A novel 17β-hydroxysteroid dehydrogenase in the fungus Cochliobolus lunatus: new insights into the evolution of steroid-hormone signalling

1999 ◽  
Vol 337 (3) ◽  
pp. 425-431 ◽  
Author(s):  
Tea LANIŠNIK RIŽNER ◽  
Gabriele MOELLER ◽  
Hubert H. THOLE ◽  
Marija ŽAKELJ-MAVRIČ ◽  
Jerzy ADAMSKI
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnaporthe Grisea ◽  
Hydroxysteroid Dehydrogenase ◽  
Cdna Clones ◽  
The Novel ◽  
Reading Frame ◽  
Cochliobolus Lunatus ◽  
Acid Protein ◽  
History Of

17β-Hydroxysteroid dehydrogenase (17β-HSD) from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) catalyses the reduction of steroids and of several o- and p-quinones. After purification of the enzyme, its partial amino acid sequence was determined. A PCR fragment amplified with primers derived from peptide sequences was generated for screening the Coch. lunatus cDNA library. Three independent full-length cDNA clones were isolated and sequenced, revealing an 810-bp open reading frame encoding a 270-amino-acid protein. After expression in Escherichia coli and purification to homogeneity, the enzyme was found to be active towards androstenedione and menadione, and was able to form dimers of Mr 60000. The amino acid sequence of the novel 17β-HSD demonstrated high homology with fungal carbonyl reductases, such as versicolorin reductase from Emericella nidulans (Aspergillus nidulans; VerA) and Asp. parasiticus (Ver1), polyhydroxynaphthalene reductase from Magnaporthe grisea, the product of the Brn1 gene from Coch. heterostrophus and a reductase from Colletotrichum lagenarium, which are all members of the short-chain dehydrogenase/reductase superfamily. 17β-HSDcl is the first discovered fungal 17β-hydroxysteroid dehydrogenase belonging to this family. The primary structure of this enzyme may therefore help to elucidate the evolutionary history of steroid dehydrogenases.

Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1435-1442 ◽  
Author(s):  
Edward M. Conway ◽  
Saskia Pollefeyt ◽  
Jan Cornelissen ◽  
Inky DeBaere ◽  
Marta Steiner-Mosonyi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Messenger Rna ◽  
Coiled Coil ◽  
Cdna Clones ◽  
Exon 2 ◽  
Reading Frame ◽  
Coiled Coil Domain ◽  
Acid Protein ◽  
Apoptosis Protein

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murinesurvivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin140, similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin121 that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin40, lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin140 and survivin121 inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin140 mRNA, whereas survivin121-specific transcripts were detected in all tissues, while those representing survivin40 were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis.

scholarly journals The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

1999 ◽  
Vol 65 (12) ◽  
pp. 5546-5553 ◽  
Author(s):  
Kazuhiro Iwashita ◽  
Tatsuya Nagahara ◽  
Hitoshi Kimura ◽  
Makoto Takano ◽  
Hitoshi Shimoi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Enzyme Assays ◽  
Partial Amino Acid Sequence ◽  
Reading Frame ◽  
Acid Protein ◽  
Aspergillus Kawachii ◽  
Amino Acid Protein

ABSTRACT We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA inSaccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast.A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase.A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that thebglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii.

scholarly journals Molecular Characterization of the β-N-Acetylglucosaminidase of Escherichia coliand Its Role in Cell Wall Recycling

2000 ◽  
Vol 182 (17) ◽  
pp. 4836-4840 ◽  
Author(s):  
Qiaomei Cheng ◽  
Hongshan Li ◽  
Keith Merdek ◽  
James T. Park
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sequence Data ◽  
Degradation Products ◽  
Predicted Amino Acid Sequence ◽  
The Novel ◽  
Sequence Comparisons ◽  
Reading Frame ◽  
Vibrio Furnissii

ABSTRACT The β-N-acetylglucosaminidase of Escherichia coli was found to have a novel specificity and to be encoded by a gene (nagZ) that maps at 25.1 min. It corresponds to an open reading frame, ycfO, whose predicted amino acid sequence is 57% identical to that of Vibrio furnissiiExoII. NagZ hydrolyzes the β-1,4 glycosidic bond betweenN-acetylglucosamine and anhydro-N-acetylmuramic acid in cell wall degradation products following their importation into the cell during the process for recycling cell wall muropeptides. From amino acid sequence comparisons, the novel β-N-acetylglucosaminidase appears to be conserved in all 12 gram-negative bacteria whose complete or partial genome sequence data are available.

Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation

1985 ◽  
Vol 5 (10) ◽  
pp. 2684-2696
Author(s):  
D H Smith ◽  
D M Kegler ◽  
E B Ziff
Keyword(s):  
Amino Acid ◽  
Simian Virus 40 ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Acid Protein ◽  
Dna Replication Origin ◽  
E1a Protein

We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.

scholarly journals Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

1996 ◽  
Vol 40 (3) ◽  
pp. 616-620 ◽  
Author(s):  
A Bauernfeind ◽  
I Stemplinger ◽  
R Jungwirth ◽  
P Mangold ◽  
S Amann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Beta Lactamase ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
The Family ◽  
Extended Spectrum Beta Lactamases ◽  
Lactamase Gene

Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.

scholarly journals Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

2003 ◽  
Vol 50 (1) ◽  
pp. 269-278
Author(s):  
Amr M Shabaan ◽  
Magdy M Mohamed ◽  
Mohga S Abdallah ◽  
Hayat M Ibrahim ◽  
Amr M Karim
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Vaccine Development ◽  
Complete Sequence ◽  
Cdna Clones ◽  
Western Blots ◽  
Calculated Molecular Mass ◽  
Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

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