Polypeptides encoded by the mer operon.

HgCl2-induced polypeptides synthesized by Escherichia coli minicells containing recombinant or natural HgR plasmids were labeled with [35S]methionine and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All plasmids examined encoded two heavily labeled, HgCl2-inducible polypeptides of 69,000 and 12,000 daltons. Most plasmids also encoded two additional HgCl2-inducible proteins in the 14,000- to 17,000-dalton range. Antiserum prepared against a purified mercuric ion reductase reacts with the 69,000-dalton polypeptide and a minor 66,000-dalton protein seen in several different HgR minicells. Recombinant plasmids constructed from portions of mer DNA from the IncFII plasmid NR1 were also analyzed in the minicell system. Five HgCl2-inducible polypeptides (69,000, 66,000, 15,100, 14,000, and 12,000 daltons) were synthesized in minicells carrying pRR130, a recombinant derivative containing the EcoRI-H and EcoRI-I restriction fragments of NR1. The EcoRI-H fragment of NR1 encodes the three small mer proteins of 15,100, 14,000, and 12,000 daltons and the amino-terminal 40,000 daltons of the mercuric ion reductase monomer.     

Polypeptides encoded by cryptic plasmids from Neisseria gonorrhoeae

Almost all clinical isolates of Neisseria gonorrhoeae harbor a small, phenotypically cryptic plasmid of approximately 4.1 kb. In this study several polypeptides encoded by two variants of such plasmids, one (pSB01C) having a deletion of approximately 50 bp as compared to the other (p31788C), have been identified, and the position of the genes for two of the proteins determined. The cryptic plasmids were cloned into the HindIII site of the vectors pBR322 and pACYC184. The resulting recombinant plasmids were transformed into the Escherichia coli minicell producing strain DS410 (minA, minB) and the plasmid-encoded proteins analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pSB01C derivatives express two distinct proteins of 22 and 16 kDa and p31788C two other proteins of 24 and 18.5 kDa. Additionally, both plasmids express common proteins of 32.5, 9, and 7.5 kDa. The genes coding for the 24- and the 7.5 kDa proteins have been mapped by restriction enzyme analysis of Tn5 insertions suppressing the expression. The additional 50 bp in p31788C are localized to the coding region of the 24-kDa protein, and the 22-kDa protein of pSB01C is possibly a shortened form of the former due to the lacking 50 bp.     

Plasmids containing 2 new variants of the E.coli lacZ gene

New plasmids containing partially deleted lacZ genes were obtained. These genes determine high-level synthesis of polypeptides of molecular mass 43-45 and 49-51 kD under the control of the lambda phage PR-promoter; inspite of the deletion, E. coli cells carrying new plasmids were found to possess beta-galactosidase activity. Use of these plasmids as new expression vectors is suggested.     

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.     

Gene cluster of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans: characterization of four structural gene products.

In previous reports from our laboratory, the three structural genes (NQO1, NQO2, and NQO3) of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans were characterized [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991) Biochemistry 30, 6422-6428; (1991) Biochemistry 30, 8678-8684; (1992) Arch. Biochem. Biophys. 296, 40-48]. In this report, the four structural genes NQO4, NQO5, NQO6, and NQO7 of the same Paracoccus denitrificans oxidoreductase were cloned and sequenced. On the basis of sequence homology and immunological cross-reactivity, these genes encode counterparts of the 49-, 30-, and 20-kDa polypeptides and the mitochondrial DNA ND3 polypeptides of bovine mitochondrial complex I. These seven structural genes were found to be located in the same gene cluster. The order of the seven structural genes of the Paracoccus NADH-quinone oxidoreductase in the gene cluster is NQO7, NQO6, NQO5, NQO4, NQO2, NQO1, and NQO3. Upstream of the NQO7 gene, an open reading frame encoding a predicted polypeptide homologous to the UV repair enzyme A of Escherichia coli and Micrococcus lysodeikticus was detected. The 5'-terminus of the gene cluster carrying the Paracoccus NADH-quinone oxidoreductase was studied, and the possible promoter region is discussed. The NQO4 and NQO5 genes appear to code for the M(r) 48,000 and 21,000 polypeptides of the isolated Paracoccus NADH dehydrogenase complex [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311] on the basis of amino acid analyses and N-terminal protein sequence analyses. The antisera to the bovine complex I 49- and 30-kDa polypeptides cross-reacted with the Paracoccus 48- and 21-kDa subunits, respectively.     

