chromosome 20q15.1-q15.2.

Dystroglycan is a laminin binding protein, which provides a structural link between the subsarcolemmal cytoskeleton and the extracellular matrix. It is also involved in the organization of basement membranes. So far the genomic organization of the dystroglycan gene DAG1 has not been completely investigated. Here we report the cloning and sequencing of 162 kb of dog genomic DNA containing the complete approximately 71-kb canine DAG1 gene, which consists of three exons, with the translation start codon located in exon 2. Its 2679-nucleotide ORF encodes a polypeptide of 892 amino acids, which is highly similar to human, rabbit, and bovine orthologs. To further characterize the dog DAG1 gene we determined the transcription start site and several naturally occurring polymorphisms, which partially result in amino acid substitutions of the dystroglycan protein. The dog DAG1 gene was assigned to chromosome 20q15.1-q15.2 by FISH analysis. The analysis of the entire reported sequence revealed that the genes for aminomethyltransferase (AMT), bassoon (BSN), TCTA (T-cell leukemia translocation-associated) gene, and an as yet uncharacterized protein are located very close to the DAG1 gene. Therefore, this study defines a novel syntenic region among dog chromosome 20q15, human chromosome 3p21, and murine chromosome 9F.

Animals↗

PubMed1998

Complete cDNA sequence of the Ki-ras proto-oncogene in the liver of wild English sole (Pleuronectes vetulus) and mutation analysis of hepatic neoplasms and other toxicopathic liver lesions.

A complete copy of Ki-ras b cDNA from English sole (Pleuronectes vetulus), a benthic marine flatfish, was cloned and sequenced. The percent identity between the predicted amino acid sequence of English sole and human Ki-ras b was 97%, whereas the percent identity between the English sole gene and rainbow trout or Rivulus Ki-ras b was 98%. Areas of amino-acid sequence conservation included codons 12, 13, and 61, the positions in which mutations are observed in ras cellular oncogenes in other species. The 5' untranslated region (UTR), consisting of 217 nt, was not highly GC rich but contained four ATG start codons upstream of the major open reading frame. The 3' UTR, containing 26 nt, was AU rich. Analysis of Ki-ras mutations was performed on a variety of necrotic, preneoplastic, and neoplastic lesions in livers from 13 English sole collected from contaminated waterways in Puget Sound, WA. Despite reports of Ki-ras mutations in hepatic tumors from other fish, no mutations in codons 12, 13, or 61 were found in hepatic lesions from English sole by direct DNA sequencing of polymerase chain reaction-amplified genomic DNA. Although mutations could exist at levels below the detection limits of this analysis, the results suggest that Ki-ras has a role in liver carcinogenesis that varies according to the fish species or carcinogen. Furthermore, future studies of the etiology of chemically induced cancer in feral English sole should consider mutations in other cancer-related genes, such a5p53, Ha-ras, and N-ras.

Amino Acid Sequence↗

PubMed2000

5'-Flanking sequence and promoter activity of the rabbit neuronal nitric oxide synthase (nNOS) gene.

We have isolated a rabbit neuronal nitric oxide synthase (nNOS) cDNA encoding a protein of 1,435 amino acids. Using the cDNA clones as probes, the 5'-flanking region of the nNOS gene was isolated from a rabbit genomic DNA library. 5'RACE and primer extension analysis of rabbit brain total RNA mapped multiple transcription initiation sites localized 474-487 bp upstream from the translation start codon. Analysis of 5,197 bp of the 5'-flanking sequence revealed that the rabbit nNOS gene promoter lacks canonical TATA or CCAAT boxes and, instead, contains a GC-rich region and multiple Sp1 sites. Farther from the +1start, various putative cis-elements including AP-1, AP-4, NF-kappaB, STAT, CREB, C/EBP and c-Myc were observed. The functional promoter activity of the 5'-flanking region was demonstrated by its ability to drive the expression of a beta-galactosidase reporter gene in several cell types. Serial deletion analysis of the promoter region revealed that the -291 to -172 region, which contains two Sp1 sites, is essential for basal transcriptional activity. These results suggest that the rabbit nNOS promoter contains characteristics of inducible genes.

5' Untranslated Regions↗

PubMed1997

T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like mechanism.

