Nucleotide and Amino Acid Sequences of the Metallo-β-Lactamase, ImiS, from Aeromonas veronii bv. sobria

1998 ◽  
Vol 42 (2) ◽  
pp. 436-439 ◽  
Author(s):  
T. R. Walsh ◽  
W. A. Neville ◽  
M. H. Haran ◽  
D. Tolson ◽  
D. J. Payne ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Aeromonas Veronii ◽  
Putative Active Site ◽  
Lactamase Gene ◽  
Active Site Sequence

ABSTRACT The Aeromonas veronii bv. sobria metallo-β-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-β-lactamases.

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1711-1721
Author(s):  
E M McIntosh ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Northern Analysis ◽  
Hindiii Site ◽  
Reading Frame ◽  
Hindiii Restriction Site ◽  
Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

scholarly journals Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (5) ◽  
pp. 1711-1721 ◽  
Author(s):  
E M McIntosh ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Northern Analysis ◽  
Hindiii Site ◽  
Reading Frame ◽  
Hindiii Restriction Site ◽  
Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

scholarly journals New Mg2+-Dependent Oxytetracycline Resistance Determinant Tet 34 in Vibrio Isolates from Marine Fish Intestinal Contents

2002 ◽  
Vol 46 (5) ◽  
pp. 1550-1552 ◽  
Author(s):  
Lisa Nonaka ◽  
Satoru Suzuki
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Open Reading Frame ◽  
Purine Nucleotide ◽  
Chromosomal Dna ◽  
Reading Frame ◽  
Intestinal Contents ◽  
Seriola Quinqueradiata

ABSTRACT A new oxytetracycline (OTC) resistance (Otcr) determinant, Tet 34, was cloned from chromosomal DNA of Vibrio sp. no. 6 isolated from intestinal contents of cultured yellowtail (Seriola quinqueradiata). The transformant, containing cloned Tet 34, could grow in broth containing 25 μg of drug per ml with 10 mM MgCl2. Tet 34 encoded an open reading frame (ORF) 154 amino acids long. The amino acid sequence of the ORF was homologous to sequences of several bacterial xanthine-guanine phosphoribosyltransferases (XPRTs), which act in purine nucleotide salvage synthesis. Mg2+ binding site residues and the active site were highly conserved in XPRT and the ORF of Tet 34. The results suggest that Tet 34 encodes a new Mg2+-dependent Otcr mechanism.

scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

PREDICTION OF THE THREE-DIMENSIONAL STRUCTURE OF THE ENZYMATIC PART OF T-PA

1987 ◽  
Author(s):  
A Heckel ◽  
K M Hasselbach
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Serine Proteases ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Salt Bridges ◽  
Three Dimensional Structure ◽  
Insertions And Deletions

Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.

scholarly journals Sequence of the Structural Gene for Xanthine Dehydrogenase (rosy Locus) in Drosophila melanogaster

Genetics ◽  
1987 ◽  
Vol 116 (1) ◽  
pp. 67-73
Author(s):  
Tim P Keith ◽  
Margaret A Riley ◽  
Martin Kreitman ◽  
R C Lewontin ◽  
Daniel Curtis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Structural Gene ◽  
Xanthine Dehydrogenase ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Eco Ri

ABSTRACT We determined the nucleotide sequence of a 4.6-kb Eco RI fragment containing 70% of the rosy locus. In combination with information on the 5′ sequence, the gene has been sequenced in entirety. rosy cDNAs have been isolated and intron/exon boundaries have been determined. We find an open reading frame which spans four exons and would encode a protein of 1335 amino acids. The molecular weight of the encoded protein (xanthine dehydrogenase), based on the amino acid translation, is 146,898 daltons which agrees well with earlier biophysical estimates. Characteristics of the protein are discussed.

scholarly journals Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts

1991 ◽  
Vol 277 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R Dumas ◽  
M Lebrun ◽  
R Douce
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Single Gene ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Open Reading Frame ◽  
Amino Acid Residues ◽  
Protein Precursor ◽  
Reading Frame ◽  
Full Length Cdna

