NOP3 is an essential yeast protein which is required for pre-rRNA processing.

The four nucleolar proteins NOP1, SSB1, GAR1, and NSR1 of Saccharomyces cerevisiae share a repetitive domain composed of repeat units rich in glycine and arginine (GAR domain). We have cloned and sequenced a fifth member of this family, NOP3, and shown it to be essential for cell viability. The NOP3 open reading frame encodes a 415 amino acid protein with a predicted molecular mass of 45 kD, containing a GAR domain and an RNA recognition motif. NOP3-specific antibodies recognize a 60-kD protein by SDS-PAGE and decorate the nucleolus and the surrounding nucleoplasm. A conditional lethal mutation, GAL::nop3, was constructed; growth of the mutant strain in glucose medium represses NOP3 expression. In cells depleted of NOP3, production of cytoplasmic ribosomes is impaired. Northern analysis and pulse-chase labeling indicate that pre-rRNA processing is inhibited at the late steps, in which 27SB pre-rRNA is cleaved to 25S rRNA and 20S pre-rRNA to 18S rRNA.

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Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein

Journal of Cell Science ◽

10.1242/jcs.105.3.777 ◽

1993 ◽

Vol 105 (3) ◽

pp. 777-785 ◽

Cited By ~ 4

Author(s):

A.B. Vojtek ◽

J.A. Cooper

Keyword(s):

Adenylyl Cyclase ◽

Leading Edge ◽

Northern Analysis ◽

Reading Frame ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Acid Protein ◽

Carboxy Terminal ◽

Amino Acid Protein

CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cerevisiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxy-terminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.

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The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome

eLife ◽

10.7554/elife.16381 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 4

Author(s):

Kathleen L McCann ◽

Takamasa Teramoto ◽

Jun Zhang ◽

Traci M Tanaka Hall ◽

Susan J Baserga

Keyword(s):

Protein Interactions ◽

Molecular Basis ◽

Large Subunit ◽

Ribosomal Subunit ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Rrna Processing ◽

Protein Protein Interactions ◽

Recognition Motif ◽

Processing Defects

ANE syndrome is a ribosomopathy caused by a mutation in an RNA recognition motif of RBM28, a nucleolar protein conserved to yeast (Nop4). While patients with ANE syndrome have fewer mature ribosomes, it is unclear how this mutation disrupts ribosome assembly. Here we use yeast as a model system and show that the mutation confers growth and pre-rRNA processing defects. Recently, we found that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome. Here we demonstrate that the ANE syndrome mutation disrupts Nop4’s hub function by abrogating several of Nop4’s protein-protein interactions. Circular dichroism and NMR demonstrate that the ANE syndrome mutation in RRM3 of human RBM28 disrupts domain folding. We conclude that the ANE syndrome mutation generates defective protein folding which abrogates protein-protein interactions and causes faulty pre-LSU rRNA processing, thus revealing one aspect of the molecular basis of this human disease.

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The mouse has a Polycomb-like chromobox gene

Development ◽

10.1242/dev.114.4.921 ◽

1992 ◽

Vol 114 (4) ◽

pp. 921-929 ◽

Cited By ~ 11

Author(s):

J.J. Pearce ◽

P.B. Singh ◽

S.J. Gaunt

Keyword(s):

Cellular Proliferation ◽

Expression Patterns ◽

Northern Analysis ◽

Heterochromatin Protein 1 ◽

Heterochromatin Protein ◽

Drosophila Gene ◽

Acid Protein ◽

Temporal And Spatial ◽

Amino Acid Protein

The Drosophila gene Polycomb (Pc) has been implicated in the clonal inheritance of determined states and is a trans-regulator of the Antennapedia-like homeobox genes. Pc shares a region of homology (the chromobox) with the Drosophila gene Heterochromatin Protein 1 (HP1), a component of heterochromatin. The Pc chromobox has been used to isolate a mouse chromobox gene, M33, which encodes a predicted 519 amino acid protein. The M33 chromodomain is more similar to that in the Pc protein, than that in the HP1 protein. In addition to the chromodomain, the M33 and Pc proteins also share a region of homology at their C termini. The temporal and spatial expression patterns of M33 have been studied by in situ hybridization and northern analysis. During the final 10 days of embryonic development, M33 expression mirrors that of the cell-cycle-specific cyclin B gene. It is therefore suggested that the rate of cellular proliferation controls M33 expression. From comparisons of the characteristics of M33 with those of Pc it is proposed that M33 is a Pc-like chromobox gene. The roles of M33 and Pc in models of cellular memory are examined and implications of the memory models addressed.

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

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5546-5553.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5546-5553 ◽

Cited By ~ 37

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.