Premature termination codons (PTCs) are known to decrease mRNA levels. Here, we report our investigation of the mechanism for this downregulation using the TCR-beta gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic development. We found that a mini-gene version of this gene, which contains only three TCR-beta exons, exhibited efficient downregulation in response to PTCs. This demonstrates that the full coding sequence is not necessary for appropriate regulation. Mutation of the translation start AUG and a downstream in-frame AUG that displayed similarity to the Kozak consensus sequence reversed the downregulatory response to PTCs. Thus, an AUG start codon is required to define the reading frame of a PTC. Specific suppressor tRNAs also reversed the downregulatory response, strongly implicating the involvement of a translation-like process. Remarkably, the addition of suppressor tRNAs or the inactivation of the start AUGs caused a dramatic rise in the levels of PTC-bearing transcripts in the nuclear fraction prepared by two independent methods. Collectively, our results provide evidence for a codon-based surveillance mechanism associated with the nucleus that downregulates aberrant transcripts encoding potentially toxic polypeptides from nonproductively rearranged genes.

Animals↗

PubMed2000

Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter.

The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5' and 3' ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.

Animals↗

PubMed2001

Nucleotide sequence and transcriptional analysis of a putative basic DNA-binding protein of Helicoverpa armigera nucleopolyhedrovirus.

A putative basic DNA-binding protein (BDBP) gene was identified in the fragment EcoRI-K of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) genome. The ORF is 330 nucleotides long encoding a basic protein of 109 amino acids with a molecular mass of 11.6 kDa. It is the first BDBP identified in single nucleocapsid NPVs and a homologue of Autographa californica MNPV (AcMNPV) P6.9. A consensus late transcription motif, ATAAG, was found at 57 nt upstream of the translational start codon and a polyadenylation signal was observed at 172 nt downstream of the stop codon. A major transcript of 620 nt was first observed in HearNPV-infected Hz2e5 cells 16 h post infection. Primer extension analysis revealed that this transcript initiated from the first residue of the consensus ATAAG late transcription start motif. Comparison with other baculoviral BDBPs showed that they all contained two conserved cAMP- and cGMP-dependent protein kinase phosphorylation motifs, R-R-R-S. The HearNPV P6.9 homologue is the longest BDBP found so far in baculoviruses.

Amino Acid Sequence↗

PubMed1991

The sequence between nucleotides 161 and 512 of cowpea mosaic virus M RNA is able to support internal initiation of translation in vitro.

Cowpea mosaic virus M RNA is translated in vitro as well as in vivo into two C-coterminal polyproteins of Mr 105K and 95K. Initiation of translation of the 95K protein gene occurs at an AUG codon at position 512 of M RNA, 351 nucleotides downstream of the initiation codon of the 105K protein gene at position 161. By employing an in vitro transcription and translation system it was determined that this 351 nucleotide sequence has the capacity to direct ribosomes to initiate translation at a downstream start codon. This effect is independent of the position of this sequence in an mRNA. Furthermore, evidence has been obtained that scanning ribosomes can bypass the AUG at position 161. Thus, both leaky scanning and internal entry are mechanisms for the initiation of translation of the 95K protein gene.

Base Sequence↗

PubMed2003

Forespore-specific expression of Bacillus subtilis yqfS, which encodes type IV apurinic/apyrimidinic endonuclease, a component of the base excision repair pathway.

The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the Esigma(G) regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the sigma(G) regulon. Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.

Bacillus subtilis↗

PubMed2025

The curious case of a heterozygous loss-of-function PSEN1 variant associated with early-onset Alzheimer's disease.

BACKGROUND: Over 300 mutations in PSEN1 have been identified as causes of early-onset Alzheimer's disease (EOAD). While these include missense mutations and a few insertions, deletions, or duplications, none result in open reading frame shifts, and all alter γ-secretase function to increase the long/short Aβ ratio. METHODS: We identified a novel heterozygous PSEN1 nonsense variant, c.325A > T, in a patient and his father, both presenting with EOAD, resulting in the substitution of lysine 109 with a premature stop codon at position (p.K109*). This produces a truncated 109 amino acid (aa) N-terminal PSEN1 fragment. Functional characterization was performed using overexpression models and a heterozygous mouse model (Psen1K109*/+). RESULTS: In overexpression models, downstream ATGs serve as alternative starting codons, generating a > 37 kDa and a > 27 kDa PSEN1 C-terminal fragment (PSEN1-CTFA and PSEN1-CTFB, respectively) that retain the two catalytic aspartates of γ-secretase. Heterozygous Psen1K109*/+ mice exhibited subtle phenotypic defects, including reduced Pen2 expression and mild APP-CTF accumulation. Notably, aged mice demonstrated significantly increased Psen2 protein expression, potentially contributing to an elevated Aβ42/Aβ38 ratio. CONCLUSIONS: These findings indicate that PSEN1 c.325A > T (p.K109*) is not a complete loss-of-function mutation. However, to what extent and by what mechanism it contributes to EOAD pathogenesis remains unclear. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s44477-025-00004-x.

