scholarly journals Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase

1992 ◽  
Vol 286 (1) ◽  
pp. 117-123 ◽  
Author(s):  
P C Byrne ◽  
P G Sanders ◽  
K Snell
Keyword(s):  
Nucleotide Sequence ◽  
Sequence Similarity ◽  
Protein Band ◽  
Serine Hydroxymethyltransferase ◽  
Rabbit Liver ◽  
Reading Frame ◽  
Transfected Cells ◽  
Liver Cdna Library ◽  
Sds Page ◽  
Cdna Nucleotide Sequence

A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5′ end and 653 bp at the 3′ end. The full-length cDNA was cloned into an expression vector and transfected into COS-1 cells. Serine hydroxymethyltransferase activity was increased by 33% in the transfected cells and a new protein band of the appropriate size was seen by SDS/PAGE analysis of proteins extracted from transfected cells. The protein sequence for rabbit cytosolic serine hydroxymethyltransferase derived from the cDNA nucleotide sequence was compared with three other derived or known prokaryotic and eukaryotic sequences. An overall sequence similarity of 34% was noted between all four sequences, whereas the similarity between the rabbit cytosolic and mitochondrial isoforms was 62%.

scholarly journals Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver

1994 ◽  
Vol 301 (2) ◽  
pp. 471-476 ◽  
Author(s):  
E Maellaro ◽  
B Del Bello ◽  
L Sugherini ◽  
A Santucci ◽  
M Comporti ◽  
...  
Keyword(s):  
Rat Liver ◽  
Protein Identification ◽  
Reductase Activity ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Dehydroascorbic Acid ◽  
Protein Band ◽  
Dehydroascorbate Reductase ◽  
Apparent Homogeneity ◽  
Sds Page

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

scholarly journals Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae

1996 ◽  
Vol 317 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Kenneth A. CORNELL ◽  
R. W. WINTER ◽  
Paula A. TOWER ◽  
Michael K. RISCOE
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Sds Page ◽  
High Degree ◽  
Putative Translation Product ◽  
Substrate Kinetics

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiellapneumoniae. Chromatography using a novel 5´-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46–50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

Identification of thecrygene inBacillus thuringiensisstrain WZ-9 and its toxicity againstHenosepilachna vigintioctomaculata

2008 ◽  
Vol 5 (3) ◽  
pp. 245-250 ◽  
Author(s):  
Song Ping ◽  
Wang Qin-Ying ◽  
Wu Hui-Xian ◽  
Lu Xiu-Jun ◽  
Wang Yong
Keyword(s):  
Amino Acid ◽  
Culture Media ◽  
Stationary Phases ◽  
Protein Band ◽  
Growth Cycle ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Gene Sequence Analysis ◽  
Sds Page ◽  
Encoding Gene

AbstractBacillus thuringiensisstrain WZ-9, isolated from soil in Hebei province, China, was effective againstHenosepilachna vigintioctomaculatalarvae. The strain presented bipyramidal crystals with a protein band of 130 kDa in SDS–PAGE. The pH changes of the culture media showed important fluctuations during the 24 h growth cycle. The pH varied less in log and stationary phases than it did in the exponential phase. Bioassay results showed that the WZ-9 strain was only harmful to larvae ofH. vigintioctomaculataand not to either adults ofH. vigintioctomaculataor other several lepidopteran and coleopteran insects. LC50to second-instar larvae ofH. vigintioctomaculatawas 2.95×107cells/ml after 72 h. Genotypic investigations showed that this strain possessed thecry7gene. Sequence analysis demonstrated that the encoding gene contained an open reading frame (ORF) of 3414 bp and encoded 1138 amino acid residues. The deduced amino acid sequence was 99.65% identical to that of the reported Cry7Ab2 sequences. This gene was designated by the Bt δ-endotoxin nomenclature committee as Cry7Ab3 with accession number BI 1015188 in the GenBank database.

