scholarly journals Primary structure of rat liver alkaline phosphatase deduced from its cDNA

1988 ◽  
Vol 249 (3) ◽  
pp. 661-668 ◽  
Author(s):  
Y Misumi ◽  
K Tashiro ◽  
M Hattori ◽  
Y Sakaki ◽  
Y Ikehara
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Rat Liver ◽  
Blot Hybridization ◽  
Fold Increase ◽  
Single Species ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Duct Ligation

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.

Differential regulation of mRNAs encoding for rat intestinal alkaline phosphatase

1990 ◽  
Vol 259 (1) ◽  
pp. G93-G98 ◽  
Author(s):  
R. Eliakim ◽  
S. Seetharam ◽  
C. C. Tietze ◽  
D. H. Alpers
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Cdna Probe ◽  
Fold Increase ◽  
Maximal Activity ◽  
Amino Acid Residues ◽  
Intestinal Alkaline Phosphatase ◽  
Mrna Accumulation ◽  
Dihydroxyvitamin D3 ◽  
Fat Feeding

A cDNA probe encoding the entire structural region of the 62-kDa rat intestinal alkaline phosphatase from amino acid residues 1 to 531 detected multiple mRNA species (3.0, 2.7, and 2.2 kb) in rat intestinal RNA. The 3.0-kb species was most evident in duodenum but could be easily detected in jejunum using a 48-mer oligonucleotide encoding amino acid residues 492-508. This 48-mer oligonucleotide bound preferentially to the 3.0-kb mRNA, suggesting that the 2.7-kb mRNA differed in this region. To determine whether each of the mRNAs encoding rat intestinal alkaline phosphatase responded coordinately to physiological stimuli, the full-length cDNA and the 48-mer oligonucleotide were used as probes for the 2.7- and 2.2-kb and the 3.0-kb mRNAs, respectively. Intestinal mRNA concentration was measured by Northern blot analysis in acute (single feed, 17 kcal) and chronic (3 wk, 30% fat diet) fat feeding and in rachitic rats after 1,25-dihydroxyvitamin D3 therapy. There was a large increase (8- to 25-fold) in the 3.0-kb mRNA 7 h after acute fat feeding, with a much smaller increase (1.4- to 5.0-fold) in the 2.7- and 2.2-kb species. The peak in 3.0-kb mRNA accumulation correlated in time with the maximal activity of serum phosphatase activity after acute fat feeding (4- to 5-fold increase). In contrast, there was a much smaller increase in all mRNAs and in tissue and serum enzyme activity after chronic fat feeding.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Replication Specificity Elements of the Worland Strain of Beet Curly Top Virus Are Compatible with Those of the CFH Strain But Not Those of the Cal/Logan Strain

1998 ◽  
Vol 88 (11) ◽  
pp. 1174-1178 ◽  
Author(s):  
Drake C. Stenger
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Separate Species ◽  
Rep Protein ◽  
Reading Frame ◽  
Replication Assay ◽  
Beet Curly Top Virus ◽  
Rep Proteins ◽  
Cis And Trans

Cloned genomes of the CFH, Worland, and Cal/Logan strains of beet curly top virus (BCTV) served as helper viruses to trans-replicate defective (D) DNAs that are incapable of self-replication due to deletions within the C1 open reading frame encoding the replication initiator (Rep) protein. The Logan Rep protein could trans-replicate a Logan-derived D DNA in a transient replication assay conducted in Nicotiana benthamiana leaf disks. However, the Logan Rep protein was unable to trans-replicate D DNAs derived from the CFH or Worland strains. In contrast, the Rep proteins of the CFH and Worland strains could trans-replicate CFH or Worland D DNAs, but not a Logan D DNA. These results indicate that the cis- and trans-acting replication specificity elements of the CFH and Worland strains are compatible and that the three strains of BCTV may be divided into two groupings based upon replication specificity determinants. A comparison of amino acid sequences of the Rep protein for the three BCTV strains suggests that the trans-acting replication specificity element may reside in one or more of 12 amino acid residues that are identical; in two amino acid residues that are chemically similar among the CFH and Worland Rep proteins, yet are different in the Logan Rep protein; or in both. Properties including replication specificity, nucleotide sequence identity, and symptom expression were used as criteria to propose separate species designations for each of the three BCTV strains. In this proposal, the Cal/ Logan strain retains the name BCTV, CFH and the closely related Iranian isolate are designated beet severe curly top virus, and Worland is designated beet mild curly top virus.

