Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene

The nucleotide sequence of the mtlA gene, which codes for the mannitol-specific Enzyme II of the Escherichia coli phosphotransferase system, is presented. From the gene sequence, the primary translation product is predicted to consist of 637 amino acids (Mr = 67,893). This result is compared to the amino acid composition and molecular weight of the purified mannitol Enzyme II protein. The hydrophobic and hydrophilic properties of the enzyme were evaluated along its amino acid sequence using a computer program (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). The computer analysis predicts that the NH2-terminal half of the enzyme resides within the membrane, whereas the COOH-terminal half of the enzyme has the properties of a soluble protein. The possible functions of such a protein structure are discussed. RNA mapping has identified the promoter and mRNA start point for the mtl operon.     

Chicken liver H-protein, a component of the glycine cleavage system. Amino acid sequence and identification of the N epsilon-lipoyllysine residue

The complete amino acid sequence of H-protein from chicken liver was determined by aligning peptides obtained by cyanogen bromide, endoproteinase Lys-C, Staphylococcus aureus V8 protease, and chymotrypsin cleavage together with the partial NH2- and COOH-terminal sequence of the intact protein. H-protein consists of 125 amino acids and a lipoic acid moiety linked to lysine 59. The sequence is: (sequence in text). The lysyl residue involved in lipoic acid attachment is indicated with an asterisk. The molecular weight including lipoic acid is calculated to be 13,883. From the secondary structure predicted by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1978) Adv. Enzymol. 47, 45-148) the lipoic acid binding region shows alpha-helical structure and is predicted to be an interior portion of the protein from the hydropathic profile according to Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132).     

The nucleotide sequence of the gene for malF protein, an inner membrane component of the maltose transport system of Escherichia coli. Repeated DNA sequences are found in the malE-malF intercistronic region

The malF gene product is an inner membrane component of the maltose transport system in Escherichia coli. Some gene fusions between malF and lacZ (encoding the normally cytoplasmic enzyme beta-galactosidase) produce hybrid proteins which are membrane-bound while other fusions produce hybrid proteins which are cytoplasmic (Silhavy, T. J., Casadaban, M. J., Shuman, H. A., and Beckwith, J. R. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3423-3427). To further analyze the localization properties of the different classes of fusion proteins and of the intact MalF protein, we have obtained the DNA sequence of 5 malF-lacZ fusions and the wild type malF gene. From the predicted amino acid sequence, MalF protein contains 514 amino acids and has a molecular weight of 56,947. Analysis of the hydropathic character of MalF using the Kyte-Doolittle assignments (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132), indicates that the protein may have 2 or 3 amino-terminal membrane-spanning segments and 4 or 5 carboxy-terminal membrane-spanning segments separated by a region of 181 hydrophilic residues. Localization properties of the different fusion proteins correspond with degree of hydrophobicity. By sequencing upstream from malF, the malE-malF intercistronic region was found to be 153 base pairs in length and to contain inverted repeats, homologous to intercistronic repeats of many other operons. Further analysis of this region may help in understanding the observed step-down in synthesis of the MalF protein.     

A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence

A heparin-binding peptide within antithrombin III (ATIII) was identified by digestion of ATIII with Staphylococcus aureus V8 protease followed by purification on reverse-phase high pressure liquid chromatography using a C-4 column matrix. The column fractions were assayed for their ability to bind heparin by ligand blotting with 125I-fluoresceinamine-heparin as previously described (Smith, J. W., and Knauer, D. J. (1987) Anal. Biochem. 160, 105-114). This analysis identified at least three fractions with heparin binding ability of which the peptide eluting at 25.4 min gave the strongest signal. Amino acid sequence analysis of this peptide gave a partially split sequence which was consistent with regions encompassing amino acids 89-96 and 114-156. These amino acids are present in a 1:1 molar ratio which is consistent with a disulfide linkage between Cys-95 and Cys-128. High affinity heparin competed more effectively for the binding of 125I-fluoresceinamine-heparin to this peptide than low affinity heparin. Chondroitin sulfate did not block the binding of 125I-fluoresceinamine-heparin to the peptide. These data strongly suggest that the isolated peptide represents a native heparin-binding region within intact ATIII. Computer generation of a plot of running charge density of ATIII confirms that the region encompassing amino acid residues 123-141 has the highest positive charge density within the molecule. A hydropathy plot of ATIII was generated using a method similar to that of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). This plot indicates that amino acid residues 126-140 are exposed to the exterior surface of the molecule. Based on these data, we suggest that the region corresponding to amino acid residues 114-156 is a likely site for the physiological heparin-binding domain of ATIII. We also conclude that the proposed disulfide bridges within the protein are suspect and should be re-examined (Petersen, T. E., Dudek-Wojiechowska, G., Sottrup-Jensen, L., and Magnussun, S. (1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen, D., Wiman, B., and Verstaeta, M., eds) pp. 43-54, Elsevier Scientific Publishing Co., Amsterdam).     

