Nucleotide sequences and expression in Escherichia coli of the in-phase overlapping Pseudomonas aeruginosa plcR genes.

The translation products of chromosomal DNAs of Pseudomonas aeruginosa encoding phospholipase C (heat-labile hemolysin) have been examined in T7 promoter plasmid vectors and expressed in Escherichia coli cells. A plasmid carrying a 4.7-kilobase (kb) DNA fragment was found to encode the 80-kilodalton (kDa) phospholipase C as well as two more proteins with an apparent molecular mass of 26 and 19 kDa. Expression directed by this DNA fragment with various deletions suggested that the coding region for the two smaller proteins was contained in a 1-kb DNA region. Moreover, the size of both proteins was reduced by the same amount by an internal BglII-BglII DNA deletion, suggesting that they were translated from overlapping genes. Similar results were obtained with another independently cloned 6.1-kb Pseudomonas DNA, which in addition coded for a 31-kDa protein of opposite orientation. The nucleotide sequence of the 1-kb region above revealed an open reading frame with a signal sequence typical of secretory proteins and a potential in-phase internal translation initiation site. Pulse-chase and localization studies in E. coli showed that the 26-kDa protein was a precursor of a secreted periplasmic 23-kDa protein (PlcR1) while the 19-kDa protein (PlcR2) was mostly cytoplasmic. These results indicate the expression of Pseudomonas in-phase overlapping genes in E. coli.     

Identification and mapping of Epstein-Barr virus early antigens and demonstration of a viral gene activator that functions in trans.

The BamHI M DNA fragment of the Epstein-Barr virus (EBV) genome was inserted in two orientations into a simian virus 40-based expression vector, and the EBV-specific proteins produced in COS-7 monkey cells were examined. In one orientation, termed BamHI-M rightward reading frame 1 (BMRF1), a set of phosphoproteins ranging in size from 47,000 to 54,000 daltons was synthesized. These proteins reacted with monoclonal and polyclonal antisera, defining them as components of the EBV early antigen diffuse set of proteins (EA-D). The BamHI M DNA fragment in the opposite orientation, termed BamHI-M leftward reading frame 1 (BMLF1), directed the synthesis of a nuclear antigen detected by antibodies in serum from a patient with nasopharyngeal carcinoma. The BMLF1 antigen was not detected by monoclonal or polyclonal antibodies directed against the EA-D complex. A series of deletion mutants were constructed in the BamHI M DNA fragment, and the EA-D complex and BMLF1 antigen were mapped to discrete open reading frames in this DNA fragment. A test for several possible functions of these antigens showed that the BMLF1 antigen had the ability to activate or enhance, in trans, the level of expression of a gene under the control of the adenovirus early region 3 promoter or the simian virus 40 early promoter in the absence of its cis-acting enhancer. These experiments demonstrate a new gene function, encoded by EBV, that may be important in the positive regulation of viral or cellular genes.     

Three forms of rat basic fibroblast growth factor are made from a single mRNA and localize to the nucleus

The molecular weight of rat basic fibroblast growth factor is predicted to be 18 kDa when the amino acid sequence is read from the single AUG initiation codon found in the cDNA. DNA sequencing upstream of this AUG codon indicated, however, that there was an extended open reading frame. In vitro translation of the rat cDNA for basic FGF gave three proteins of 18.0, 21.5, and 22.0 kDa in equal abundance. The same proteins were produced in vivo by COS cells transfected with the rat cDNA. Deletion of 81 base pairs from the reading frame upstream of the AUG codon resulted in the expression of only one protein observed at 18.0 kDa. These results indicated that the 22.0 and 21.5 kDa forms of rat basic FGF were formed when translation initiates at the alternative upstream non-AUG codons. Rat cell lines and tissues were found to express all three forms of basic FGF protein. The cDNA was used to analyze the subcellular distribution of the different forms of rat basic FGF. Subcellular fractionation and immunofluorescence of transfected COS cells showed that all three forms of the protein localized preferentially in the nucleus. Expression of a truncated cDNA from which 81 base pairs (27 amino acids) of the upstream reading frame had been deleted, showed localization of the smaller form of bFGF alone in the nucleus. These results demonstrated that although the amino acids that were deleted from the N-terminus of rat basic fibroblast growth factor have a sequence characteristic of nuclear localization motifs, they are not obligatory for the transport of the growth factor into the nucleus. Nuclear extracts taken from transfected cells also contained two smaller proteins of 16 and 12 kDa that were detected by Western blot analysis. It is possible that these are proteolytic products of bFGF.     

Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains.

