Cloning and characterization of the cDNA encoding human adenylosuccinate synthetase.

Adenylosuccinate synthetase (AS) catalyzes the first committed step in the conversion of IMP to AMP. A cDNA was isolated from a human liver library which encodes a protein of 455 amino acids (M(r) of 49,925). Alignments of human, mouse, Dictyostelium discoideum and E. coli AS sequences identify a number of invariant residues which are likely to be important for structure and/or catalysis. The human AS sequence was also 19% identical to the human urea cycle enzyme, argininosuccinate synthetase (ASS), which catalyzes a chemically similar reaction. Both human liver and HeLa AS mRNA showed signals of 2.3 and 2.8 kb. An unmodified N-terminus is required for function of the human AS enzyme in E. coli mutants lacking the bacterial enzyme. The human cDNA provides a means to assess the possible role of AS abnormalities in unclassified, idiopathic cases of gout.     

Nucleotide sequence of the dihydrofolate reductase gene of Saccharomyces cerevisiae

The nucleotide sequence of a DNA fragment that contained the Saccharomyces cerevisiae gene DFR coding for dihydrofolate reductase (DHFR) was determined. The DHFR was encoded by a 633-bp open reading frame, which specified an Mr24264 protein. The polypeptide was significantly related to the DHFRs of chicken liver and Escherichia coli. The yeast enzyme shared 60 amino acid (aa) residues with the avian enzyme and 51 aa residues with the bacterial enzyme. DHFR was overproduced about 40-fold in S. cerevisiae when the cloned gene was present in the vector YEp24. As isolated from the Saccharomyces library, the DFR gene was not expressed in E. coli. When the gene was present on a 1.8-kb BamHI-SalI fragment subcloned into the E. coli vector, pUC18, weak expression in E. coli was observed.     

The primary structure of human H-protein of the glycine cleavage system deduced by cDNA cloning

A full-length cDNA encoding the human H-protein of the glycine cleavage system has been isolated from a lambda gt11 human fetal liver cDNA library. The cDNA insert was 1091 base pairs with an open reading frame of 519 base pairs which encoded a 125-amino acid mature human H-protein with a 48-amino acid presequence. Human H-protein is 97%, 86%, and 46% identical to the bovine, chicken, and pea H-protein, respectively.     

Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli.

The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.     

Iron superoxide dismutase. Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures

The nucleotide sequence of the iron superoxide dismutase gene from Escherichia coli K12 has been determined. Analysis of the DNA sequence and mapping of the mRNA start reveal a unique promoter and a putative rho-independent terminator, and suggest that the Fe dismutase gene constitutes a monocistronic operon. The gene encodes a polypeptide product consisting of 192 amino acid residues with a calculated Mr of 21,111. The published N-terminal amino acid sequence of E. coli B Fe dismutase (Steinman, H. M., and Hill, R. L. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3725-3729), along with the sequences of seven other peptides reported here, was located in the primary structure deduced from the K12 E. coli gene sequence. A new molecular model for iron dismutase from E. coli, based on the DNA sequence and x-ray data for the E. coli B enzyme at 3.1 A resolution, allows detailed comparison of the structure of the iron enzyme with manganese superoxide dismutase from Thermus thermophilus HB8. The structural similarities are more extensive than indicated by earlier studies and are particularly striking in the vicinity of the metal-ligand cluster, which is surrounded by conserved aromatic residues. The combined structural and sequence information now available for a series of Mn and Fe superoxide dismutases identifies variable regions in these otherwise very similar molecules; the principal variable site occurs in a surface region between the two long helices which dominate the N-terminal domain.     

Nucleotide sequence and linkage map position of the secX gene in maize chloroplast and evidence that it encodes a protein belonging to the 50S ribosomal subunit

The nucleotide sequence of the segment of maize chloroplast DNA lying between the map coordinate positions 32.59 and 32.98 Kb and containing the secX gene has been determined. The derived amino acid sequence of maize chloroplast secX is 95%, 87% and 62% identical to the corresponding derived amino acid sequences from two plant chloroplasts and Escherichia coli, respectively. It is also 70% identical to the experimentally determined amino acid sequence of a protein isolated from Bacillus stearothermophilus ribosomes. Separation of the 50S ribosomal subunit proteins of E. coli by reversed phase HPLC gave a peak which contained pure secX protein, as determined by N-terminal amino acid sequencing. Spinach chloroplast 50S subunit proteins separated by HPLC also gave a peak corresponding to pure secX protein. From these results we conclude that the secX gene in E. coli and in plant chloroplasts encodes a small (37-38 amino acid residues) ribosomal protein belonging to the 50S subunit. The same conclusion has been reached recently by A. Wada with respect to E. coli secX. In agreement with Wada, we name the secX protein L36. Its chloroplast gene is designated rpL36.     

The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.