Synaptonemal complex proteins

Synaptonemal complexes were isolated from rate spermatocytes for the purpose of biochemical and morphological analysis. Several monoclonal antibodies were elicited against purified synaptonemal complexes to study the composition and assembly of these structures. Four classes of antibodies could be discriminated according to the polypeptides that they recognize on Western blots of purified synaptonemal complexes, namely antibodies recognizing (i) a 190-kDa polypeptide; (ii) a 30- and a 33-kDa polypeptide; (iii) two polypeptides with molecular weights of about 120 kDa; and (iv) polypeptides with molecular weights of 66-55 kDa. The localization of these antigens within spermatocytes was analyzed light microscopically, by means of the immunoperoxidase technique and ultrastructurally, by immunogold labelling of surface-spread spermatocytes. The 66- to 55-kDa polypeptides are not confined to synaptonemal complexes; rather, these polypeptides appear to be chromosomal components. The 190-, 30-, and 33-kDa polypeptides make part of the lateral elements of paired as well as unpaired segments of synaptonemal complexes. The 120-kDa polypeptides were localized on the inner edge of the lateral elements, specifically in paired segments of synaptonemal complexes. The distribution of the 190-, 120-, 30-, and 33-kDa polypeptides within the testis was analyzed by immunofluorescence staining of cryostat sections. All these polypeptides turned out to be specific for nuclei of zygotene up to and including diplotene spermatocytes. Only in some early spermatids could the 190-, 30-, and 33-kDa polypeptides be detected, presumably in remnants of synaptonemal complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gene amplification on mercuric ion reduction activity of Escherichia coli.

The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold for the 47-fold gene amplification. In contrast, the rate of the cytoplasmic reduction reaction, measured in permeabilized cells, increased linearly with increasing gene copy number, resulting in a 6.8-fold overall amplification. RNA hybridizations indicated that mRNA of the cytoplasmic mercuric reductase (merA gene product) increased 11-fold with the 47-fold gene amplification, while mRNA of the transport protein (merT gene product) increased only 5.4-fold. Radiolabeled proteins produced in maxicells were used to correlate the expression levels of the mer polypeptides with the measured reduction rates. The results indicated that, with increasing gene copy number, there was an approximately 5-fold increase in the merA gene product compared with a 2.5-fold increase in the merT gene product. These data demonstrate a parallel increase of Hg2+ reduction activity and transport protein expression in intact cells with plasmids with different copy numbers. In contrast, the expression level of the mercuric reductase gene underwent higher amplification than that of the transport genes at both the RNA and protein levels as plasmid copy number increased.     

Effect of gene amplification on mercuric ion reduction activity of Escherichia coli.

The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold for the 47-fold gene amplification. In contrast, the rate of the cytoplasmic reduction reaction, measured in permeabilized cells, increased linearly with increasing gene copy number, resulting in a 6.8-fold overall amplification. RNA hybridizations indicated that mRNA of the cytoplasmic mercuric reductase (merA gene product) increased 11-fold with the 47-fold gene amplification, while mRNA of the transport protein (merT gene product) increased only 5.4-fold. Radiolabeled proteins produced in maxicells were used to correlate the expression levels of the mer polypeptides with the measured reduction rates. The results indicated that, with increasing gene copy number, there was an approximately 5-fold increase in the merA gene product compared with a 2.5-fold increase in the merT gene product. These data demonstrate a parallel increase of Hg2+ reduction activity and transport protein expression in intact cells with plasmids with different copy numbers. In contrast, the expression level of the mercuric reductase gene underwent higher amplification than that of the transport genes at both the RNA and protein levels as plasmid copy number increased.     

