Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei

The nucleotide sequence of the dihydrofolate reductase (DHFR) gene of a methotrexate-resistant strain of Lactobacillus casei, which is the source of DHFR for nuclear magnetic resonance (NMR) studies, has been determined. The derived amino acid sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at position 8 and proline instead of leucine at position 90. The nucleotide sequences of 320-bp 5' and 335-bp 3' flanking regions of this gene have also been determined.     

Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.     

Cloning and nucleotide sequencing of sheep growth hormone cDNA.

cDNA was prepared from the mRNA isolated from sheep anterior pituitary glands. On cloning cDNA in E. coli, a clone coding full sequence of sheep pre-growth hormone was determined. The sequence for the sheep growth hormone (GH) is in agreement with the amino acid sequence of the protein determined previously except for the asparagine residue at position 99 rather than aspartic acid and the arginine residue at position 146 in place of threonine. The cDNA sequence presented is also in accordance with the genomic sequence for the sheep GH gene that has been reported.     

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.     

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.     

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.     

Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli

The nucleotide sequence of the alkaline phosphatase (APase) gene (phoA) of Escherichia coli strain 294 has been determined. Pre-APase has a total of 471 amino acids (aa) including a signal sequence of 21 aa. The derived aa sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at positions 16 and 36, and glutamic acid instead of glutamine at position 197. Two open reading frames (ORF1 and ORF2) located downstream from phoA or upstream from proC have been found. ORF1 encodes a putative presecretory protein of 106 aa with a signal sequence of 21 or 22 aa. If this protein is actually produced, it may be one of the smallest periplasmic proteins in E. coli.     

Studies on the mutator gene, mutT of Escherichia coli. Molecular cloning of the gene, purification of the gene product, and identification of a novel nucleoside triphosphatase

The mutator gene, mutT, has been cloned into an expression vector and overproduced in Escherichia coli. The gene product has been purified to over 90% homogeneity as judged by gel electrophoresis and amino acid analysis. The amino acid composition of the protein and the sequence of the 20 amino acids of the N-terminal region agree well with the nucleotide sequence of the gene reported by Akiyama et al. (Akiyama, M., Horiuchi, T., and Sekiguchi, M. (1987) Mol. Gen. Genet. 206, 9-16) and indicate that the first of the potential initiation codons (position 164) of the open reading frame in the PvuII fragment carrying the mutT gene is the site of initiation of translation of the 15,000-Da polypeptide. A novel nucleoside triphosphatase activity which has a preference for dGTP is associated with the purified protein, and preliminary experiments are consistent with the notion that the mutT gene product is the enzyme responsible for this activity.     

Molecular cloning and sequence analysis of the ribosomal ‘A’ protein gene from the archaebacterium,Halobacterium halobium

The ribosomal ‘A’ protein gene of Halobacterium halobium has been cloned and the nucleotide sequence of the DNA fragment containing the ‘A’ protein gene has been determined. The amino-acid sequence of the protein deduced from the nucleotide sequence was established from manual sequence analysis of the protein and structural data provided by peptides derived from cleavage of the protein with various proteinases. The ‘A’ protein consisted of 114 amino acids with a molecular weight of 11562 and was characterized mainly by a high amounts of alanine and acidic amino acid in the C-terminal half of the molecule. The coding sequence of the gene was preceded by a predicted Shine-Dalgarno sequence and two terminal codons. There was no intron or insertion sequence in the coding sequence. Following the terminal codon of the ‘A’ gene, there was a structure reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 71%. Inspection of the codon usage for the ‘A’ gene revealed 85% preference for G or C at the third codon position.     

Nucleotide sequence of the phoS gene, the structural gene for the phosphate-binding protein of Escherichia coli.

phoS is the structural gene for the phosphate-binding protein, which is localized in periplasm and involved in active transport of phosphate in Escherichia coli. It is also a negative regulatory gene for the pho regulon, and the gene expression is inducible by phosphate starvation. The complete nucleotide sequence of the phoS gene was determined by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). The amino acid sequences at the amino termini of the pre-PhoS and PhoS proteins and at the carboxy terminus of the PhoS protein were determined by using the purified proteins. Furthermore, the amino acid sequence of enzymatically digested peptide fragments of the PhoS protein was determined. The combined data established the nucleotide sequence of the coding region and the amino acid sequence of the pre-PhoS and the PhoS proteins. The pre-PhoS protein contains an extension of peptide composed of 25 amino acid residues at the amino terminus of the PhoS protein, which has the general characteristics of a signal peptide. The mature PhoS protein is composed of 321 amino acid residues, with a calculated molecular weight of 34,422, and lacks the disulfide bond and methionine. The regulatory region of phoS contains a characteristic Shine-Dalgarno sequence at an appropriate position preceding the translational initiation site, as well as three possible Pribnow boxes and one -35 sequence. the nucleotide sequence of the regulatory region of phoS was compared with those of phoA and phoE, the genes constituting the pho regulon.     

Genomic DNA sequence for human C-reactive protein

The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C-reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site.     

Nucleotide sequence of the gene encoding the two-subunit pilin of Bacteroides nodosus 265.

