Giardia gene predicts a 183 kDa nucleotide-binding head-stalk protein

1995 ◽  
Vol 108 (7) ◽  
pp. 2683-2692
Author(s):  
J. Marshall ◽  
D.V. Holberton
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Coiled Coil ◽  
Nucleotide Binding ◽  
Amino Acid Sequences ◽  
Phase Changes ◽  
Predicted Amino Acid Sequence ◽  
Body Protein ◽  
Reading Frame ◽  
Amino Terminal

Previously described extended proteins from the cytoskeleton of Giardia lamblia (beta-giardin, median body protein) have been found to be segmented coiled coils with regular structural repeat patterns in their amino acid sequences. Screening a lambda ZAPII library derived from Giardia genomic DNA with an antibody directed against a 34 × 10(3) M(r) giardin isoform selected a gene encoding a much larger polypeptide chain (HPSR2), the sequence of which was determined by chromosome walking the open reading frame. The complete gene has been cloned and expressed as a recombinant protein of 183 × 10(3) M(r). The predicted amino acid sequence of the protein has identifiable features suggesting that it might be a motor protein with an amino-terminal hydrolytic domain attached to a long coiled coil stalk. The presumed head domain is 211 residues and contains a P-loop sequence conserved in purine nucleotide-binding proteins. The remaining 1409 amino acids mainly make up a region of heptad repeats such as in myosin or the kinesin stalk, ending in a small (67 amino acids) carboxy-terminal domain. Fourier analysis of the predicted stalk shows the presence of a strong physical repeat created by regular heptad phase changes dividing the coil into segments of 25 residues. This structure most closely resembles the smaller microtubule-associated median body protein which has segments of 24 residues.

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).

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.

scholarly journals Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

1995 ◽  
Vol 6 (2) ◽  
pp. 171-183 ◽  
Author(s):  
H Yu ◽  
C V Nicchitta ◽  
J Kumar ◽  
M Becker ◽  
I Toyoshima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Hydrophobic Region ◽  
Reading Frame

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

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).

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.

The v-ski oncogene encodes a truncated set of c-ski coding exons with limited sequence and structural relatedness to v-myc

1989 ◽  
Vol 9 (9) ◽  
pp. 4038-4045
Author(s):  
E Stavnezer ◽  
D Brodeur ◽  
L A Brennan
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Avian Leukosis Virus ◽  
Amino Acid Sequences ◽  
Biologically Active ◽  
Predicted Amino Acid Sequence ◽  
Base Sequence ◽  
Reading Frame ◽  
Metal Binding Domain ◽  
Virus C

The nucleotide sequence of a biologically active v-ski gene from a cloned proviral segment shows that ski is a 1,312-base sequence embedded in the p19 region of the avian leukosis virus gag gene. The v-ski sequence contains a single open translational reading frame that encodes a polypeptide with a molecular mass of 49,000 daltons. The predicted amino acid sequence includes nuclear localization motifs that have been identified in other nuclear oncoproteins. It also contains a proline-rich region and a set of cysteine and histidine residues that could constitute a metal-binding domain. Two regions of the amino acid sequences of v-ski and v-myc are related, and the two proteins exhibit similar distributions of hydrophobic and hydrophilic amino acids. Cloned segments of the chicken c-ski proto-oncogene totaling 65 kilobases have been analyzed, and regions related to v-ski have been sequenced. The results indicate that v-ski is derived from at least five coding exons of c-ski, that it is correctly spliced, and that it is missing c-ski coding sequences at both its 5' and 3' ends. The c-ski and avian leukosis virus sequences that overlap the 5' virus/v-ski junction in Sloan-Kettering virus contain an 18-of-20-base sequence match that presumably played a role in the transduction of ski by facilitating virus/c-ski recombination.

scholarly journals Studies On Reduced Wool Ix. The N-Terminal Sequence of A Fragment Produced by Cleavage of Component 8 With Cyanogen Bromide

1969 ◽  
Vol 22 (2) ◽  
pp. 471 ◽  
Author(s):  
IJ O'donnell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cyanogen Bromide ◽  
Amino Acid Sequences ◽  
Chemical Environment ◽  
Terminal Sequence ◽  
Terminal Residue ◽  
Amino Terminal ◽  
Fundamental Sequence ◽  
Methionine Residues

Component 8 is a major component extracted from reduced and carboxy-methylated wool. Further study of its reaction with cyanogen bromide and of the fractions obtained under carefully controlled disaggregating conditions has rev<\laled that while most of the methionine residues are in the same position relative to the ends of the chains, at least 30% of them appear to be in a different chemical environment from the rest. The evidence can be interpreted in terms of variations in some of the amino acids at particular points in a fundamental sequence of the 60 residues (CNBr3) at the amino terminal end. Further amino acid sequences near the acetylated terminal residue have been determined and provide examples of the amino acid variatioIl along the chain. One sequeIlce with variants is: N-acetyISer-(Tyr or Phe Or Pro)-Asp-(Phe or Leu)-SCMCySH-Leu-Pro-Asp-Leu-Ser-Phe-Arg-. There is a region in component 8 where many of the S-carboxymethylcysteine residues are congregated.