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Two states of the conformation of 21S outer arm dynein coupled with ATP hydrolysis

Tryptic digestion of 21S outer arm dynein from sea urchin sperm flagella in the presence of ATP (or ADP) and vanadate produced quite different polypeptides from those obtained in the absence of ATP (ADP) and/or vanadate (Inaba and Mohri (1989) J. Biol. Chem. 264, 8384-8388). The 21S dynein heavy chains were consistently digested into 165- and 135-kDa polypeptides in the absence of both ATP (ADP) and vanadate. In the presence of 2 mM ADP and 100 microM vanadate, 300-kDa polypeptide, which appeared to be a precursor of 165- and 135-kDa polypeptides, became less accessible to trypsin, and 165- and 135-kDa polypeptides were digested into 150-/148-kDa and 96-kDa polypeptides, respectively. Quantitative analysis of the degradation of 165- and 135-kDa polypeptides showed that the conformations of these polypeptides change remarkably in the presence of ATP (ADP) and vanadate, and slightly in the presence of ATP gamma S. Photoaffinity labeling with 8-azidoadenosine 5'-triphosphate and vanadate-mediated photocleavage of dynein heavy chains revealed that both adenine- and gamma-Pi-binding sites were located on 165- and 150-/148-kDa polypeptides, but not on 135-kDa polypeptide. These results suggest that the conformational change occurring in the 165-kDa region on binding ATP spreads to the 135-kDa region and causes the conformational change of the 135-kDa region.     

Cold-stress response in the amphibian oocyte: changes in synthesis and nucleocytoplasmic distribution of some proteins

In vivo cold stress was found to induce characteristic changes in the protein synthesis pattern of Pleurodeles waltl oocytes, as analyzed by two-dimensional gel electrophoresis. The nature of the response varied with the duration and intensity of stress. After a short period of cold stress (12 h at 8°C), synthesis and intracellular distribution of polypeptides were dramatically disturbed. There occured: 1) a reduction in synthesis of several polypeptides, including two major polypeptides (54-kDa and 47-kDa); 2) changes in distribution of polypeptides in oocyte, ie some polypeptides (185- and 96-kDa) were blocked in the cytoplasm, while other polypeptides (82-, 74-, 72- and 68-kDa, actin and nucleoplasmin) continued to enter the nucleus, but were quantitatively reduced; 3) no changes in the distribution of two nuclear polypeptides (53- and 43-kDa); 4) changes in the relative quantities of β- and γ-actin and preferential migration of γ-actin towards the nucleus. After a long period of in vivo cold stress (5 days at 8°C), we noted a partial recuperation of synthesis and nuclear migration (except for a 96-kDa polypeptide), but a persistent perturbation at the level of actin. For more drastic stress conditions (4°C), such a recuperation of protein synthesis was never observed.     

Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant.

The mer operon from a strain of Thiobacillus ferrooxidans (C. Inoue, K. Sugawara, and T. Kusano, Mol. Microbiol. 5:2707-2718, 1991) consists of the regulatory gene merR and an operator-promoter region followed by merC and merA structural genes and differs from other known gram-negative mer operons. We have constructed four potential shuttle plasmids composed of a T. ferrooxidans-borne cryptic plasmid, a pUC18 plasmid, and the above-mentioned mer determinant as a selectable marker. Mercury ion-sensitive T. ferrooxidans strains were electroporated with constructed plasmids, and one strain, Y4-3 (of 30 independent strains tested), was found to have a transformation efficiency of 120 to 200 mercury-resistant colonies per microgram of plasmid DNA. This recipient strain was confirmed to be T. ferrooxidans by physiological, morphological, and chemotaxonomical data. The transformants carried a plasmid with no physical rearrangements through 25 passages under no selective pressure. Cell extracts showed mercury ion-dependent NADPH oxidation activity.     

Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg2+ transport systems.