The nucleotide sequence of the gene encoding pilin from Bacteroides nodosus 265 has been determined. The pilin is encoded by a single-copy gene, from which can be predicted a prepilin comprising a single protein chain of Mr 16,637. The prepilin sequence differs in several respects from the mature protein sequence. Seven additional N-terminal amino acid residues are present in prepilin, whereas residue 8, phenylalanine, undergoes posttranslational modification to become the N-methylated amino-terminal residue of mature pilin. In addition, further processing occurs through internal cleavage to produce two noncovalently linked subunits characteristic of pilins from serogroup H of B. nodosus, of which strain 265 is a member. The position of cleavage has been identified between alanine residues at positions 72 and 73 of the mature 149-residue pilin protein. The predicted pilin sequence of B. nodosus 265 shows extensive N-terminal amino acid sequence homology with other pilins of the N-methylphenylalanine type. In addition this sequence also shows homology with these N-methylphenylalanine-type pilins in the C-terminal region of the molecule, especially with pilin from Pseudomonas aeruginosa PAK.     

Determination of the cleavage site of the phage T4 prohead protease in gene product 68. Influence of protein secondary structure on cleavage specificity

The cleavage site of the T4 prohead protease in gene product 68 of bacteriophage T4 has been determined by direct protein sequencing. It is located close to the carboxy-terminal end of a predicted alpha-helix in the sequence Asn-Val-Glu-Ala between the Glu and Ala residues. Secondary structure seems to be more important in determining cleavage than the presence of an aliphatic amino acid three residues before the cleavage site that was proposed earlier. In this case, that position is occupied by Asn, a hydrophilic residue. A second potentially cleavable Glu-Ala is found five residues after the cleaved sequence and this is preceded by an Ile at the -3 position. Despite this, the sequences of the amino and carboxyl termini of the uncleaved protein are identical to those previously proposed from an analysis of the DNA sequence of the gene.     

The nucleotide sequence of the protein subunit of the K88ac fimbriae of porcine enterotoxigenic Escherichia coli

Abstract The nucleotide sequence of the gene encoding the K88ac fimbrial subunit has been determined and the amino acid sequence was derived. In comparison with the two other, previously determined sequences of the K88ab and K88ad sequences, the most striking features of the K88ac protein sequence are the insertion of a lys residue at position 104 and the deletion of three amino residues at positions 165. The differences between the three sequences are discussed with respect to possible structure-functions relationships and antigenic determinants.     

Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases

Summary The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta- 1,3-1,4-glucanase ofBacillus macerans has been determined. ThebglM gene comprises an open reading frame (ORF) of 711 by (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta- 1,3-1,4-glucanases fromB. subtilis andB. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilicBacillus endo-beta-glucanases. TheB. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm inE. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 ofB. macerans endo-beta-glucanase could be identified.     

Characterization of V protein in measles virus-infected cells.

An edited mRNA transcribed from the phosphoprotein (P) gene of measles virus (MV) has been predicted to encode a cysteine-rich protein designated V. This mRNA contains a single additional nontemplated G residue which permits access to an additional protein-coding reading frame. Such an edited P gene-specific mRNA has been detected in MV-infected cells, but no corresponding protein has yet been identified in vivo. We report the use of antisera directed against synthetic peptides corresponding to five different regions of the predicted MV V protein amino acid sequence to analyse MV-specific proteins synthesized in vivo and in vitro. The MV V protein (40 kDa) was detected in MV-infected cells in a diffuse cytoplasmic distribution, a predominant subcellular localization distinct from that of virus nucleocapsids. The protein was found to be phosphorylated and to be maximally synthesized at 16 h postinfection, when MV-specific structural protein synthesis was also maximal. Antiserum directed against a peptide (PV2) corresponding to amino acids 65 to 87 of the V protein amino acid recognized the P protein but not the V protein, indicating that the P and V proteins may be folded differently at or near this region so that the PV2 sequence is in an exposed position at the surface of the P protein but not at the surface of the V protein.     

Lectin from sainfoin (Onobrychis viciifolia scop.). Complete amino acid sequence

The complete amino acid sequence of a lectin from sainfoin ( Onobrychis viciifolia Scop . var. Eski ) has been determined by sequential Edman analyses of the intact protein and peptides derived from digests with trypsin and thermolysin. Peptides were purified by pH fractionation, by gel filtration, and by cation-exchange and reverse-phase high-performance liquid chromatography. Seven segments of continuous sequence, accounting for the entire protein, were aligned through sequence comparison with several homologous leguminous lectins to give the final structure. Sainfoin lectin monomer, a glycoprotein which contains a single polypeptide chain of 236 amino acid residues with a molecular weight of 26 509, has amino- and carboxyl-terminal residues of alanine and threonine, respectively. A single residue of cysteine, located at position 33, is the only sulfur-containing amino acid present. Asparagine-118 is the single oligosaccharide attachment site. At least two apparent allelomorphic forms of the protein, having valine or isoleucine at position 49 in equal amounts, were detected. The amino acid sequence of sainfoin lectin exhibits circular permutation relative to that of the homologous protein concanavalin A.