Alzheimer’s disease↗

PubMed1990

Thiobacillus ferrooxidans mer operon: sequence analysis of the promoter and adjacent genes.

The merA upstream nucleotide (nt) sequence (1378 bp) of the Hg2(+)-resistance-encoding gene system (mer) in Thiobacillus ferrooxidans, was determined. The region contains two open reading frames: unidentified reading frame 1 (URF1) and merC. URF1 has 63-73% homology with those of Tn501, R100 and pDU1358, although the corresponding product has not yet been identified. Thiobacillus merC had 61% and 55% homology with R100 merC at the nt and amino acid (aa) sequence levels, respectively, and its product, consisting of 143 aa, was highly hydrophobic. No sequence homologous to merR, merT. merP or merD of R100 were observed on either strand. Within the 70-100-bp sequence upstream from the merC start codon. there was a sequence highly homologous to the promoter of merT of other Gram- mer systems. From primer extension and Northern-blot analyses, it became clear that merC and merA were co-transcribed from this putative transcription start point. The mer transcript in T. ferrooxidans was only detected in Hg2(+)-induced cells. Therefore, it was concluded that the T. ferrooxidans mer system is an inducible operon.

Amino Acid Sequence↗

PubMed1998

Genetic analysis of the central untranslated genome region and the proximal coding part of the F gene of wild-type and vaccine canine distemper morbilliviruses.

Located between the open reading frames encoding the matrix (M) and the fusion (F) protein the morbillivirus genome contains an unusually large non-coding intercistronic region (M-F UTR) of up to 5.6% of the full length genome. Any function(s) of this region have largely remained obscure. Here, we analyze the M-F UTR and the proximal coding part of the downstream F gene of several recent canine distemper morbillivirus (CDV) wild-type (wt) isolates and vaccine strains. While the F gene coding part appeared to be highly conserved (about 93% homology), a considerable degree of strain-specific variation of up to 21.4% was evident when comparing the M-F UTR. Phylogenetic analysis revealed a co-circulation of several contemporary CDV genotypes within a close geographic range (central Europe). A remarkably distinct CDV wt lineage, so far detected only in mustelids, is displayed. A rather non-scattered pattern of mutations within the M-F UTR suggested superimposition of RNA sequence and/or secondary structure constraints. Extensive folding in the long (460 nt) and moderately GC-rich 5'-UTR of the F mRNA was evident, particularly around the putative F protein translation initiation codon (AUG461 of the Onderstepoort vaccine strain). The region immediately preceding the putative F initiation site also harbored the only mutation unique to both vaccine strains within the F-5'UTR (position 455: Awt vs. Cvac). The putative F protein start codon, AUG461, was found to be mutated to AUA or GUA in all wt isolates analyzed and in another vaccine strain (Rockborn). Possible consequences for F protein translation initiation in wt CDV are discussed.

Animals↗

PubMed2005

Characterization of an exchangeable gene trap using pU-17 carrying a stop codon-beta geo cassette.

We have developed a new exchangeable gene trap vector, pU-17, carrying the intron-lox71-splicing acceptor (SA)-beta geo-loxP-pA-lox2272-pSP73-lox511. The SA contains three stop codons in-frame with the ATG of beta galactosidase/neomycin-resistance fusion gene (beta geo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre-mutant lox system, we successfully replaced the beta geo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp-deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the beta geo gene. Thus, using this pU-17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain-of-function mutation by replacing the beta geo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre-mediated recombination.

Animals↗

PubMed1998

The gene and processed pseudogenes of the rat mitochondrial single-strand DNA-binding protein: structure and promoter strength analyses.

The gene for the rat mitochondrial single-stranded DNA-binding protein (mtSSB) was amplified by PCR and isolated as several overlapping genomic clones. The clones encompassed the 5' untranslated sequence and all intron/exon junctions. The gene contained seven exons and six introns. The first exon contained only 5' untranslated sequence. The 16-amino acid mitochondrial targeting presequence, encoded by the second and third exons, was precisely bisected by intron 2. All intron donor and acceptor sites were consistent with the GT/AG consensus. The transcription start site was determined by primer-extension analysis to be 69bp upstream of the translation start codon. The upstream sequence lacked TATA and CCAAT boxes at the expected locations, but did contain several other potential regulatory elements including a GC box (Sp1-binding site) and three NRF-2 sites, one of which was located precisely beside the transcription start site. A 10 out of 12 imperfect NRF-1 site was located within the first exon. The 5' flanking sequence (-546 to +30) was shown to have strong promoter activity in transient transfection assays in primary rat hepatocytes and HepG2 cells. In addition, evidence for the existence of several mtSSB processed pseudogenes was obtained. These pseudogenes lacked introns and contained substitution and deletion mutations compared to the cDNA sequence. The 5' upstream region of one of the pseudogenes was analyzed and found to contain negligible promoter activity.

