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PubMed1989

Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding p39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata.

The gene encoding the 39-kDa major structural protein (p39) of Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) was sequenced and transcriptionally mapped, and its expression was examined at various times postinfection. By Northern hybridization, primer extension, and S1 nuclease analysis, we identified p39 mRNAs of approximately 2600 nt. By primer extension analysis, we identified two major sets of transcripts which initiated around -48 and -96 nt upstream of the translation start codon. The transcription start sites were located within the conserved baculovirus late gene consensus sequence, ATAAG, which is duplicated in the p39 5' flanking region. In OpMNPV-infected Lymantria dispar cells, the p39 mRNAs were expressed abundantly at 24 and 36 hr p.i. but were present in lower quantities at 48 hr p.i. The p39 gene contained an open reading frame of 1053 nt which encodes a predicted protein of 351 amino acids with an estimated molecular weight of 39.5 kDa. Three repeats of the amino acid sequence Ala-Pro-Ala-Ala-Pro were identified at the C-terminus of the predicted p39 protein.

Amino Acid Sequence↗

PubMed1992

Development of a prokaryotic expression vector that exploits dicistronic gene organization.

For unknown reasons, levels of expression of foreign genes inserted into expression vectors in Escherichia coli have frequently been undetectable. The most critical step in the successful production of foreign proteins seems to be the initiation of translation. Since most prokaryotic genes are transcribed in a polycistronic form, we have devised a new prokaryotic expression system utilizing dicistronic gene organization. Downstream from a strong promoter and the gene encoding glutathione S-transferase from Schistosoma japonicum, various foreign genes were connected via a ribosome-binding site, a stop codon and a start codon. The VH domain of an immunoglobulin fused to the alpha subunit of tryptophan synthase, FK506-binding protein, cyclophilin, and a domain of a major histocompatibility complex antigen were successfully produced in E. coli as discrete polypeptides by this method.

Amino Acid Isomerases↗

PubMed1993

Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2.

We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from the previously reported cDNA, where it was suggested that translation might initiate at a CTG codon instead of at an ATG codon. Using the vaccinia virus (Vv) expression system, we demonstrate that translation starts at the conventional ATG start codon and not at the CTG codon. The protein is synthesized as a pro-protein of 282 amino acids (aa) that is post-translationally processed by removal of the initial 73 aa to a mature protein of 209 aa.

Amino Acid Sequence↗

PubMed1995

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

Amino Acid Sequence↗

PubMed2000

Assembly and functional expression of murine glutathione reductase cDNA: a sequence missing in expressed sequence tag libraries.

Glutathione reductase (GR) is a chemotherapeutic target. Murine GRcDNA, which contains 85% GC in the 38 codons following the start codon, was assembled from the PCR-amplified exon 1 and a downstream cDNA prior to expression in Escherichia coli as a His(6)-tagged protein. Recombinant GR, an FAD-containing homodimer, corresponds in its enzymic and spectral properties to GR isolated from murine Ehrlich ascites tumor cells. Another cDNA, representing GR with a mitochondrial targeting sequence, yielded two distinct enzymically active expression products.

Animals↗

PubMed1990

Characterization of the human N-CAM promoter.

In contrast with the complex series of splicing choices that generate the various membrane-associated isoforms of the neural cell-adhesion molecule alternative splicing of 5' exons does not contribute to additional molecular diversity. A single regulatory unit in genomic DNA, mapping to a 5 kb restriction-endonuclease-HindIII fragment, controls the expression of all major RNA size classes. DNA sequence analysis of a 2 kb fragment spanning the two major identified transcriptional initiation sites (194 and 188 bp from the ATG codon) and translation start codon indicates that the regulatory unit does not possess classical TATA or CCAAT motifs. The region of the putative promoter exhibits a GC-rich content and a high frequency of the dinucleotide CpG, both characteristics of a HTF(HpaII tiny fragments)-island. Introduction of deletion-mutant chimaeric-gene constructs into human and rodent N-CAM-expressing cell lines defines an active promoter region of 467 bp (-144 to -611 bp from the ATG codon). This region of genomic DNA contains consensus sites for the interaction of known transcriptional factors.

Animals↗

PubMed2000

In vivo studies of upstream regulatory cis-acting elements of the alcR gene encoding the transactivator of the ethanol regulon in Aspergillus nidulans.