Structure of the lac carrier protein of Escherichia coli

Circular dichroic measurements on the lac carrier protein purified from the cytoplasmic membrane of Escherichia coli indicate that 85 +/- 5% of the amino acid residues comprising this integral membrane protein are arranged in helical secondary structures. Analysis of the sequential hydropathic character of this protein by the method of Kyte and Doolittle (J. Mol. Biol. (1982) 157, 105-132) indicates that the protein is composed of at least 12 hydrophobic segments with a mean length of 24 +/- 4 residues/segment. Approximately 70% of the 417 amino acids in the lac carrier are found in these domains. The hydropathic profile, together with the circular dichroic measurements, suggest that the 12 hydrophobic segments are largely in a helical conformation. If the segments are assumed to be alpha-helical, the mean length of each domain approximates the thickness of the most hydrophobic portion of the lipid bilayer. Based on these considerations, it is proposed that the lac carrier protein consists of at least 12 alpha-helical segments that traverse the membrane in a perpendicular sense, i.e. in a fashion similar to bacteriorhodopsin.     

Sequence of the exfoliative toxin B gene of Staphylococcus aureus.

We sequenced the Staphylococcus aureus exfoliative toxin B gene contained on a 1.7-kilobase HindIII fragment of plasmid pRW001. The gene was located by comparison of the amino acid sequences of open reading frames with the amino-terminal sequence of exfoliative toxin B and the total amino acid composition of the protein (A.D. Johnson, L. Spero, J.S. Cades, and B.T. De Cicco, Infect. Immun. 24:679-684, 1979). The primary translation product consists of 274 amino acids and contains a 31-amino-acid N-terminal peptide presumably necessary for transport.     

Data-sieving hydrophobicity plots

Hydrophobicity plots provide clues to the tertiary structure of proteins (J. Kyte and R. F. Doolittle, 1982, J. Mol. Biol. 157, 105; C. Chothia, 1984, Annu. Rev. Biochem. 53, 537; T. P. Hopp and K. R. Woods, 1982, Proc. Natl. Acad. Sci. USA 78, 3824). To render domains more visible, the raw data are usually smoothed using a running mean of between 5 and 19 amino acids. This type of smoothing still incorporates two disadvantages. First, peculiar residues that do not share the properties of most of the amino acids in the domain may prevent its identification. Second, as a low-pass frequency filter the running mean smoothes sudden transitions from one domain, or phase, to another. Data-sieving is described here as an alternative method for identifying domains within amino acid sequences. The data-sieve is based on a running median and is characterized by a single parameter, the mesh size, which controls its resolution. It is a technique that could be applied to other series data and, in multidimensions, to images in the same way as a median filter.     

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.     

Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.     

Activin B: precursor sequences, genomic structure and in vitro activities

We report here the complete amino acid sequence of the human inhibin beta B-subunit as deduced from the sequence of cDNA and genomic clones. The primary translation product of the beta B mRNA predicts a protein of 407 amino acids, containing a prepro region of 292 amino acids separated by basic amino acids from the mature C-terminal 115 amino acids. Mammalian tissue culture cells transfected with a beta B-subunit expression plasmid secreted an activin B homodimer of approximately 22K mol wt. Coexpression of the beta A- and beta B-subunit mRNAs resulted in the secretion of the three forms of activin, A, AB, and B. Purified activin B was shown to elicit FSH release in an in vitro pituitary assay and trigger the accumulation of hemoglobin in K562 cells. The potency of activin B in both of these assays (ED50 approximately 2 ng/ml) was indistinguishable from that observed for activin A.     

Rat major acute-phase protein: biosynthesis and characterization of cDNA clone

The major acute-phase protein (alpha 1-MAP) of rat serum is induced in response to inflammation. This induction may be attributed to a corresponding increase in the level of translatable mRNA for the protein. Using in vitro and in vivo systems, various biosynthetic processing intermediates of this glycoprotein have been isolated. alpha 1-MAP is translated in a rabbit reticulocyte system as a preprotein with an amino-terminal signal peptide and an apparent molecular weight of 51,000. Translation of rough microsomes yields a product with a mass of 57,000 Da, representing the core glycosylated form of alpha 1-MAP. Cotranslational glycosylation appears to occur in a stepwise fashion, since three glycosylated forms of alpha 1-MAP (51,000, 54,000, and 57,000 Da) were detected in polysome translations; these products were digested by endoglycosidase H to a 48,000-Da protein. Two intracellular forms of alpha 1-MAP were observed in vivo, a 57,000-Da (core carbohydrate sidechains) and a 66,000-Da protein (mature complex carbohydrate side-chains); the latter was the only component secreted into the culture medium. To extend our studies on this protein, a cDNA clone specific for alpha 1-MAP was isolated. The recombinant was positively identified by hybrid selection procedures and contains a 1.55-kb insert. Partial radiosequence analysis of the primary translation product indicated the distribution of Leu, Ile, Cys, and Met in the amino-terminal region of this protein. To relate the location of these amino acids with the nucleotide sequence, cDNA was analyzed by the method of Maxam and Gilbert. These results indicate that the cDNA insert contains the 3' poly(A) tail, and alignment of the 5' end of the cDNA with the available amino acid sequence of the primary translation product corroborated that the insert encodes the entire alpha 1-MAP protein except for the first four amino acids of the signal peptide.     