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

1999 ◽  
Vol 65 (7) ◽  
pp. 3001-3007 ◽  
Author(s):  
Frederic Chavagnat ◽  
Michael G. Casey ◽  
Jacques Meyer
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Streptococcus Thermophilus ◽  
Maximal Activity ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
T7 Promoter ◽  
Reading Frame ◽  
Endopeptidase Activity

ABSTRACT The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of thepepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.

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 Primary structure of the cytosolic β-glucosidase of guinea pig liver

1996 ◽  
Vol 319 (3) ◽  
pp. 829-837 ◽  
Author(s):  
William S HAYS ◽  
Steven A. JENISON ◽  
Takashi YAMADA ◽  
Andrzej PASTUSZYN ◽  
Robert H. GLEW
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Guinea Pig ◽  
Mammalian Species ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Pig Liver ◽  
Reading Frame ◽  
Degenerate Oligonucleotide ◽  
Guinea Pig Liver

The cytosolic β-glucosidase (EC 3.2.1.21) present in the livers of mammalian species is distinguished by its broad specificity for sugars and its preference for hydrophobic aglycones. We purified the cytosolic β-glucosidase from guinea pig liver and sequenced 142 amino acid residues contained within 12 trypsin digest fragments. Using degenerate oligonucleotide primers deduced from the peptide sequences, a 622 bp cytosolic β-glucosidase cDNA was amplified by reverse-transcriptase PCR, using total guinea pig liver RNA as template. The ‘rapid amplification of cDNA ends (RACE)’ method [Frohman (1993) Methods Enzymol. 218, 340–356] was used to synthesize the remaining segments of the full-length cDNA. The complete cDNA contained 1671 nucleotides with an open reading frame coding for 469 amino acid residues. The amino acid sequence deduced from the cDNA sequence included the amino acid sequences of all 12 trypsin digest fragments derived from the purified enzyme. Amino acid sequence analysis indicates that the guinea pig liver cytosolic β-glucosidase is a Family 1 β-glycosidase and that it is most closely related to mammalian lactase-phlorizin hydrolase. These results suggest that the cytosolic β-glucosidase and lactase-phlorizin hydrolase diverged from a common evolutionary precursor.

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).

Two Nearly Identical Aromatic Compound Hydrolase Genes in a Strong Polychlorinated Biphenyl Degrader,Rhodococcus sp. Strain RHA1

1998 ◽  
Vol 64 (6) ◽  
pp. 2006-2012 ◽  
Author(s):  
Akihiro Yamada ◽  
Hidekazu Kishi ◽  
Katsumi Sugiyama ◽  
Takashi Hatta ◽  
Kanji Nakamura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polychlorinated Biphenyl ◽  
Blot Hybridization ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Gene Encoding ◽  
Amino Terminal ◽  
Gene Assay ◽  
Amino Terminal Sequence

ABSTRACT The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes,etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. TheetbD2 gene was located in the vicinity of bphAgene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in α/β hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD,cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphDgenes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product ofbphD was very specific to HPDA, and the products ofetbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against themeta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphDgene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.

