Knockdown of β-N-acetylglucosaminidase 2 Impairs Molting and Wing Development in Lasioderma serricorne (Fabricius)

β-N-acetylglucosaminidases (NAGs) are carbohydrate enzymes that degrade chitin oligosaccharides into N-acetylglucosamine monomers. This process is important for chitin degradation during insect development and metamorphosis. We identified and evaluated a β-N-acetylglucosaminidase 2 gene (LsNAG2) from the cigarette beetle, Lasioderma serricorne (Fabricius). The full-length open reading frame of LsNAG2 was 1776 bp and encoded a 591 amino acid protein. The glycoside hydrolase family 20 (GH20) catalytic domain and an additional GH20b domain of the LsNAG2 protein were highly conserved. Phylogenetic analysis revealed that LsNAG2 clustered with the group II NAGs. Quantitative real-time PCR analyses showed that LsNAG2 was expressed in all developmental stages and was most highly expressed in the late larval and late pupal stages. In the larval stage, LsNAG2 was predominantly expressed in the integument. Knockdown of LsNAG2 in fifth instar larvae disrupted larval–pupal molting and reduced the expression of four chitin synthesis genes (trehalase 1 (LsTRE1), UDP-N-acetylglucosamine pyrophosphorylase 1 and 2 (LsUAP1 and LsUAP2), and chitin synthase 1 (LsCHS1)). In late pupae, LsNAG2 depletion resulted in abnormal adult eclosion and wing deformities. The expression of five wing development-related genes (teashirt (LsTSH), vestigial (LsVG), wingless (LsWG), ventral veins lacking (LsVVL), and distal-less (LsDLL)) significantly declined in the LsNAG2-depleted beetles. These findings suggest that LsNAG2 is important for successful molting and wing development of L. serricorne.

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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 Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

Journal of Bacteriology ◽

10.1128/jb.184.20.5753-5761.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5753-5761 ◽

Cited By ~ 63

Author(s):

Sophie Bozonnet ◽

Marguerite Dols-Laffargue ◽

Emeline Fabre ◽

Sandra Pizzut ◽

Magali Remaud-Simeon ◽

...

Keyword(s):

Leuconostoc Mesenteroides ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Carboxy Terminus ◽

Reading Frame ◽

Average Mass ◽

Catalytic Domains ◽

Acid Protein ◽

Amino Acid Protein

ABSTRACT A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of α-1,6 and α-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and α-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.

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Cloning, Characterization, and RNA Interference Effect of the UDP-N-Acetylglucosamine Pyrophosphorylase Gene in Cnaphalocrocis medinalis

Genes ◽

10.3390/genes12040464 ◽

2021 ◽

Vol 12 (4) ◽

pp. 464

Author(s):

Yuan-Jin Zhou ◽

Juan Du ◽

Shang-Wei Li ◽

Muhammad Shakeel ◽

Jia-Jing Li ◽

...

Keyword(s):

Developmental Disorders ◽

Developmental Stages ◽

Phylogenetic Analyses ◽

Digital Pcr ◽

Cnaphalocrocis Medinalis ◽

Chitin Synthesis ◽

Reading Frame ◽

Rnai Technology ◽

Glyphodes Pyloalis ◽

Key Enzyme

The rice leaf folder, Cnaphalocrocis medinalis is a major pest of rice and is difficult to control. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin synthesis pathway in insects. In this study, the UAP gene from C. medinalis (CmUAP) was cloned and characterized. The cDNA of CmUAP is 1788 bp in length, containing an open reading frame of 1464 nucleotides that encodes 487 amino acids. Homology and phylogenetic analyses of the predicted protein indicated that CmUAP shared 91.79%, 87.89%, and 82.75% identities with UAPs of Glyphodes pyloalis, Ostrinia furnacalis, and Heortia vitessoides, respectively. Expression pattern analyses by droplet digital PCR demonstrated that CmUAP was expressed at all developmental stages and in 12 tissues of C. medinalis adults. Silencing of CmUAP by injection of double-stranded RNA specific to CmUAP caused death, slow growth, reduced feeding and excretion, and weight loss in C. medinalis larvae; meanwhile, severe developmental disorders were observed. The findings suggest that CmUAP is essential for the growth and development of C. medinalis, and that targeting the CmUAP gene through RNAi technology can be used for biological control of this insect.

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

The Journal of Cell Biology ◽

10.1083/jcb.119.4.737 ◽

1992 ◽

Vol 119 (4) ◽

pp. 737-747 ◽

Cited By ~ 57

Author(s):

I D Russell ◽

D Tollervey

Keyword(s):

Northern Analysis ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Rrna Processing ◽

Reading Frame ◽

Repeat Units ◽

Nucleolar Proteins ◽

Acid Protein ◽

Sds Page ◽

Amino Acid Protein

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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