The alcR gene of Aspergillus nidulans, which encodes the specific transactivator of the ethanol utilization pathway, is positively autoregulated and carbon catabolite repressed. Regulation by these two circuits occurs at the transcriptional level via the binding of the two regulators, AlcR and CreA, to their cognate targets respectively. We demonstrate here that out of two clustered putative AlcR repeated consensus sequences, only the palindromic target is functional in vivo. Hence, it is solely responsible for the alcR positive autogenous activation loop. Transcript mapping of the alcR gene showed that transcription initiation can occur at 553 bp and at or near 86 bp upstream of the start codon. These transcription start sites yield a transcript of 3.0 kb, which appears only under induced growth conditions, and of 2.6 kb, which is present under both induced and non-induced growth conditions respectively. Nine CreA consensus sites are present in the alcR promoter but only two pairs of two sites are functional in vivo. One of them is located in close proximity to the AlcR functional target. Within this pair, both sites are necessary to mediate a partial repression of alcR transcription. Disruption of either site results in an overexpression of alcR due to the absence of direct competition between AlcR and CreA for the same DNA region. The second functional pair of CreA sites is located between the two transcription initiation sites. Disruption of either of the two sites results in a totally derepressed alcR transcription, showing that they work as a pair constituting the more efficient repression mechanism. Thus, CreA acts by two different mechanisms: by competing with AlcR for the same DNA region and by an efficient direct repression. The latter mechanism presumably interfers with the general transcriptional machinery.

Aspergillus nidulans↗

PubMed2001

Structural and functional characterization of the 5' upstream promoter of the human Phox2a gene: possible direct transactivation by transcription factor Phox2b.

The specification of neurotransmitter identity is a critical step in neural development. Recent progresses have indicated that the closely related homeodomain factors Phox2a and 2b are essential for development of noradrenergic (NA) neuron differentiation, and may directly determine the neurotransmitter identity. With a long-term goal of understanding the regulatory cascade of NA phenotype determination, we isolated and characterized a hPhox2a genomic clone encompassing approximately 7.5 kb of the 5' upstream promoter region, the entire exon-intron structure, and approximately 4 kb of the 3' flanking region. Using mRNAs isolated from the Phox2a-expressing human cell line, both primer extension and 5'-rapid amplification of cDNA ends analyses identified a single transcription start site that resides 172 nucleotides upstream of the start codon. The transcription start site was preceded by a TATA-like sequence motif and transcripts from this site contained an additional G residue at the 5' position, supporting the authenticity of this site as the transcriptional start site of hPhox2a. We assembled hPhox2a-luciferase reporter constructs containing different lengths of the 5' upstream sequences. Transient transfection assays of these reporter constructs in both hPhox2a-positive and -negative cell lines show that 1.3-kb or longer upstream sequences of the hPhox2a gene may confer NA cell-specific reporter gene expression. Furthermore, cotransfection assays in the Phox2a-negative HeLa cell line show that forced expression of Phox2b, but not that of Phox2a or MASH1, significantly transactivates the transcriptional activity of hPhox2a. This study will provide a frame to further delineate the regulatory cascade of NA neuron differentiation.

Amino Acid Sequence↗

PubMed1989

Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation.

We present genetic and structural analyses of a fix operon conserved among rhizobia, fixGHI from Rhizobium meliloti. The nucleotide sequence of the operon suggests it may contain a fourth gene, fixS. Adjacent open reading frames of this operon showed an overlap between TGA stop codons and ATG start codons in the form of an ATGA motif suggestive of translational coupling. All four predicted gene products contained probable transmembrane sequences. FixG contained two cysteine clusters typical of iron-sulfur centers and is predicted to be involved in a redox process. FixI was found to be homologous with P-type ATPases, particularly with K+ pumps from Escherichia coli and Streptococcus faecalis but also with eucaryotic Ca2+, Na+/K+, H+/K+, and H+ pumps, which implies that FixI is a pump of a specific cation involved in symbiotic nitrogen fixation. Since prototrophic growth of fixI mutants appeared to be unimpaired, the predicted FixI cation pump probably has a specifically symbiotic function. We suggest that the four proteins FixG, FixH, FixI, and FixS may participate in a membrane-bound complex coupling the FixI cation pump with a redox process catalyzed by FixG.

Adenosine Triphosphatases↗

PubMed1989

Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12.

The bacterioferritin (BFR) of Escherichia coli K-12 is an iron-storage hemoprotein, previously identified as cytochrome b1. The bacterioferritin gene (bfr) has been cloned, sequenced, and located in the E. coli linkage map. Initially a gene fusion encoding a BFR-lambda hybrid protein (Mr 21,000) was detected by immunoscreening a lambda gene bank containing Sau3A restriction fragments of E. coli DNA. The bfr gene was mapped to 73 min (the str-spc region) in the physical map of the E. coli chromosome by probing Southern blots of restriction digests of E. coli DNA with a fragment of the bfr gene. The intact bfr gene was then subcloned from the corresponding lambda phage from the gene library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The bfr gene comprises 474 base pairs and 158 amino acid codons (including the start codon), and it encodes a polypeptide having essentially the same size (Mr 18,495) and N-terminal sequence as the purified protein. A potential promoter sequence was detected in the 5' noncoding region, but it was not associated with an "iron box" sequence (i.e., a binding site for the iron-dependent Fur repressor protein). BFR was amplified to 14% of the total protein in a bfr plasmid-containing strain. An additional unidentified gene (gen-64), encoding a relatively basic 64-residue polypeptide and having the same polarity as bfr, was detected upstream of the bfr gene.