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Molecular cloning, characterization, and expression analysis of a novel sema-2a homologue in Polyrhachis vicina (Hymenoptera: Formicidae)

The Canadian Entomologist ◽

10.4039/n09-046 ◽

2010 ◽

Vol 142 (1) ◽

pp. 1-13 ◽

Cited By ~ 2

Author(s):

Jing Luo ◽

Geng-Si Xi ◽

Shu-Min Lü ◽

Ke Li ◽

Qing Li

Keyword(s):

Untranslated Region ◽

Axonal Guidance ◽

Mrna Levels ◽

Chain Reaction ◽

Base Pairs ◽

Reading Frame ◽

Acid Protein ◽

Polymerase Chain ◽

Polyrhachis Vicina ◽

Amino Acid Protein

AbstractThe semaphorin gene family plays important roles in axonal guidance in vertebrates and invertebrates. Semaphorin 2a, a member of the semaphorin family, belongs to class 2, which is found only in invertebrates. In our study, semaphorin 2a was cloned from the ant Polyrhachis vicina Roger. The full length of P. vicina semaphorin 2a (Pv-sema-2a) is 2763 base pairs (bp) and it contains a 5′-untranslated region (UTR) 92 bp long and a 3′-UTR 521 bp long. The open reading frame of Pv-sema-2a encodes a 716-amino-acid protein with a predicted molecular mass of 81.1 kilodaltons. Real-time quantitative reverse-transcription – polymerase chain reaction indicated that Pv-sema-2a mRNA is differentially expressed during P. vicina development, in the whole bodies as well as the heads of different castes. The high mRNA levels in embryos and pupae suggest that Pv-sema-2a plays an important role in ant development.

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New Carbenicillin-Hydrolyzing β-Lactamase (CARB-7) from Vibrio cholerae Non-O1, Non-O139 Strains Encoded by the VCR Region of the V. cholerae Genome

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.7.2162-2168.2002 ◽

2002 ◽

Vol 46 (7) ◽

pp. 2162-2168 ◽

Cited By ~ 30

Author(s):

Roberto Melano ◽

Alejandro Petroni ◽

Alicia Garutti ◽

Héctor Alex Saka ◽

Laura Mange ◽

...

Keyword(s):

Nucleotide Sequence ◽

Vibrio Cholerae ◽

Gene Families ◽

Chromosome 2 ◽

Reading Frame ◽

West Region ◽

Acid Protein ◽

Lactamase Gene ◽

Flanking Regions ◽

Amino Acid Protein

ABSTRACT In a previous study, an analysis of 77 ampicillin-nonsusceptible (resistant plus intermediate categories) strains of Vibrio cholerae non-O1, non-O139, isolated from aquatic environment and diarrheal stool, showed that all of them produced a β-lactamase with a pI of 5.4. Hybridization or amplification by PCR with a probe for bla TEM or primers for bla CARB gene families was negative. In this work, an environmental ampicillin-resistant strain from this sample, ME11762, isolated from a waterway in the west region of Argentina, was studied. The nucleotide sequence of the structural gene of the β-lactamase was determined by bidirectional sequencing of a Sau3AI fragment belonging to this isolate. The gene encodes a new 288-amino-acid protein, designated CARB-7, that shares 88.5% homology with the CARB-6 enzyme; an overall 83.2% homology with PSE-4, PSE-1, CARB-3, and the Proteus mirabilis N29 enzymes; and 79% homology with CARB-4 enzyme. The gene for this β-lactamase could not be transferred to Escherichia coli by conjugation. The nucleotide sequence of the flanking regions of the bla CARB-7 gene showed the occurrence of three 123-bp V. cholerae repeated sequences, all of which were found outside the predicted open reading frame. The upstream fragment of the bla CARB-7 gene shared 93% identity with a locus situated inside V. cholerae's chromosome 2. These results strongly suggest the chromosomal location of the bla CARB-7 gene, making this the first communication of a β-lactamase gene located on the VCR island of the V. cholerae genome.

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Cloning and characterization of a 2,3-oxidosqualene cyclase from Eleutherococcus trifoliatus

Holzforschung ◽

10.1515/hf-2012-0120 ◽

2013 ◽

Vol 67 (4) ◽

pp. 463-471 ◽

Cited By ~ 1

Author(s):

Li-Ting Ma ◽

Sheng-Yang Wang ◽

Yen-Hsueh Tseng ◽

Yi-Ru Lee ◽

Fang-Hua Chu

Keyword(s):

Physiological Role ◽

Active Site Residues ◽

Reading Frame ◽

Oxidosqualene Cyclases ◽

Oxidosqualene Cyclase ◽

Acid Protein ◽

Leaf Tissues ◽

New Perspective ◽

Amino Acid Protein

Abstract The 2,3-oxidosqualene cyclases (OSCs) are a family of enzymes that have an important role in plant triterpene biosynthesis. In this study, an OSC gene designed EtLUS from Eleutherococcus trifoliatus has been cloned. EtLUS includes a 2292-bp open reading frame and encodes a 763-amino acid protein. EtLUS has an MLCYCR motif, which is conserved in lupeol synthases. Comparison of active-site residues and gene expression in yeast showed that EtLUS synthesizes lupeol. However, EtLUS has the highest sequence identity with β-amyrin synthases from Araliaceae rather than lupeol synthases, adding new perspective to the evolution of the OSCs of Araliaceae. Furthermore, EtLUS is upregulated in leaf tissues under methyl jasmonate treatment, which can be interpreted that lupeol and its derivatives play an ecological and physiological role in plant defense against pathogens and insect herbivores.