scholarly journals Cloning and Characterization of a Cryptic Haloacid Dehalogenase from Burkholderia cepacia MBA4

1999 ◽  
Vol 181 (19) ◽  
pp. 6003-6009 ◽  
Author(s):  
Jimmy S. H. Tsang ◽  
Laiju Sam
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Burkholderia Cepacia ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Haloacid Dehalogenase ◽  
Reading Frame ◽  
Relative Molecular Weight

ABSTRACT Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards thel-isomer of 2-monochloropropionic acid only. The structural gene, chd1, was isolated on a 1.7-kb PstI fragment. This fragment contains a functional promoter, because expression of chd1 in E. coli is orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence of chd1 did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.

scholarly journals The v-ski oncogene encodes a truncated set of c-ski coding exons with limited sequence and structural relatedness to v-myc.

1989 ◽  
Vol 9 (9) ◽  
pp. 4038-4045 ◽  
Author(s):  
E Stavnezer ◽  
D Brodeur ◽  
L A Brennan
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Avian Leukosis Virus ◽  
Amino Acid Sequences ◽  
Biologically Active ◽  
Predicted Amino Acid Sequence ◽  
Base Sequence ◽  
Reading Frame ◽  
Metal Binding Domain ◽  
Virus C

The nucleotide sequence of a biologically active v-ski gene from a cloned proviral segment shows that ski is a 1,312-base sequence embedded in the p19 region of the avian leukosis virus gag gene. The v-ski sequence contains a single open translational reading frame that encodes a polypeptide with a molecular mass of 49,000 daltons. The predicted amino acid sequence includes nuclear localization motifs that have been identified in other nuclear oncoproteins. It also contains a proline-rich region and a set of cysteine and histidine residues that could constitute a metal-binding domain. Two regions of the amino acid sequences of v-ski and v-myc are related, and the two proteins exhibit similar distributions of hydrophobic and hydrophilic amino acids. Cloned segments of the chicken c-ski proto-oncogene totaling 65 kilobases have been analyzed, and regions related to v-ski have been sequenced. The results indicate that v-ski is derived from at least five coding exons of c-ski, that it is correctly spliced, and that it is missing c-ski coding sequences at both its 5' and 3' ends. The c-ski and avian leukosis virus sequences that overlap the 5' virus/v-ski junction in Sloan-Kettering virus contain an 18-of-20-base sequence match that presumably played a role in the transduction of ski by facilitating virus/c-ski recombination.

STRUCTURE OF HUMAN α2-PLASMIN INHIBITOR DEDUCED FROM THAT OF cDNA

1987 ◽  
Author(s):  
Y Sumi ◽  
Y Nakamura ◽  
M Sakai ◽  
M Muramatsu ◽  
N Aoki
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Stop Codon ◽  
Amino Acid Sequences ◽  
The Other ◽  
Terminal Sequence ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Plasmin Inhibitor

The complete amino acid sequence of α2-plasmin inhibitor (α2PI) was determined by cDNA cloning. A Agt 10 cDNA library was prepared from poly(A)+mRNA isolated from cultured human liver cells. The labeled oligonucleotides, corresponding to the reported partial amino acid sequences of α2PI, were used as probes to screen the library. One of the positive clones was subcloned into plasmid pUC8. A 2.2 kilobase cDNA clone thus isolated contains a region coding for a portion of a leader sequence, the mature protein, a stop codon (TGA), a 3' noncoding region (733 nucleotides), and a poly(A)tail (37 nucleotides). The amino acid sequence deduced from the cDNA is composed of 452 amino acids starting with an amino-terminal sequence of Asn-Gln-Glu-Gln and ending with a carboxyl-terminal sequence of Gly-Ser-Pro-Lys. The sequence shows approximately 30% homology with those of other plasma serine protease inhibitors. However, α2PI extends 50-52 amino acids beyond the carboxyl-terminal ends of the other inhibitors. This 50-52 carboxyl-terminal amino acid sequence is therefore specific to α2PI, and contains the sequence that is exactly the same as that of the peptide containing the plasminogen binding site. There are three lysine residues possibly involved in the binding to plasminogen in this region. From the homology with the other inhibitors, the inhibitor's reactive-site peptide bond was suggested to be Met-Ser and the same as that of ai-antitrypsin. The Met residue is located at the 362 position from the amino-terminal end.

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