scholarly journals Complete Genome Sequence of a Novel Human Papillomavirus Isolated from Oral Rinse

2017 ◽  
Vol 5 (45) ◽  
Author(s):  
Zigui Chen ◽  
Martin C. S. Wong ◽  
Po Yee Wong ◽  
Wendy C. S. Ho ◽  
Miaoyin Liang ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Nucleotide Sequence ◽  
Complete Genome Sequence ◽  
Sequence Similarity ◽  
Species Group ◽  
Open Reading Frame ◽  
Male Resident ◽  
Reading Frame ◽  
Oral Rinse ◽  
Chinese Male

ABSTRACT A novel human papillomavirus (HPV TG550) isolated from the oral rinse of a Chinese male resident was fully characterized. The L1 open reading frame of HPV TG550 shares 82.5% nucleotide sequence similarity with its closest relative, HPV166, and clusters within the species group Gammapapillomavirus 19.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals The primary structure of rabbit liver cytosolic serine hydroxymethyltransferase.

1987 ◽  
Vol 262 (12) ◽  
pp. 5499-5509 ◽  
Author(s):  
F. Martini ◽  
S. Angelaccio ◽  
S. Pascarella ◽  
D. Barra ◽  
F. Bossa ◽  
...  
Keyword(s):  
Primary Structure ◽  
Serine Hydroxymethyltransferase ◽  
Rabbit Liver

An ubiquitously expressed gene 3.5 kilobases upstream of the glycerol-3-phosphate dehydrogenase gene in mice

1989 ◽  
Vol 9 (3) ◽  
pp. 935-945
Author(s):  
L A Johnston ◽  
M A Kotarski ◽  
D J Jerry ◽  
L P Kozak
Keyword(s):  
Sequence Similarity ◽  
Single Copy ◽  
Transcriptional Unit ◽  
Chromosome 15 ◽  
Glycerol Phosphate ◽  
Reading Frame ◽  
Dehydrogenase Gene ◽  
New Gene ◽  
Or Gene ◽  
Glycerol 3 Phosphate Dehydrogenase

While studying the organization of the mouse glycerol-phosphate dehydrogenase gene (Gdc-1 on chromosome 15), we identified a novel transcriptional unit located only 3.4 kilobases (kb) upstream of the 5' end of the Gdc-1 gene. This gene has been provisionally named D15Kz1. The unusual proximity of these two genes led us to investigate the pattern of expression and sequence characteristics of the new gene for comparison with those of Gdc-1. D15Kz1 was found to have transcripts of 3.2 and 3.4 kb in length. The 3.4-kb transcript was expressed at low levels in all tissues examined, whereas the 3.2-kb transcript was detected only in the cerebral cortex and the brown fat. D15Kz1 and Gdc-1 are not coordinately regulated, as evidenced by the characteristics of their expression in several tissues and in differentiating 3T3-F442A adipocyte cultures. A cDNA sequence of 3,105 bases isolated from an embryonal carcinoma lambda gt10 cDNA library had a large open reading frame of 461 amino acids at one end followed by 1.6 kb of sequence with multiple stop codons. Algorithms used to search the protein and nucleic acid data bases detected no significant sequence similarity to any other protein or gene. Southern blot analysis of genomic DNA using the D15Kz1 cDNA as a probe indicated that D15Kz1 is a single-copy gene in the mouse genome and that it is conserved in humans, rats, and chickens. This conservation of gene sequences suggests that D15Kz1 encodes a protein with an important cellular function.

scholarly journals Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from Clostridium botulinum Strain 213B

2000 ◽  
Vol 66 (12) ◽  
pp. 5480-5483 ◽  
Author(s):  
Sean S. Dineen ◽  
Marite Bradshaw ◽  
Eric A. Johnson
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Clostridium Botulinum ◽  
Shuttle Vector ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Heat Stable ◽  
Group I

ABSTRACT Boticin B is a heat-stable bacteriocin produced byClostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene,btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing theHindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.

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