We report characterization of the component proteins and molecular cloning of the genes encoding the two subunits of the carboxyltransferase component of the Escherichia coli acetyl-CoA carboxylase. Peptide mapping of the purified enzyme component indicates that the carboxyltransferase component is a complex of two nonidentical subunits, a 35-kDa alpha subunit and a 33-kDa beta subunit. The alpha subunit gene encodes a protein of 319 residues and is located immediately downstream of the polC gene (min 4.3 of the E. coli genetic map). The deduced amino acid composition, molecular mass, and amino acid sequence match those determined for the purified alpha subunit. Six sequenced internal peptides also match the deduced sequence. The amino-terminal sequence of the beta subunit was found within a previously identified open reading frame of unknown function called dedB and usg (min 50 of the E. coli genetic map) which encodes a protein of 304 residues. Comparative peptide mapping also indicates that the dedB/usg gene encodes the beta subunit. Moreover, the deduced molecular mass and amino acid composition of the dedB/usg-encoded protein closely match those determined for the beta subunit. The deduced amino acid sequences of alpha and beta subunits show marked sequence similarities to the COOH-terminal half and the NH2-terminal halves, respectively, of the rat propionyl-CoA carboxylase, a biotin-dependent carboxylase that catalyzes a similar carboxyltransferase reaction reaction. Several conserved regions which may function as CoA-binding sites are noted.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

Molecular cloning and expression of the spsB gene encoding an essential type I signal peptidase from Staphylococcus aureus.

The gene, spsB, encoding a type I signal peptidase has been cloned from the gram-positive eubacterium Staphylococcus aureus. The gene encodes a protein of 191 amino acid residues with a calculated molecular mass of 21,692 Da. Comparison of the protein sequence with those of known type I signal peptidases indicates conservation of amino acid residues known to be important or essential for catalytic activity. The enzyme has been expressed to high levels in Escherichia coli and has been demonstrated to possess enzymatic activity against E. coli preproteins in vivo. Experiments whereby the spsB gene was transferred to a plasmid that is temperature sensitive for replication indicate that spsB is an essential gene. We identified an open reading frame immediately upstream of the spsB gene which encodes a type I signal peptidase homolog of 174 amino acid residues with a calculated molecular mass of 20,146 Da that is predicted to be devoid of catalytic activity.     

Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene

The gene that codes for xylose isomerase in Escherichia coli has been cloned by complementation of a xylose isomerase-negative E. coli mutant. The structural gene is 1320 nucleotides in length and codes for a protein of 440 amino acids. An additional 209 nucleotides 5' and 82 nucleotides 3' to the structural gene were also sequenced. To verify that the cloned gene encodes E. coli xylose isomerase, the enzyme was purified to homogeneity and the sequence of the first 25 amino acid residues was determined by a semimicromanual Edman procedure. These results establish that the NH2-terminal methionine of xylose isomerase is specified by an ATG which is 7 nucleotides downstream from a Shine-Dalgarno sequence.     

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases.

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence

Glutamine synthetase is encoded by the glnA gene of Escherichia coli and catalyzes the formation of glutamine from ATP, glutamate, and ammonia. A 1922-base pair fragment from a cDNA containing the glnA structural gene for E. coli glutamine synthetase has been sequenced. An open reading frame of 1404 base pairs encodes a protein of 468 amino acid residues with a calculated molecular weight of 51,814. With few exceptions, the amino acid sequence deduced from the DNA sequence agreed very well with the amino acid sequences of several peptides reported previously. The secondary structure predicted for the E. coli enzyme has approximately 36% of the residues in alpha-helices which is in agreement with calculations of approximately 39% based on optical rotatory dispersion data. Comparison of the amino acid sequences of glutamine synthetase from E. coli (468 amino acids) and Anabaena (473 amino acids) (Turner, N. E., Robinson, S. T., and Haselkorn, R. (1983) Nature 306, 337-342) indicates that 260 amino acids are identical and 80 are of the same type (polar or nonpolar) when aligned for maximum homology. Several homologous regions of these two enzymes exist, including the sites of adenylylation and oxidative modification, but the regulation of each enzyme is different.     

The primary structure of rabbit liver cytosolic serine hydroxymethyltransferase

The complete amino acid sequence of cytosolic serine hydroxymethyltransferase from rabbit liver was determined. The sequence was determined from analysis of peptides isolated from tryptic and cyanogen bromide cleavages of the enzyme. Special procedures were used to isolate and sequence the C-terminal and blocked N-terminal peptides. Each of the four identical subunits of the enzyme consists of 483 residues. The sequence could be easily aligned with the sequence of Escherichia coli serine hydroxymethyltransferase. The primary structural homology between the rabbit and E. coli enzymes is about 42%. The importance of the primary and predicted secondary structural homology between the two enzymes is discussed.