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Molecular Cloning and Characterization of a cDNA Encoding Sedoheptulose-1,7-bisphosphatase from Mulberry (Morus alba var. multicaulis)

Silvae Genetica ◽

10.1515/sg-2008-0023 ◽

2008 ◽

Vol 57 (1-6) ◽

pp. 152-157 ◽

Cited By ~ 3

Author(s):

X. Ji ◽

Y. Gai ◽

J. Ma ◽

C. Zheng ◽

Z. Mu

Keyword(s):

Morus Alba ◽

Evolutionary Analysis ◽

Comparison Analysis ◽

Reading Frame ◽

Acid Protein ◽

Fructose 1,6 Bisphosphatase ◽

Rapid Amplification ◽

Molecular Evolutionary Analysis ◽

Amino Acid Protein

Abstract A full-length cDNA encoding sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) was cloned from mulberry (Morus alba var. multicaulis) by rapid amplification of cDNA ends (RACE). The cDNA consisted of 1,527 nucleotides with an open reading frame (ORF) of 1,179 nucleotides encoding a 393 amino acid protein of approximately 42.6 kDa. Sequence comparison analysis showed that mulberry SBPase (MSBPase) had high homology to other plant counterparts. Phylogenetic and molecular evolutionary analysis revealed that MSBPase fell into plant SBPase group. Moreover, SBPase and fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) shared 28-32% identical residues, suggesting that the two enzymes originated from the same evolution branch. Molecular modeling indicated that each subunit of MSBPase was composed of α-helices and β-sheets joined by turns and loops, and folded into a structure of hexahedron shape which was very similar to FBPase.

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Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893

Applied and Environmental Microbiology ◽

10.1128/aem.01733-07 ◽

2008 ◽

Vol 74 (8) ◽

pp. 2379-2383 ◽

Cited By ~ 16

Author(s):

Hitomi Ichinose ◽

Toshihisa Kotake ◽

Yoichi Tsumuraya ◽

Satoshi Kaneko

Keyword(s):

Signal Sequence ◽

Recombinant Enzyme ◽

Streptomyces Avermitilis ◽

Glycoside Hydrolase Family ◽

Reading Frame ◽

Secretion Signal ◽

Acid Protein ◽

Hydrolysis Of ◽

Amino Acid Protein

ABSTRACT The putative endo-β-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of β-1,6-linked galacto-oligosaccharides, predominantly β-1,6-galactobiose, from β-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-β-1,6-galactanase from a prokaryote.

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Molecular cloning and expression of a new putative inositol 1,4,5-trisphosphate 3-kinase isoenzyme

Biochemical Journal ◽

10.1042/bj2780883 ◽

1991 ◽

Vol 278 (3) ◽

pp. 883-886 ◽

Cited By ~ 46

Author(s):

K Takazawa ◽

J Perret ◽

J E Dumont ◽

C Erneux

Keyword(s):

Amino Acid ◽

Kinase Activity ◽

Cloning And Expression ◽

Fusion Product ◽

Molecular Evidence ◽

Reading Frame ◽

Acid Protein ◽

Sds Page ◽

Inositol 1,4,5 Trisphosphate ◽

Kinase A

A human hippocampus cDNA library in lambda ZAP II was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Two clones (hh6 and hh3) were isolated and sequenced. The insert of clone hh6 was shown to correspond to the 3′ end of the coding sequence of 50,000-Mr InsP3 3-kinase (referred to as 3-kinase-A). Sequencing of the clone hh3 insert yielded an open reading frame encoding a 472-amino acid protein with a calculated Mr of 53,451 (referred to as 3-kinase-B). The C-terminal part of 3-kinase-B (residues 187-462) was 68% identical with 3-kinase-A in amino acid sequence. The cDNA of clone hh3 was rescued as a Bluescript plasmid and expressed in Escherichia coli as a beta-galactosidase fusion product. It showed InsP3 3-kinase activity that was stimulated in the presence of Ca2+/calmodulin (more than 7-fold in a crude bacterial lysate from expressed plasmid). Regeneration of InsP3 3-kinase activity after SDS/PAGE identified a major polypeptide (Mr 60,000-65,000). The Km for InsP3 of expressed 3-kinase-B was 1.6 microM. These data provide molecular evidence for the existence of InsP3 3-kinase isoenzymes.

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