Animals↗

PubMed1999

Isochore specificity of AUG initiator context of human genes.

The efficiency of AUG start codon recognition in translation initiation is modulated by its sequence context. Here we investigated a non-redundant set of 5914 human genes and show that this context is different in genes located in different isochores. In particular, of the two main consensus start sequences, RCCaugR is five-fold more represented than AARaugR in genes from the GC-rich H3 isochores compared to genes from the GC-poor L isochores. Furthermore, genes located in GC-rich isochores have shorter 5' UTRs and stronger avoidance of upstream AUG than genes located in GC-poor isochores. This suggests that genes requiring highly efficient translation are located in GC-rich isochores and genes requiring fine modulation of expression are located in GC-poor isochores. This is in agreement with independent data from the literature concerning the location of housekeeping and tissue-specific genes, respectively.

5' Untranslated Regions↗

PubMed1996

Identification of a novel splice acceptor in the HIV-1 genome: independent expression of the cytoplasmic tail of the envelope protein.

Multiple splicing sites exist in the RNA genome of the human immunodeficiency virus type 1 (HIV-1). In a screen for subgenomic forms of the HIV-1 genome that could be transferred to fresh cells by virus infection, we identified a novel spliced variant of HIV-1 RNA that uses a hitherto unknown splice acceptor site within the envelope (Env) gene. We demonstrate that this splice acceptor is infrequently used in HIV-infected T cells. Interestingly, an AUG initiator codon is created at this splice junction which has the potential to direct the synthesis of the cytoplasmic tail of the Env gp41 protein. Transient transfection experiments with the new cDNA cloned in an expression vector demonstrated efficient utilization of this start codon and the C-terminus of the Env open reading frame. Independent expression of the 152 amino acid long, intracellular Env domain provides novel regulatory mechanisms for modulating viral infectivity and perhaps pathogenicity.

Base Sequence↗

PubMed1997

Evidence for gene conversion between tandemly duplicated cytoplasmic actin genes of Helicoverpa armigera (Lepidoptera:Noctuidae)

Tandemly duplicated actin genes have been isolated from a Helicoverpa armigera genomic library. Sequence comparisons with actin genes from other species suggest they encode cytoplasmic actins, being most closely related to the Bombyx mori A3 actin gene. The duplicated H. armigera actin genes, termed A3a and A3b, share 98.3% nucleotide sequence identity over their entire putative coding region. Analysis of the distribution of nucleotide differences shows the first 763 bp are identical between the two coding regions, with the 18 nucleotide changes occurring in the remaining 366 bp. This observation suggests a gene conversion event has taken place between the duplicated H. armigera A3a and A3b actin genes. Translation of the open-reading frames indicates the products of these genes are identical, apart from a single amino acid difference at codon 273. Polymerase chain reaction and northern blot analysis have shown both H. armigera A3a and A3b genes are expressed during pupal development and in the brain of newly enclosed adults. A region 5' of the H. armigera A3a actin gene start codon has been identified which contains regulatory sequences commonly found in the promoter region of actin genes, including TATA, CAAT, and CArG motifs.

Actins↗

PubMed1997

Involvement of two Sp1 elements in basal endothelial prostaglandin H synthase-1 promoter activity.

Prostaglandin H synthase-1 (PGHS-1) is a constitutively expressed key enzyme in the biosynthesis of physiologically important prostanoids. The promoter of the human PGHS-1 gene lacks a TATA box, has a very GC-rich region, and contains multiple transcription start sites. To identify the elements involved in the constitutive expression of the PGHS-1 gene, we constructed a 2075-base pair fragment (-2095 to -21 relative to the translation start codon) and a series of 5'-deletion mutants into a promoterless luciferase expression vector, which was transfected in HUVEC. Two important regions were identified. DNase I footprinting identified a protected segment, which contains an Sp1 binding site proximal to the transcription start sites. Band shift assays confirmed specific binding of Sp1 to this segment. Band shift assays further revealed specific binding of Sp1 to a distal region containing a canonical Sp1 site. Mutation of either Sp1 binding site significantly reduced the promoter activity. When both sites were mutated, the activity was reduced to 29% of that of the wild type. Mutation of Sp1 sites did not abrogate promoter activity stimulated by phorbol ester. These results indicate that binding of Sp1 or its related proteins to two widely separated Sp1 sites on the promoter region activates the basal PGHS-1 gene transcription.

Base Sequence↗