Amino Acid Sequence↗

PubMed1995

Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus.

The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined. The sequence was found to contain an open reading frame (ORF) with a G+C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension. The ORF encodes a protein with an M(r) of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site. The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G+C content in the third position. The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence. Neither ATG nor GTG initiator codons were identified for the protein. However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon. The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension. The -10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 region was less similar. Comparison of the primary amino acid sequence of PHS of S. antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain. A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843. This phsA promoter-dependent xylE expression could be repressed by glucose in S. antibioticus when the organism was grown on glucose or galactose plus glucose. Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production.

Amino Acid Sequence↗

PubMed1998

Promoter characterization and constitutive expression of the Escherichia coli gcvR gene.

The Escherichia coli glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (gcv). In this study, the gcvR translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of the gcvR promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a gcvR-lacZ fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of gcv, respectively. In addition, it was shown that gcvR-lacZ expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream bcp gene overlaps the gcvR stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of gcvR had no effect on the expression of a downstream bcp-lacZ fusion, and primer extension analysis of the bcp promoter region demonstrated that bcp has its own promoter within the gcvR coding sequence. These results show that gcvR and bcp do not form an operon. Furthermore, the deletion of bcp from the chromosome had no effect on gcv-lacZ expression.

Amino Acid Sequence↗

PubMed2002

Cloning, molecular characterization, and transcriptional analysis of dnaK operon in a methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726.

Three structural genes that consist of a dnaK operon in a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 were cloned and characterized. The genes were clustered in the transcription order grpE-dnaK-dnaJ. The cloned grpE, dnaK, and dnaJ genes had open-reading frames of 474, 1,926, and 1,116 nucleotides, coding for proteins with calculated molecular masses of 17,390, 69,761, and 41,050, respectively. The overall identities in the deduced amino acid sequences of GrpE, DnaK, and DnaJ with those of the Escherichia coli homologs were 45.2, 74.5, and 61.2%, respectively. Northern blot analyses with grpE-, dnaK-, and dnaJ-specific probes revealed that the three genes are co-transcribed as a 4.0-kb mRNA. A primer extension analysis revealed that the transcription of the dnaK operon started at the nucleotide A that is located 28 bp upstream of the grpE start codon. The transcription start site was preceded by a putative promoter region 15'-CCCCGCTTGAA(13-bp)CCCCAATTT-3'], which is highly homologous to the consensus sequences of the E. coli sigma32-type heat shock promoter. The putative promoter worked under both normal and heat shock conditions in E. coli. The nature of the nucleotide sequence in the second half of the -35 region played a critical role during transcription. The heat shock mRNA was maximally produced at about 10 min after transfer of the Methylovorus sp. strain SS1 from 30 to 42 degrees C. The dnaK operon was also induced by ethanol, hydrogen peroxide, and NaCl shocks. The cloned dnaK operon complemented the E. coli dnaK mutant.

Bacterial Proteins↗

PubMed1994

Characterization of polyketide ketoreductase gene (MPKR) from midecamycin-producing strain (Streptomyces mycarofaciens 1748).

This paper presents the results about the expression of the polyketide ketoreductase gene from midecamycin-producing strain (S. mycarofaciens 1748), gene localization and nucleotide sequences analysis. A BamHI-BamHI 4.0 kg fragment isolated from a genomic library of midecamycin-producing strains containing the actIII homologous DNA was inserted into E. coli-Streptomyces shuttle vector pWHM3. A recombinant plasmid pCB4 was obtained and introduced into a 2-hydroxyaklavinone producer S. galilaeus ATCC 31671 that was a polyketide ketoreductase gene deficient mutant. The transformant produced aklavinone according to TLC and HPLC analyses. The BamHI-BamHI 4.0-kb fragment was inserted into pWHM3 in the reverted orientation with pCB4 and a recombinant plasmid pCBR4 was obtained. Introduction pCBR4 into S. galilaeus ATCC 31671 also resulted in the production of aklavinone. Thus we demonstrated that this gene encoded a polyketide ketoreductase which results in deoxygenation of 2-hydroxyaklavinone in C-2 position and that this gene has its own promoter. Restriction map analysis of pCB4 indicated that there were no sites for EcoRI and HindIII, but there were sites for BssHII, SalI, SphI and XhoI on the cloned gene fragment. The polyketide ketoreductase gene was located on a BssHII-BamHI 1.3-kb fragment from Southern hybridization result, using actIII gene as a probe, and that was confirmed by gene expression in S. galilaeus ATCC 31671. The nucleotide sequence analysis showed that the BssHII-BamHI 1.3-kb fragment contained an open reading frame (ORF). The protein coding region was assumed to start with an ATG start codon, end at an TAG stop codon. There was a 5nt (GGAGG) SD sequence at the upstream of start codon. A presumptive promoter consisted of 7 nt AACCGGA at the -10 region and 5 nt TTCGA at the -35 region. The deduced amino acid sequences of MPKR gene consisted of 261 aa residues. Its amino acid were compared with actIII gene coding protein sequences. The similarity was 77.4% and the identity was 66.7%.