In Salmonella typhimurium, the corA, mgtA, and mgtB loci are involved in active transport of Mg2+ (S. P. Hmiel, M. D. Snavely, C. G. Miller, and M. E. Maguire, J. Bacteriol. 168:1444-1450, 1988; S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). In this study, the gene products coded for by the corA, mgtA, and mgtB genes were identified by using plasmid expression in Escherichia coli maxicells. Complementation was assessed by introducing plasmids into a Mg2+-dependent corA mgtA mgtB strain and determining the ability of the plasmid to restore growth on medium without a Mg2+ supplement. Complementing plasmids containing corA expressed a 42-kilodalton (kDa) protein. This protein was not expressed by plasmids containing insertions or deletions that eliminated complementation. A plasmid containing mgtA expressed 37- and 91-kDa gene products. Data obtained with subclones and insertions in this plasmid indicated that plasmids expressing only the 91-kDa polypeptide complemented; plasmids that did not express this protein did not complement regardless of whether they expressed the 37-kDa protein. Plasmids carrying mgtB expressed a single protein of 102 kDa whose presence or absence correlated with the ability of the plasmid to complement the Mg2+-dependent triple mutant. Fractionation of labeled maxicells demonstrated that the 42-kDa corA, the 91-kDa mgtA, and the 102-kDa mgtB gene products are all tightly associated with the membrane, a location consistent with involvement in a transport process. These data provide further support the for existence of three distinct systems for Mg2+ transport in S. typhimurium.     

Amino-terminal sequence and structure of monoclonal antibody immunopurified cytochromes P-450

Six hepatic cytochromes P-450 were isolated from 3-methylcholanthrene-treated animals by immunopurification with monoclonal antibodies. The purified cytochromes P-450 include 57- and 56-kDa polypeptides from Sprague-Dawley rats, 57- and 56-kDa polypeptides from C57BL/6 mice, a 56-kDa polypeptide from DBA/2 mice, and a 53-kDa polypeptide from guinea pigs. These isozymes were structurally compared by peptide mapping using both sodium dodecyl sulfate--polyacrylamide gel electrophoresis and high-pressure liquid chromatography and by amino acid and NH2-terminal sequence analyses. The 57-kDa polypeptides from rats and mice have similar but nonidentical peptide maps and amino acid compositions and are about 80% homologous in their NH2-terminal amino acid sequence. The 56-kDa polypeptides from rats and both mice strains have very similar peptide maps and amino acid compositions and identical NH2-terminal sequences. The NH2-terminal sequence of the mice 56-kDa polypeptides corresponds to that reported for the mouse P1-450 isozyme except that we identified two additional residues, proline and serine, at the NH2 terminus in the 57-kDa polypeptide from C57BL/6 mice that were not deduced from the cDNA sequence of the mouse P1-450 isozyme. The guinea pig 53-kDa polypeptide has a distinct peptide map relative to the other polypeptides studied and an NH2-terminal sequence with only partial homology to the 56- and 57-kDa polypeptides from rats and mice. This report shows the varying degree of structural relatedness among the isozymes examined and demonstrates the suitability and advantage of immunopurified cytochromes P-450 for sequencing and structural studies.     

Analysis of Major Surface Polypeptides of Rickettsia japonica

Major surface polypeptides of Rickettsia japonica migrated to the position of 120, 135, and 145 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, when the organisms were solubilized at room temperature. Two major bands at the position of 135 and 185 kDa were seen, when the organisms were solubilized by heating before electrophoresis. Heat-denaturation of the 120- and 145-kDa polypeptides in excised gel bands changed their mobility and caused them to migrate to 135- and 185-kDa positions, respectively. Two polypeptides at the 120-kDa position were demonstrated: one is a major heat-modifiable polypeptide and the other a minor heat-stable. Peptide mapping was performed to determine the identity between native and denatured polypeptides.     

The nuclear 42-kDa phosphoprotein preferentially binds promoter-containing single-stranded DNA

The DNA-binding activity of a 42-kDa phosphoprotein from salivary gland cells and cultured epithelial cells of Chironomus tentans have been analyzed by the Southwestern technique. Both the salivary gland and the epithelial cell 42-kDa polypeptides were found to be single-stranded DNA-binding proteins. They bind to single-stranded promoter-containing restriction fragments including sequences from -204 to +74 from the ecdysterone controlled I-18C gene as well as sequences including the joint histone H2A/H2B promoters in a sequence selective manner. By contrast, the 42-kDa polypeptides show no significant binding to intragenic restriction fragments from +71 to +351 from the I-18C gene. Previous and present data taken together suggest that the 42-kDa protein has a general role in the regulation of protein coding genes.