The replicase open reading frame lb (ORF1b) protein of equine arteritis virus (EAV) is expressed from the viral genome as an ORF1ab fusion protein (345 kDa) by ribosomal frameshifting. Processing of the ORF1b polyprotein was predicted to be mediated by the nsp4 serine protease, the main EAV protease. Several putative cleavage sites for this protease were detected in the ORF1b polyprotein. On the basis of this tentative processing scheme, peptides were selected to raise rabbit antisera that were used to study the processing of the EAV replicase ORF1b polyprotein (158 kDa). In immunoprecipitation and immunoblotting experiments, processing products of 80, 50, 26, and 12 kDa were detected. Of these, the 80-kDa and the 50-kDa proteins contain the putative viral polymerase and helicase domains, respectively. Together, the four cleavage products probably cover the entire ORF1b-encoded region of the EAV replicase, thereby representing the first complete processing scheme of a coronaviruslike ORF1b polyprotein. Pulse-chase analysis revealed that processing of the ORF1b polyprotein is slow and that several large precursor proteins containing both ORF1a- and ORF1b-encoded regions are generated. The localization of ORF1b-specific proteins in the infected cell was studied by immunofluorescence. A perinuclear staining was observed, which suggests association with a membranous compartment.     

Molecular cloning and sequence of the thdF gene, which is involved in thiophene and furan oxidation by Escherichia coli.

Our previous work resulted in the isolation of mutant strains of Escherichia coli K-12 which were able to oxidize furans and thiophenes as a result of mutations in several novel genes. Some of the genes involved in thiophene oxidation were cloned into the multicopy vector pUC19. The plasmid pKA10 carries a 3.8-kb chromosomal fragment which encodes a previously undiscovered gene involved in thiophene oxidation. Three proteins with approximate molecular sizes of 48, 30, and 26 kDa were overproduced by cells carrying pKA10. Maxicell experiments and DNA sequence analysis indicated that the 48- and 26-kDa proteins are encoded by pKA10, whereas the 30-kDa protein is apparently chromosomally derived. A cassette specifying kanamycin resistance was inserted into various sites on pKA10. An insertion which abolished the 48-kDa protein also abolished thiophene oxidation. Chromosomal integration of pKA10::Kan allowed us to locate the chromosomal insert of pKA10 at 84 min on the E. coli genetic map by transduction. Since no previously identified genes involved in thiophene metabolism are located in this region, we designated the gene for the 48-kDa protein as thdF. Sequencing of the 3.8-kb insert revealed an overlap of several hundred bases with the regulatory and structural regions of the tnaA gene, which is also located at 84 min. The 26-kDa protein is probably truncated tnaA protein. An open reading frame corresponding to the 48-kDa thdF protein was located next to the tnaA gene, which encodes tryptophanase, but was transcribed in the opposite sense.     

Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter.

The human cytomegalovirus (HCMV) XbaI E cloned DNA fragment of approximately 20 kilobases can complement an adenovirus mutant (dl312) defective in the E1a viral gene product (D. J. Spector and M. J. Tevethia, Virology 151:329-338, 1986). This viral DNA fragment contains three immediate-early (IE) genes between 0.709 and 0.751 map units (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). Two of the IE genes, IE1 and IE2, were isolated and tested for a role in regulating viral gene expression. Since HCMV early and late promoters require additional characterization, the chloramphenicol acetyl transferase (cat) gene, driven by the adenovirus E2 promoter, was used as an indicator of gene expression. cat expression from this heterologous viral promoter was shown to be stimulated by HCMV at early times after infection. The IE1 gene product did not function independently in activating this promoter. The IE2 gene products could independently stimulate the expression of a plasmid of a plasmid when the cat gene was placed downstream of the inducible E2 promoter (E2CAT). Five proteins of different sizes have been predicted to originate from IE2, depending on mRNA splicing. The protein products specified by the IE2 gene were characterized with an antibody to a synthetic peptide according to the open reading frame of exon 2. Three of the five proteins are encoded by exon 2. Three viral proteins of 82, 54, and 28 kilodaltons (kDa) were detected. The exons contained in the region designated as IE2a have open reading frames that could code for two of the smaller proteins of 27 and 30 kDa. This region, when driven by the HCMV enhancer, could independently stimulate gene expression from E2CAT to a high level. A plasmid with the HCMV enhancer upstream of exons, that could code for the HCMV IE2 proteins of 48 and 51 kDa, as well as 27- and 30-kDa proteins, also stimulated E2CAT expression but at a lower level. The activity of this plasmid was augmented by the IE1 gene product, despite the fact that the latter gene product alone was inactive. It is proposed that the HCMV IE region 2 gene products are involved in the regulation of viral or host cell promoters either independently or in combination with other HCMV IE proteins.     

