Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5'' leader.

The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.     

Sulfide oxidation in gram-negative bacteria by expression of the sulfide-quinone reductase gene of Rhodobacter capsulatus and by electron transport to ubiquinone.

The oxidation of sulfide was studied in recombinant bacteria expressing the sulfide-quinone reductase gene (sqr) from Rhodobacter capsulatus. Sulfide was oxidized by the Escherichia coli strain W3110 harboring the sqr construct (pKKSQ) under anaerobic conditions and nitrate was utilized as a terminal electron acceptor. Following the oxidation, elemental sulfur and nitrite were produced as the final reaction products. This activity was retained in the membrane preparation and was sensitive towards antimycin A, stigmatellin, and azide. As a consequence of the ubiquinone deficiency, this activity was markedly decreased. In additon, by recovery of ubiquinone, the oxidation was also restored to rates similar to those of the wild-type strain. These results indicate that sulfide oxidation in this strain occurs via the quinone pool in vivo, and that this sulfide-quinone reductase (SQR) in particular utilizes ubiquinone as a more appropriate electron acceptor than menaquinone or demetylmenaquinone. To our knowledge, this is the first study to show a direct interaction between SQR and ubiquinone in cells. When expressed in Pseudomonas putida and Rhizobium meliloti, the SQR conferred on these organisms the ability to oxidize sulfide as well as E. coli in vivo.     

The herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate.

The herpes simplex virus 1 open reading frames UL26 and UL26.5 are 3' coterminal. The larger, UL26 open reading frame encodes a protein approximately 80,000 in apparent molecular weight and contains the promoter and coding sequence of the UL26.5 gene, which specifies a capsid protein designated infected cell protein 35. The larger product contains in its entirety the amino acid sequence of the smaller protein. We report that the UL26 gene encodes a protease which catalyzes its own cleavage and that of the more abundant product of UL26.5. By inserting the coding sequence of an epitope to a cytomegalovirus monoclonal antibody and homologs of the immunoglobulin G binding domain of staphylococcal protein A into the 3' termini of the coding domains of the two open reading frames, we identified both products of the cleavage and determined that the cleavage site is approximately 20 amino acids from the carboxyl termini of both proteins.     

Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli

The nucleotide sequence of a 6.5 kilobasepair chromosomal DNA fragment encoding the anaerobic dimethylsulphoxide (DMSO) reductase operon of Escherichia coli has been determined. The DMSO reductase structural operon was shown to consist of three open reading frames, namely dmsABC, encoding polypeptides with predicted molecular weights of 87,350, 23,070, and 30,789 Daltons, respectively. The DMS A polypeptide displayed a high degree of amino acid sequence homology with the single-subunit enzyme, biotin sulphoxide reductase (bisC) and with formate dehydrogenase (fdhF), suggesting that the active site and molybdopterin cofactor binding site that is common to these enzymes is located in the DMS A subunit. A comparison of the predicted N-terminal amino acids of the dmsA gene product to those of the 82,600 subunit of purified DMSO reductase indicated that post-translational processing of a 16 amino acid peptide at the amino terminus of DMS A had occurred. The DMS B polypeptide contains 16 cysteine residues organized in four clusters, two of which are typical of 4Fe-4S binding domains. The DMS C polypeptide is composed of eight segments of hydrophobic amino acids of appropriate length to cross the cytoplasmic membrane, suggesting that this subunit functions to anchor the enzyme to the membrane.     

Dihydrofolate reductase of the extremely halophilic archaebacterium Halobacterium volcanii. The enzyme and its coding gene

Halobacterium volcanii mutants that are resistant to the dihydrofolate reductase inhibitor trimethoprim contain DNA sequence amplifications. This paper describes the cloning and nucleic acid sequencing of the amplified DNA sequence of the H. volcanii mutant WR215. This sequence contains an open reading frame that codes for an amino acid sequence that is homologous to the amino acid sequences of dihydrofolate reductases from different sources. As a result of the gene amplification, the trimethoprim-resistant mutant overproduces dihydrofolate reductase. This enzyme was purified to homogeneity using ammonium sulfate-mediated chromatographies. It is shown that the enzyme comprises 5% of the cell protein. The amino acid sequence of the first 15 amino acids of the enzyme fits the coding sequence of the gene. Preliminary biochemical characterization shows that the enzyme is unstable at salt concentrations lower than 2 M and that its activity increases with increase in the KCl or NaCl concentrations.     

Cloning and characterization of sulfite dehydrogenase, two c-type cytochromes, and a flavoprotein of Paracoccus denitrificans GB17: essential role of sulfite dehydrogenase in lithotrophic sulfur oxidation.