Identification of the genes for the lactose-specific components of the phosphotransferase system in the lac operon of Staphylococcus aureus

The nucleotide and deduced amino acid sequences of the lacE and lacF genes, which code for the lactose-specific Enzyme II and Enzyme III of the Staphylococcus aureus phosphotransferase system, are presented. The primary translation products consist of a hydrophobic protein of 572 amino acids (Mr = 62,688) and a polypeptide of 103 amino acids (Mr = 11,372), respectively. The assignment of lacF as the gene for Enzyme IIIlac was based upon the known amino acid sequence of the protein. The identity of lacE as encoding Enzyme IIlac was based upon immunoreactivity of the cloned gene product with antibodies raised against purified Enzyme IIlac from S. aureus and an assay of biological function of the protein expressed in Escherichia coli. The order of the known genes of the S. aureus lac operon is lacF-lacE-lacG, the latter encoding phospho-beta-galactosidase.     

Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding beta-xylosidase from the thermophilic fungus Talaromyces emersonii

The gene coding for beta-xylosidase, bxl1, has been cloned from the thermophilic filamentous fungus, Talaromyces emersonii. This is the first report of a hemicellulase gene from this novel source. At the genomic level, bxl1 consists of an open reading frame of 2388 nucleotides with no introns that encodes a putative protein of 796 amino acids. The bxl1 translation product contains a signal peptide of 21 amino acids that yields a mature protein of 775 amino acids, with a predicted molecular mass of 86.8 kDa. The deduced amino acid sequence of bxl1 exhibits considerable homology with the primary structures of the Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Trichoderma reesei beta-xylosidase gene products, and with some beta-glucosidases, all of which have been classified as Family 3 glycosyl hydrolases. Northern blot analysis of the bxl1 gene indicates that it is induced by xylan and methyl-beta-D-xylopyranoside. D-Xylose induced expression of bxl1 but was shown to repress induction of the gene at high concentrations. The presence of six CreA binding sites in the upstream regulatory sequence (URS) of the bxl1 gene indicates that the observed repression by D-glucose may be mediated, at least partly, by this catabolite repressor.     

A single amino acid substitution in an ectopic alpha subunit of a human carcinoma choriogonadotropin

Human choriogonadotropin [hCG] has two dissimilar noncovalently associated subunits, designated alpha and beta. An ectopically secreted hCG alpha subunit that fails to associate with the beta subunit and displays an anomalously high molecular weight on molecular sieve chromatography but not on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been sequenced. A single substitution of Glu56 by Ala56 has been found in the altered subunit. No evidence for conformational differences between normal and ectopic alpha could be found using circular dichroism or intrinsic fluorescence as measures of secondary and tertiary structure, respectively. Hydrophobicity profiles as determined by the method of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132) predicted, however, that the hydrophilic segment, Thr54-Ser55-Glu56-Ser57-Thr58, becomes an extension of the preceding hydrophobic segment when Glu56 is substituted with Ala. This solitary hemoglobin S-like mutation may lead to an altered tertiary structure, self dimerization, or an alteration in glycosylation that could be responsible for the ectopic alpha subunit's failure to associate with the beta subunit.     

Rat gastric prepepsinogen: in vitro synthesis and partial amino-terminal signal sequence

The major translation product of rat gastric mucosa RNA in a wheat germ cell-free system was identified as prepepsinogen by electrophoretic analysis of its immunoprecipitate on sodium dodecyl sulfate (SDS)-polyacrylamide gels and amino-terminal sequence determination. The translation product containing radioactive amino acids, purified by SDS-polyacrylamide gel electrophoresis, was shown to have an amino-terminal extension peptide comprising 16 amino acid residues. A partial amino acid sequence of this extension peptide is as follows: Met-X-X-Met-Val-Val-X-Leu-Leu-X-Leu-X-Leu-Leu-X-X-pepsinogen.     

Regulation and structure of an Escherichia coli gene coding for an outer membrane protein involved in export of K88ab fimbrial subunits.

The nucleotide sequence of the faeD gene of Escherichia coli and the amino acid sequence of its product is presented. The faeD product is an outer membrane protein required for transport of K88ab fimbrial subunits across the outer membrane. The protein is synthesized as a precursor containing a signal peptide, and the tentative mature protein comprises 777 amino acid residues. The distribution of amino acids in the faeD protein is similar to that of other outer membrane proteins; showing a fairly even distribution of charged residues and the absence of extensive hydrophobic stretches. Secondary structure predictions revealed a region of 250 amino acid residues which might be embedded in the outer membrane. The 5'-end of faeD is located within a region showing dyad symmetry. This region serves to couple translation of faeD to the translation of the gene preceding it (faeC). The 3'-end of faeD shows an overlap of 5 bases with the next gene (faeE).     

The nucleotide sequence of the Escherichia coli K12 dnaJ+ gene. A gene that encodes a heat shock protein

The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures. It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth. We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions. Here we present the primary DNA sequence of the dnaJ gene. It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973. The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein. We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product.     

Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation.     

Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.