Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

ABSTRACT The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

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Molecular cloning and expression of an intracellular serpin: an elastase inhibitor from horse leucocytes

Biochemical Journal ◽

10.1042/bj2930187 ◽

1993 ◽

Vol 293 (1) ◽

pp. 187-193 ◽

Cited By ~ 7

Author(s):

T Kordula ◽

A Dubin ◽

H Schooltink ◽

A Koj ◽

P C Heinrich ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Oligonucleotide Probe ◽

Protein Sequencing ◽

Cloning And Expression ◽

Coding Region ◽

Elastase Inhibitor ◽

Escherichia Coli Cells ◽

Degenerate Oligonucleotide ◽

Horse Blood

Horse blood leucocytes contain an elastase inhibitor (HLEI) belonging to the serpin family. Poly(A)+RNA isolated from these cells was used to construct a cDNA library in lambda gt10, which was first screened with a synthetic degenerate oligonucleotide probe corresponding to the amino acid sequence of the reactive centre of the inhibitor. Three clones were obtained covering the entire coding region of the protein. Sequencing of these clones showed identity with the amino acid sequence obtained from Edman degradation of the elastase inhibitor. The coding sequence of the HLEI cDNA was cloned into the bacterial expression vector pKK233-2 and expressed in Escherichia coli cells. Transformed bacteria expressed significant amounts of the protein, which was immunoprecipitated with a specific anti-HLEI antiserum. Furthermore, HLEI expressed in bacteria inhibited the activity of elastase but not trypsin.

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Murine erythropoietin gene: cloning, expression, and human gene homology.

Molecular and Cellular Biology ◽

10.1128/mcb.6.3.849 ◽

1986 ◽

Vol 6 (3) ◽

pp. 849-858 ◽

Cited By ~ 131

Author(s):

C B Shoemaker ◽

L D Mitsock

Keyword(s):

Amino Acid ◽

Genomic Library ◽

Cdna Probe ◽

Amino Acid Sequences ◽

Nucleotide Sequence Analysis ◽

Coding Region ◽

Transcription Control ◽

Erythroid Progenitor ◽

Human Genes ◽

Cell Expression

The gene for murine erythropoietin (EPO) was isolated from a mouse genomic library with a human EPO cDNA probe. Nucleotide sequence analysis permitted the identification of the murine EPO coding sequence and the prediction of the encoded amino acid sequence based on sequence conservation between the mouse and human EPO genes. Both the coding DNA and the amino acid sequences were 80% conserved between the two species. Transformation of COS-1 cells with a mammalian cell expression vector containing the murine EPO coding region resulted in secretion of murine EPO with biological activity on both murine and human erythroid progenitor cells. The transcription start site for the murine EPO gene in kidneys was determined. This permitted tentative identification of the transcription control region. The region included 140 base pairs upstream of the cap site which was over 90% conserved between the murine and human genes. Surprisingly, the first intron and much of the 5'- and 3'-untranslated sequences were also substantially conserved between the genes of the two species.

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Nucleotide and derived amino acid sequences of a cDNA coding for pre-uteroglobin from the lung of the hare (Lepus capensis)

Biochemical Journal ◽

10.1042/bj2350895 ◽

1986 ◽

Vol 235 (3) ◽

pp. 895-898 ◽

Cited By ~ 12

Author(s):

M S López de Haro ◽

A Nieto

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Untranslated Regions ◽

Coding Region ◽

Homologous Proteins ◽

Lepus Capensis ◽

Rabbit Gene

An almost full-length cDNA coding for pre-uteroglobin from hare lung was cloned and sequenced. The derived amino acid sequence indicated that hare pre-uteroglobin contained 91 amino acids, including a signal peptide of 21 residues. Comparison of the nucleotide sequence of hare pre-uteroglobin cDNA with that previously reported for the rabbit gene indicated five silent point substitutions and six others leading to amino acid changes in the coding region. The untranslated regions of both pre-uteroglobin mRNAs were very similar. The amino acid changes observed are discussed in relation to the different progesterone-binding abilities of both homologous proteins.

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No polymorphisms in the coding region of the prion-like protein gene in Thoroughbred racehorses

Acta Veterinaria Hungarica ◽

10.1556/004.2019.019 ◽

2019 ◽

Vol 67 (2) ◽

pp. 174-182 ◽

Cited By ~ 4

Author(s):

Min-Ju Jeong ◽

Byung-Hoon Jeong

Keyword(s):