A 13-kb genomic region of Paracoccus dentrificans GB17 is involved in lithotrophic thiosulfate oxidation. Adjacent to the previously reported soxB gene (C. Wodara, S. Kostka, M. Egert, D. P. Kelly, and C. G. Friedrich, J. Bacteriol. 176:6188-6191, 1994), 3.7 kb were sequenced. Sequence analysis revealed four additional open reading frames, soxCDEF. soxC coded for a 430-amino-acid polypeptide with an Mr of 47,339 that included a putative signal peptide of 40 amino acids (Mr of 3,599) with a RR motif present in periplasmic proteins with complex redox centers. The mature soxC gene product exhibited high amino acid sequence similarity to the eukaryotic molybdoenzyme sulfite oxidase and to nitrate reductase. We constructed a mutant, GBsoxC delta, carrying an in-frame deletion in soxC which covered a region possibly coding for the molybdenum cofactor binding domain. GBsoxC delta was unable to grow lithoautotrophically with thiosulfate but grew well with nitrate as a nitrogen source or as an electron acceptor. Whole cells and cell extracts of mutant GBsoxC delta contained 10% of the thiosulfate-oxidizing activity of the wild type. Only a marginal rate of sulfite-dependent cytochrome c reduction was observed from cell extracts of mutant GBsoxC delta. These results demonstrated that sulfite dehydrogenase was essential for growth with thiosulfate of P. dentrificans GB17. soxD coded for a periplasmic diheme c-type cytochrome of 384 amino acids (Mr of 39,983) containing a putative signal peptide with an Mr of 2,363. soxE coded for a periplasmic monoheme c-type cytochrome of 236 amino acids (Mr of 25,926) containing a putative signal peptide with an Mr of 1,833. SoxD and SoxE were highly identical to c-type cytochromes of P. denitrificans and other organisms. soxF revealed an incomplete open reading frame coding for a peptide of 247 amino acids with a putative signal peptide (Mr of 2,629). The deduced amino acid sequence of soxF was 47% identical and 70% similar to the sequence of the flavoprotein of flavocytochrome c of Chromatium vinosum, suggesting the involvement of the flavoprotein in thiosulfate oxidation of P. denitrificans GB17.     

Functional Properties of Pro Region of Escherichia coli Heat-Stable Enterotoxin

Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as the 72-amino-acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre-pro-nuclease, pre-mST-nuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.     

Expression and Export of Pseudomonas putida NTU-8 Creatinase by Escherichia coli Using the Chitinase Signal Sequence of Aeromonas hydrophila

The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.     

Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein.

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.     

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.     

Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5

The "P" gene of the paramyxovirus SV5 encodes two known proteins, P (Mr approximately equal to 44,000) and V (Mr approximately equal to 24,000). The complete nucleotide sequence of the "P" gene has been obtained and is found to contain two open reading frames, neither of which is large enough to encode the P protein. We have shown that the P and V proteins are translated from two mRNAs that differ by the presence of two nontemplated G residues in the P mRNA. These two additional nucleotides convert the two open reading frames to one of 392 amino acids. The P and V proteins are amino coterminal and have 164 amino acids in common. The unique C terminus of V consists of a cysteine-rich region that resembles a cysteine-rich metal binding domain. An open reading frame that contains this cysteine-rich region exists in all other paramyxovirus "P" gene sequences examined, which suggests that it may have important biological significance.     

Sequence organization of repetitive elements in the flanking regions of the chloroplast ribosomal unit of Chlamydomonas reinhardii.

The flanking regions and the end of the chloroplast ribosomal unit of Chlamydomonas reinhardii have been sequenced. The upstream region of the ribosomal unit contains three open reading frames coding for 111, 117 and 124 amino acids, respectively. The latter polypeptide is partially related to the ribosomal protein L16 of E. coli. Two of the open reading frames overlap each other and are oriented in opposite direction. The region between these open reading frames and the 5' end of the 16S rRNA gene contains numerous short direct and inverted repeats which can be folded into large stem-loop structures. Sequence elements that resemble prokaryotic promoters are found in the same region. Several of the repeated elements are distributed throughout the non-coding regions of the chloroplast inverted repeat. Sequence comparison between the 5S rRNA and its gene does not reveal any significant sequence heterogeneity between the chloroplast 5S rRNA genes.     

Construction, expression, and localization of a CycA::PhoA fusion protein in Rhodobacter sphaeroides and Escherichia coli.

We demonstrated the utility of Escherichia coli alkaline phosphatase, encoded by phoA, as a reporter molecule for genetic fusions in Rhodobacter sphaeroides. A portion of the R. sphaeroides cycA gene was fused to phoA, yielding a fusion protein comprising the putative signal sequence and first 10 amino acids of the cytochrome c2 apoprotein joined to the sixth amino acid of alkaline phosphatase. The fusion protein was efficiently transported to the periplasm of R. sphaeroides as determined by enzyme activity, Western immunoblot analysis, and immunogold electron microscopy. We also documented the ability of an R. sphaeroides mutant, RS104, with gross defects in photosynthetic membrane morphology to efficiently recognize and translocate the fusion protein to the periplasmic compartment. The inclusion of 500 base pairs of R. sphaeroides DNA in cis to the cycA structural gene resulted in a 2.5-fold increase in alkaline phosphatase activity in photosynthetically grown cells compared with the activity in aerobically grown cells, demonstrating that the fusion protein is regulated in a manner similar to that of cytochrome c2 regulation. We also constructed two pUC19-based plasmids suitable for the construction of translational fusions to phoA. In these plasmids, translational fusions of phoA to the gene under consideration can be made in all three reading frames, thus facilitating construction and expression of fusion protein systems utilizing phoA.     

Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon

Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ. Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.