Amino Acid Sequence↗

PubMed1995

An extranuclear expression system for analysis of cytoplasmic promoters of yeast linear killer plasmids.

Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.

Bacillus megaterium↗

PubMed2001

Studies of propionate toxicity in Salmonella enterica identify 2-methylcitrate as a potent inhibitor of cell growth.

Salmonella enterica serovar Typhimurium LT2 showed increased sensitivity to propionate when the 2-methylcitric acid cycle was blocked. A derivative of a prpC mutant (which lacked 2-methylcitrate synthase activity) resistant to propionate was isolated, and the mutation responsible for the newly acquired resistance to propionate was mapped to the citrate synthase (gltA) gene. These results suggested that citrate synthase activity was the source of the increased sensitivity to propionate observed in the absence of the 2-methylcitric acid cycle. DNA sequencing of the wild-type and mutant gltA alleles revealed that the ATG start codon of the wild-type gene was converted to the rare GTG start codon in the revertant strain. This result suggested that lower levels of this enzyme were present in the mutant. Consistent with this change, cell-free extracts of the propionate-resistant strain contained 12-fold less citrate synthase activity. This was interpreted to mean that, in the wild-type strain, high levels of citrate synthase activity were the source of a toxic metabolite. In vitro experiments performed with homogeneous citrate synthase enzyme indicated that this enzyme was capable of synthesizing 2-methylcitrate from propionyl-CoA and oxaloacetate. This result lent further support to the in vivo data, which suggested that citrate synthase was the source of a toxic metabolite.

Acyl Coenzyme A↗

PubMed1997

Initiation codon scanthrough versus termination codon readthrough demonstrates strong potential for major histocompatibility complex class I-restricted cryptic epitope expression.

Accumulating evidence shows that the repertoire of major histocompatibility complex class I-restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough translation, where the initiating AUG of a primary open reading frame is bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/34 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8(+) T cell (TCD8+) epitopes in alternative reading frames, each preceded by a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurring at the second AUG. We find that scanthrough translation has great potency in our system, with its impact being modulated, as predicted, by the base composition surrounding the first initiation codon, the number of start codons preceding the point of alternate reading frame initiation, and the efficiency with which the epitope itself is generated. Additionally, we investigated the efficiency of eukaryotic translation termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3' untranslated regions. In contrast with initiation codons, eukaryotic stop codons appear to be highly efficient at preventing expression of epitopes encoded in 3' untranslated regions, suggesting that 3' untranslated regions are not a common source of cryptic epitope substrate. We conclude that scanthrough is a powerful mechanism for the expression of epitopes encoded in upstream alternative open reading frames that may contribute significantly to TCD8+ responses and to tolerance induction.

Animals↗

PubMed1994

A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6.

The p41 DNA-binding protein of human herpesvirus 6 is an apparent processivity factor important for viral DNA replication. The p41 promoter was characterized to understand how this processivity factor is regulated. A single transcription start site and a functional TATA box are located 48 and 74 bp, respectively, upstream of the start codon. A reporter construct containing 1,027 bp of the sequence upstream of the p41 start codon was inactive in uninfected T cells but functioned as a strong promoter in human herpesvirus 6-infected cells. Mutational analysis identified a 21-bp element (the EA site) which is located at -73 to -52 bp relative to the transcription start site and is essential for promoter activity. The ability of the EA site to stimulate transcription optimally appears to be strictly dependent upon its distance from the p41 basal promoter. The EA site contains three overlapping sequences, a CAAT-enhancer-binding protein (C/EBP) transcription factor recognition site and two repeat elements. Mobility shift assays using the EA site identified four binding activities (C1 to C4). C1 and C2 are present in both uninfected and infected cells and do not contain C/EBP factors. In infected cells, point mutation of the EA site abrogates C1 and C2 binding activities and destroys transcriptional activity of the p41 promoter. C3 and C4 are present in uninfected cells only and were found to contain C/EBP factors. These findings indicate that in infected cells, transcriptional stimulation of the p41 promoter by the EA site requires C1 and C2 binding activities. These results further suggest that transcriptional activity may also depend upon the elimination of C3 and C4 binding activities.

Amino Acid Sequence↗