Cleavage of Treponema denticola PrcA polypeptide to yield protease complex-associated proteins Prca1 and Prca2 is dependent on PrtP

Analysis of potential virulence factors of oral spirochetes focuses on surface and secreted proteins. The Treponema denticola chymotrypsin-like protease (CTLP) is implicated in degradation of host cell molecules and contributes to tissue invasion. The CTLP complex, composed of the 72-kDa PrtP protein and two auxiliary proteins with molecular masses of approximately 40 and 30 kDa, is also involved in localization and oligomerization of the T. denticola major surface protein (Msp). The larger auxiliary protein was reported to be encoded by an open reading frame (ORF2) directly upstream of prtP. The deduced 39-kDa translation product of ORF2 contains a sequence matching the N-terminal sequence determined from one of the CTLP complex proteins. No proteins with significant homology are known, nor was information available on the third protein of the complex. DNA sequence analysis showed that ORF2 extended an additional 852 bp upstream of the reported sequence. The complete gene, designated prcA, encodes a predicted N-terminally-acylated polypeptide of approximately 70 kDa. Isogenic mutants with mutations in prtP, prcA, and prcA-prtP all lacked CTLP protease activity. The prcA mutant lacked all three CTLP proteins. The prcA-prtP mutant produced only a C-terminally-truncated 62-kDa PrcA protein. The prtP mutant produced a full-length 70-kDa PrcA. Immunoblot analysis of recombinant PrcA constructs confirmed that PrcA is cleaved to yield the two smaller proteins of the CTLP complex, designated PrcA1 and PrcA2. These data indicate that PrtP is required for cleavage of PrcA and suggest that this cleavage may be required for formation or stability of outer membrane complexes.     

Cloning and characterization of a new member of the T-box gene family

The T-box is a strongly conserved protein domain, 174 to 186 amino acids in length, that binds DNA. Many genes from many species have been shown to encode T-box domain-containing proteins. Here we report the cloning and characterization of a novel T-box gene, TBX21. The human cDNA contains an open reading frame encoding a 535-amino-acid protein with a predicted molecular mass of 58.3 kDa. Except for the T-box sequence, database searches revealed no significant homology to any known sequences at the nucleotide or protein level. In addition to the human cDNA sequence, we report the cDNA sequence of the murine homologue, the structure and organization of the murine Tbx21 gene, and its localization to mouse chromosome 11. TBX21 expression was detected in peripheral blood lymphocytes, spleen, lung, and natural killer cells.     

Gene 26 of bacteriophage T4 basal plate. I. Two expression products of the cloned gene

We have cloned the 1.9 kb EcoRV-BglII DNA fragment with T4 genes 51, 26, and 25 into the expression plasmid pT7-5 carrying a T7 promoter. The resulting recombinant plasmid, pRR5-3, contained T4 genes 26 and 25 in the correct orientation for expression. We expressed these genes using the T7 RNA polymerase/promoter system and the synthesis of three polypeptides with the molecular masses of approximately 24, 15, and 8-9 kDa was observed. Expression of genes from the subcloned DNA fragments and from the fragments carrying deletions was studied as well and the 15 kDa protein appeared to be the product of gene 25, while 24 kDa and 8-9 kDa proteins were identified as products of gene 26. The 8-9 kDa protein was shown to be expressed from the end region of gene 26. Having analysed the proteins expressed from the fragments carrying fusion of genes 26 and 25 we supposed two products of gene 26 to be encoded by the same open reading frame.     

Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni.

Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure.     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

Cloning and characterization of phosphoglucomutase and phosphomannomutase derived from Sphingomonas chungbukensis DJ77

The enzymes phosphoglucomutase (PGM) and phosphomannomutase (PMM) play an important role in the synthesis of extracellular polysaccharide. By colony hybridization of the fosmid library of Sphingomonas chungbukensis DJ77, an open reading frame (ORF-1) of 1,626 nucleotides, whose predicted product is highly homologous with other PGM proteins from several bacterial species, was identified. An additional open reading frame (ORF-2) of 1,437 nucleotides was identified, and its encoded protein shows a high level of similarity with the PGM/PMM protein family. The two genes were cloned into a bacterial expression vector pET-15b (+) and expressed in Escherichia coli as fusion proteins with (His)(6)-tag. Both recombinant proteins (designated as SP-1 and SP-2 for ORF-1 and ORF-2, respectively) exhibited PGM and PMM activities. The molecular masses of subunits of SP-1 and SP-2 were estimated to be around 58 and 51 kDa from SDS-PAGE, respectively. However, molecular masses of SP-1 and SP-2 in their native condition were determined to be approximately 59.5 and 105.4 kDa, according to non-denaturing PAGE, respectively. The SP-1 protein has a preference for glucose-1-phosphate rather than mannose-1-phosphate, while the preferred substrate of SP-2 is mannose-1-phosphate. Thus, the existence of two proteins with bifunctional PGM/PMM activities was first found S. chungbukensis DJ77.