Acetohydroxy acid reductoisomerase (AHRI), the second enzyme in the parallel isoleucine/valine-biosynthetic pathway, catalyses an unusual two-step reaction in which the substrate, either 2-acetolactate or 2-aceto-2-hydroxybutyrate, is converted via an alkyl migration and an NADPH-dependent reduction to give 2,3-dihydroxy-3-methylbutyrate or 2,3-dihydroxy-3-methylvalerate respectively. We have isolated and characterized a full-length cDNA from a lambda gt11 spinach library encoding the complete acetohydroxy acid reductoisomerase protein precursor. The 2050-nucleotide sequence contains a 1785-nucleotide open reading frame. The derived amino acid sequence indicates that the protein precursor consists of 595 amino acid residues including a presequence peptide of 72 amino acid residues. The N-terminal sequence of the first 16 amino acid residues of the purified AHRI confirms the identity of the cDNA. The derived amino acid sequence from this open reading frame shows 23% identity with the deduced amino acid sequences of the Escherichia coli and Saccharomyces cerevisiae AHRI proteins. There are two blocks of conserved amino acid residues in these three proteins. One of these is a sequence similar to the ‘fingerprint’ region of the NAD(P)H-binding site found in a large number of NAD(P)H-dependent oxidoreductases. The other, a short sequence (Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Ser-His-Gly-Phe) containing the amino acids lysine and histidine, could well be the catalytic site of the first step of the AHRI reaction. Southern-blot analysis indicated that AHRI is encoded by a single gene per haploid genome of about 7.5 kbp containing at least four introns.

scholarly journals Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999 ◽  
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

scholarly journals Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase

1998 ◽  
Vol 331 (3) ◽  
pp. 953-958 ◽  
Author(s):  
Hiro-omi TAMURA ◽  
Yuki HARADA ◽  
Atsushi MIYAWAKI ◽  
Katsuhiko MIKOSHIBA ◽  
Michio MATSUI
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nasal Cavity ◽  
Rat Liver ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Rt Pcr ◽  
Reading Frame ◽  
Residue Polypeptide

Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT–PCR).

A new insertion sequence, IS14999, from Corynebacterium glutamicum

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 501-508 ◽  
Author(s):  
Yota Tsuge ◽  
Kana Ninomiya ◽  
Nobuaki Suzuki ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Insertion Sequence ◽  
Genetic Manipulation ◽  
Insertion Site ◽  
Amino Acid Sequences ◽  
Transfer Event ◽  
Reading Frame ◽  
Partial Homology

A new insertion sequence from Corynebacterium glutamicum ATCC 14999 was isolated and characterized. This IS element, designated IS14999, comprised a 1149 bp nucleotide sequence with 22 bp imperfect terminal inverted repeats. IS14999 carries a single open reading frame of 345 amino acids encoding a putative transposase that appears to have partial homology to IS642, an IS630/Tc1 superfamily element, at the C-terminal region in the amino acid sequence. This indicated that IS14999 belonged to the IS630/Tc1 superfamily, which was first identified in C. glutamicum. IS14999 has a unique distance of 38 amino acid residues between the second and third amino acids in the DDE motif, which is well known as the catalytic centre of transposase. This suggested that IS14999 constituted a new subfamily of the IS630/Tc1 superfamily. A phylogenetic tree constructed on the basis of amino acid sequences of transposases revealed that this new transposable element was more similar to eukaryotic Tc1/mariner family elements than to prokaryotic IS630 family elements. Added to the fact that IS14999 was present in only a few C. glutamicum strains, this implies that IS14999 was probably acquired by a recent lateral transfer event from eukaryotic cells. Analysis of the insertion site in C. glutamicum R revealed that IS14999 appeared to transpose at random and always caused a target duplication of a 5′-TA-3′ dinucleotide upon insertion, like the other IS630/Tc1 family elements. These findings indicated that IS14999 could be a powerful tool for genetic manipulation of corynebacteria and related species.

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