Molecular cloning, characterization and antigenicity ofBabesiasp. BQ1 (Lintan) (Babesiacf.motasi) apical membrane antigen-1 (AMA-1)

Parasitology ◽  
2016 ◽  
Vol 144 (5) ◽  
pp. 641-649 ◽  
Author(s):  
QINGLI NIU ◽  
ZHIJIE LIU ◽  
JIFEI YANG ◽  
GUIQUAN GUAN ◽  
YUPING PAN ◽  
...  
Keyword(s):  
Amino Acid ◽  
Apical Membrane ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Gene Characterization ◽  
Reading Frame ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Potential Vaccine

SUMMARYApical membrane antigen-1 (AMA-1) has been described as a potential vaccine candidate in apicomplexan parasites. Here we characterize theama-1gene. The full-lengthama-1gene ofBabesiasp. BQ1 (Lintan) (BLTAMA-1) is 1785 bp, which contains an open reading frame (ORF) encoding a 65-kDa protein of 594 amino acid residues; by definition, the 5′ UTR precedes the first methionine of the ORF. Phylogenetic analysis based on AMA-1 amino acid sequences clearly separated Piroplasmida from other Apicomplexa parasites. TheBabesiasp. BQ1 (Lintan) AMA-1 sequence is most closely associated with that ofB. ovataandB. bigemina, with high bootstrap value. A recombinant protein encoding a conserved region and containing ectodomains I and II of BLTAMA-1 was constructed. BLTrAMA-1-DI/DII proteins were tested for reactivity with sera from sheep infected byBabesiasp. BQ1 (Lintan). In Western-blot analysis, nativeBabesiasp. BQ1 (Lintan) AMA-1 proteins were recognized by antibodies raised in rabbits against BLTrAMA-1in vitro. The results of this study are discussed in terms of gene characterization, taxonomy and antigenicity.

scholarly journals Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

2001 ◽  
Vol 67 (4) ◽  
pp. 1783-1787 ◽  
Author(s):  
Jun Ogawa ◽  
Sou Takeda ◽  
Sheng-Xue Xie ◽  
Haruyo Hatanaka ◽  
Toshihiko Ashikari ◽  
...  
Keyword(s):  
Amino Acid ◽  
Metal Ion ◽  
Amino Acid Sequences ◽  
Leader Peptide ◽  
Amino Acid Residues ◽  
Ochrobactrum Anthropi ◽  
Pyrimidine Nucleotides ◽  
Reading Frame ◽  
Pyrimidine Nucleosides ◽  
Nicotinamide Mononucleotide

ABSTRACT A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversibleN-riboside hydrolysis of purine nucleosides, theKm values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with Ki and Ki ′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn2+ or Co2+ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed inEscherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH2-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.

scholarly journals Expression of heparanase mRNA in bovine placenta during gestation

Reproduction ◽  
2001 ◽  
pp. 573-580 ◽  
Author(s):  
K Kizaki ◽  
H Nakano ◽  
H Nakano ◽  
T Takahashi ◽  
K Imai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Blot Hybridization ◽  
Amino Acid Sequences ◽  
Pcr Analysis ◽  
Fetal Membrane ◽  
Northern Blot Hybridization ◽  
Amino Acid Residues ◽  
Placental Development ◽  
Bovine Placenta ◽  
And Function

This study reports the identification and sequence of a partial cDNA for bovine heparanase and the expression of its mRNA in the placenta during gestation. The 364 amino acid residues deduced from the 1092 bp cDNA fragment share 81.9% and 80.5% identity with amino acid sequences of human and rat heparanase, respectively. Northern blot hybridization showed that two mRNAs (2.0 and 3.5 kb) are strongly expressed in placenta, and weakly expressed in the kidney, lung, spleen and non-pregnant uterus. In the placenta, these transcripts were detected in the cotyledon at all stages of gestation examined, and in the intercotyledonary fetal membrane and caruncle on day 60, day 120 and day 260. Quantitative real-time RT-PCR analysis showed very low expression of heparanase mRNA in the conceptus before implantation (day 17), but high expression in the cotyledon-containing fetal membrane (days 27-34) after implantation. Furthermore, heparanase mRNA was detected in the cotyledon, intercotyledonary fetal membrane and caruncle after days 60-64 of gestation. However, no significant expression of heparanase mRNA was observed in intercaruncular endometrium at all stages of gestation examined. These results demonstrate that heparanase mRNA is expressed in the placentome, indicating that heparanase may play a role in implantation, and in placental development and function.

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