Amino Acid ◽

Prion Protein ◽

Protein Gene ◽

Prion Diseases ◽

Amino Acid Sequences ◽

Prion Protein Gene ◽

Coding Region ◽

Thoroughbred Racehorses ◽

Thoroughbred Horses ◽

Protein Isoform

Prion diseases are fatal neurodegenerative diseases characterised by the accumulation of an abnormal prion protein isoform (PrPSc), which is converted from the normal prion protein (PrPC). Prion diseases have been reported in an extensive number of species but not in horses up to now; therefore, horses are known to be a species resistant to prion diseases. The prion-like protein gene (PRND) is closely located downstream of the prion protein gene (PRNP) and the prion-like protein (Doppel) is a homologue with PrP. Previous studies have shown that an association between prion diseases and polymorphisms of the PRND gene is reported in the main hosts of prion diseases. Hence, we examined the genetic variations of the PRND gene in Thoroughbred horses. Interestingly, polymorphisms of the PRND gene were not detected. In addition, we conducted a comparative analysis of the amino acid sequences of the PRND gene to identify the differences between horses and other species. The amino acid sequence of the horse PRND gene showed the highest identity to that of sheep (83.7%), followed by that of goats, cattle and humans. To the best of our knowledge, this is the first genetic study of the PRND gene in horses.

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Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase, an Enzyme Involved in the Metabolism of Daidzein, from Equol-Producing Lactococcus Strain 20-92

Applied and Environmental Microbiology ◽

10.1128/aem.01101-10 ◽

2010 ◽

Vol 76 (17) ◽

pp. 5892-5901 ◽

Cited By ~ 51

Author(s):

Yoshikazu Shimada ◽

Setsuko Yasuda ◽

Masayuki Takahashi ◽

Takashi Hayashi ◽

Norihiro Miyazawa ◽

...

Keyword(s):

Amino Acid ◽

Enteric Bacteria ◽

Amino Acid Sequences ◽

Soy Isoflavones ◽

Cloning And Expression ◽

Gene Encoding ◽

Binding Motifs ◽

Reading Frame ◽

Cofactor Binding ◽

International Patent

ABSTRACT Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.

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The cDNA and protein sequences of human lactate dehydrogenase B

Biochemical Journal ◽

10.1042/bj2480933 ◽

1987 ◽

Vol 248 (3) ◽

pp. 933-936 ◽

Cited By ~ 31

Author(s):

I Sakai ◽

F S Sharief ◽

Y C Pan ◽

S S Li

Keyword(s):

Amino Acid ◽

Lactate Dehydrogenase ◽

Amino Acid Composition ◽

Acid Composition ◽

Amino Acid Sequences ◽

British Library ◽

Amino Acid Residues ◽

Coding Region ◽

Cdna Insert ◽

Protein Coding

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5′ (54 bp) and 3′ (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].

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Isolation and characterization of the pea cytochrome c oxidase Vb gene

Genome ◽

10.1139/g06-105 ◽

2006 ◽

Vol 49 (11) ◽

pp. 1481-1489 ◽

Cited By ~ 1

Author(s):

Nakao Kubo ◽

Shin-ichi Arimura ◽

Nobuhiro Tsutsumi ◽

Koh-ichi Kadowaki ◽

Masashi Hirai

Keyword(s):

Amino Acid ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Fluorescent Proteins ◽

Amino Acid Sequences ◽

Homology Search ◽

Coding Region ◽

Angiosperm Evolution ◽

Isolation And Characterization ◽

Duplication Events

Three copies of the gene that encodes cytochrome c oxidase subunit Vb were isolated from the pea (PscoxVb-1, PscoxVb-2, and PscoxVb-3). Northern Blot and reverse transcriptase-PCR analyses suggest that all 3 genes are transcribed in the pea. Each pea coxVb gene has an N-terminal extended sequence that can encode a mitochondrial targeting signal, called a presequence. The localization of green fluorescent proteins fused with the presequence strongly suggests the targeting of pea COXVb proteins to mitochondria. Each pea coxVb gene has 5 intron sites within the coding region. These are similar to Arabidopsis and rice, although the intron lengths vary greatly. A phylogenetic analysis of coxVb suggests the occurrence of gene duplication events during angiosperm evolution. In particular, 2 duplication events might have occurred in legumes, grasses, and Solanaceae. A comparison of amino acid sequences in COXVb or its counterpart shows the conservation of several amino acids within a zinc finger motif. Interestingly, a homology search analysis showed that bacterial protein COG4391 and a mitochondrial complex I 13 kDa subunit also have similar amino acid compositions around this motif. Such similarity might reflect evolutionary relationships among the 3 proteins.

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Occurrence of PG-Lb, a leucine-rich small chondroitin/dermatan sulphate proteoglycan in mammalian epiphyseal cartilage: molecular cloning and sequence analysis of the mouse cDNA

Biochemical Journal ◽

10.1042/bj3180909 ◽

1996 ◽

Vol 318 (3) ◽

pp. 909-914 ◽

Cited By ~ 20

Author(s):

Kazuhiro KURITA ◽

Tamayuki SHINOMURA ◽

Minoru UJITA ◽

Masahiro ZAKO ◽

Daihei KIDA ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Epiphyseal Cartilage ◽

Newborn Mouse ◽

Dermatan Sulphate ◽

Cdna Fragment ◽

Sulphate Proteoglycan ◽

C Terminus ◽

Degenerate Oligonucleotide

PG-Lb is a chondroitin/dermatan sulphate proteoglycan first isolated from chick embryo limb cartilage. It had been assumed that osteoglycin represents its mammalian homologue. However, partial amino acid sequences of a novel proteoglycan from bovine epiphyseal cartilage showed high identity with those of chick PG-Lb (P. Neame, L. Rosenberg and M. Höök, personal communication). Reverse transcriptase PCR using degenerate oligonucleotide primers gave a cDNA fragment that might correspond to mouse PG-Lb. We isolated a clone from a cDNA library of newborn mouse epiphyseal cartilage using the cDNA fragment as a probe. The cloned cDNA was 1430 bp long and contained a 966 bp open reading frame which encoded the core protein consisting of 322 amino acid residues. The deduced amino acid sequence showed a high overall identity with chick PG-Lb (about 62%, reaching about 80% over the carboxyl two-thirds). In addition, the amino acid sequence contained a signal peptide, six cysteine residues at the invariant relative position to chick PG-Lb, six leucine-rich repeats at the carboxyl two-thirds, three possible glycosaminoglycan-attachment sites (two sites at the N-terminal side and one site at the C-terminus) and two possible Asn-glycosylation sites near the C-terminus. Northern-blot analysis demonstrated the specific expression of a 1.5 kb message in cartilage and testis. These structural features and the characteristic expression suggest that the cloned molecule is mouse PG-Lb.

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Characterization of the mouse thyrotrophin-releasing hormone receptor gene: an exon corresponds to a deletion in the rat cDNA

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0110141 ◽

1993 ◽

Vol 11 (2) ◽

pp. 141-149 ◽

Cited By ~ 6

Author(s):

S M Duthie ◽

P L Taylor ◽

K A Eidne

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Stop Codon ◽

Amino Acid Sequences ◽

R Gene ◽

Coding Region ◽

Thyrotrophin Releasing Hormone ◽

Exon 2 ◽

Exon 4

ABSTRACT The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)16.d(AG)21) in the 5′ untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3′ UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA. The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3′ UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3′ UTR, allowing transcription to continue into the 3′ UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA. This would then result in an alternative 412 amino acid version of the mouse TRH-R protein, with 95% homology to the rat TRH-R. This study focused on the structural differences in the intracellular COOH-terminal tail of the receptor, which is known to be a functionally important domain in other members of the G protein-coupled receptor family. We have also recently characterized the human TRH-R cDNA, which revealed a third variant at the COOH terminus. Comparisons between mouse, rat and human TRH-Rs show that the amino acid sequences are virtually identical. However, significant differences between these species exist at the COOH terminus, with each TRH-R having a unique form of the COOH-terminal tail, beginning at exactly the same site and encoding 1, 20 and 6 amino acids in the mouse, rat and human respectively.

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Cloning and expression of caprine KIT gene and associations of polymorphisms with litter size

Animal Production Science ◽

10.1071/an13497 ◽

2016 ◽

Vol 56 (10) ◽

pp. 1579 ◽

Cited By ~ 1

Author(s):

X. P. An ◽

J. X. Hou ◽

T. Y. Gao ◽

B. Y. Cao

Keyword(s):

Amino Acid ◽

Litter Size ◽

Bos Taurus ◽

Homo Sapiens ◽

Ovis Aries ◽

Cloning And Expression ◽

Nucleotide Polymorphisms ◽

Coding Region ◽

Reading Frame ◽

Kit Gene

The full coding region of KIT mRNA was cloned from the caprine ovary. The results showed the caprine KIT cDNA (GenBank accession number KF364483) contained a 2925-bp open reading frame encoding a protein with 974 amino acid residues. BLAST analysis revealed that the caprine KIT protein had high similarity with that of four species: Ovis aries (99%), Bos taurus (99%), Sus scrofa (94%) and Homo sapiens (90%). The KIT mRNA expression pattern showed that KIT mRNA was expressed highly in kidney, ovary, uterus and breast. Two single nucleotide polymorphisms (g.88430T > A and g.120466G > A) in the caprine KIT gene were detected by PCR–restriction fragment length polymorphism (RFLP) and DNA sequencing in 735 goats of Xinong Saanen, Guanzhong and Boer breeds. The g.88430T > A mutation was a missense mutation (Tyr > Asn at position 409 amino acid of KIT). The association study has been done by jointly analysing all data in one analysis. The result showed that individuals with TT and TA genotypes had their litter size increased by 0.11 and 0.09, respectively, compared with those with AA genotype at the g.88430T > A locus for three goat breeds (P < 0.05). Further analysis revealed that combined genotype TTAA was better than the others for litter size in three goat breeds. Therefore, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the KIT gene could serve as a genetic marker for litter size in goat breeding.

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