scholarly journals Functional Genomics Analysis of Singapore Grouper Iridovirus: Complete Sequence Determination and Proteomic Analysis

2004 ◽  
Vol 78 (22) ◽  
pp. 12576-12590 ◽  
Author(s):  
Wen Jun Song ◽  
Qi Wei Qin ◽  
Jin Qiu ◽  
Can Hua Huang ◽  
Fan Wang ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Complete Sequence ◽  
Nucleotide Metabolism ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Computer Assisted ◽  
Rt Pcr ◽  
Genomic Libraries ◽  
Reverse Transcription Pcr ◽  
Flight Mass Spectrometry

ABSTRACT Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products.

scholarly journals Characterization of Novel Carbazole Catabolism Genes from Gram-Positive Carbazole Degrader Nocardioides aromaticivorans IC177

2006 ◽  
Vol 72 (5) ◽  
pp. 3321-3329 ◽  
Author(s):  
Kengo Inoue ◽  
Hiroshi Habe ◽  
Hisakazu Yamane ◽  
Hideaki Nojiri
Keyword(s):  
Amino Acid ◽  
Gene Clusters ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Class Iii ◽  
Rt Pcr ◽  
Gram Positive ◽  
Mononuclear Iron ◽  
Reverse Transcription Pcr ◽  
Genes Encoding

ABSTRACT Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.

scholarly journals Cloning of two Bombyx homologues of the Drosophila rosy gene and their relationship to larval translucent skin colour mutants

1998 ◽  
Vol 71 (1) ◽  
pp. 11-19 ◽  
Author(s):  
YUJI YASUKOCHI ◽  
TOSHIO KANDA ◽  
TOSHIKI TAMURA
Keyword(s):  
Tissue Specificity ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Striking Similarity ◽  
Wild Type ◽  
Rt Pcr ◽  
Significant Difference ◽  
Phage Dna ◽  
Polymerase Chain ◽  
Reading Frames

To clone the Bombyx xanthine dehydrogenase (XDH) gene as a dominant marker for silkworm transgenesis, we performed nested reverse transcriptase–polymerase chain reaction (RT-PCR) using embryonic mRNA and primers designed from the conserved region of Drosophila and rat XDH genes. Sequencing of amplified 180 bp fragments showed that two different sequences were present in the fragments. Since both possessed striking similarity to XDH genes of other organisms, we considered these to be portions of silkworm XDH genes and designated them BmXDH1 and BmXDH2. Subsequently we cloned separately the entire region of the two cDNAs by PCR using phage DNA of an embryonic cDNA library and sequenced them. The two cDNAs were around 4 kb in size and possessed complete open reading frames. The deduced amino acid sequences of the two BmXDHs were very similar to each other and to those of other organisms. The expression pattern of wild-type larvae basically followed the tissue specificity of the enzyme and no significant difference was observed between the two XDH genes. The expression of both genes was detected in the XDH-deficient mutants, oq and og, but non-synonymous substitutions were specifically detected in the BmXDH1 of the oq mutant. In addition, a length polymorphism of the second intron of the BmXDH1 co-segregated with the oq translucent phenotype, suggesting that deficiency in BmXDH1 is the cause of the oq translucent phenotype.

scholarly journals Two C—P Lyase Operons in Pseudomonas stutzeri and Their Roles in the Oxidation of Phosphonates, Phosphite, and Hypophosphite

2004 ◽  
Vol 186 (14) ◽  
pp. 4730-4739 ◽  
Author(s):  
Andrea K. White ◽  
William W. Metcalf
Keyword(s):  
Insertion Site ◽  
Pseudomonas Stutzeri ◽  
Gene Organization ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Transposon Insertion ◽  
Intergenic Sequences ◽  
Reading Frames

ABSTRACT DNA sequencing and analysis of two distinct C—P lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes C—P lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional C—P lyase pathway in this organism. To identify the genes comprising this pathway, a Δhtx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both C—P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

scholarly journals Multiple-Subunit Genes of the Aromatic-Ring-Hydroxylating Dioxygenase Play an Active Role in Biphenyl and Polychlorinated Biphenyl Degradation in Rhodococcus sp. Strain RHA1

2006 ◽  
Vol 72 (8) ◽  
pp. 5396-5402 ◽  
Author(s):  
Takumi Iwasaki ◽  
Keisuke Miyauchi ◽  
Eiji Masai ◽  
Masao Fukuda
Keyword(s):  
Aromatic Ring ◽  
Polychlorinated Biphenyl ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Role ◽  
Amino Acid Sequences ◽  
Reverse Transcription Pcr ◽  
Substrate Preferences ◽  
Insertional Inactivation ◽  
Biphenyl Degradation ◽  
Polychlorinated Biphenyl Degradation

ABSTRACT A gram-positive strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, can degrade PCBs by cometabolism with biphenyl or ethylbenzene. In RHA1, three sets of aromatic-ring-hydroxylating dioxygenase genes are induced by biphenyl. The large and small subunits of their terminal dioxygenase components are encoded by bphA1 and bphA2, etbA1 and etbA2, and ebdA1 and ebdA2, respectively, and the deduced amino acid sequences of etbA1 and etbA2 are identical to those of ebdA1 and ebdA2, respectively. In this study, we examined the involvement of the respective subunit genes in biphenyl/PCB degradation by RHA1. Reverse transcription-PCR and two-dimensional polyacrylamide gel electrophoresis analyses indicated the induction of RNA and protein products of etbA1 and ebdA1 by biphenyl. Single- and double-disruption mutants of etbA1, ebdA1, and bphA1 were constructed by insertional inactivation. The 4-chlorobiphenyl (4-CB) degradation activities of all the mutants were lower than that of RHA1. The results indicated that all of these genes are involved in biphenyl/PCB degradation. Furthermore, we constructed disruption mutants of ebdA3 and bphA3, encoding ferredoxin, and etbA4, encoding ferredoxin reductase components. The 4-CB degradation activities of these mutants were also lower than that of RHA1, suggesting that all of these genes play a role in biphenyl/PCB degradation. The substrate preferences of etbA1A2/ebdA1A2- and bphA1A2-encoded dioxygenases for PCB congeners were examined using the corresponding mutants. The results indicated that these dioxygenase isozymes have different substrate preferences and that the etbA1A2/ebdA1A2-encoded isozyme is more active on highly chlorinated congeners than the bphA1A2-encoded one.

Purification and molecular cloning of rat 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase

2002 ◽  
Vol 361 (3) ◽  
pp. 567-575 ◽  
Author(s):  
Atsushi TANABE ◽  
Yukari EGASHIRA ◽  
Shin-Ichi FUKUOKA ◽  
Katsumi SHIBATA ◽  
Hiroo SANADA
Keyword(s):  
Amino Acid ◽  
Hepg2 Cells ◽  
Expressed Sequence Tag ◽  
Amino Acid Sequences ◽  
Rt Pcr ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Reverse Transcription Pcr ◽  
Dna Database ◽  
Liver And Kidney

2-Amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD; EC 4.1.1.45) is one of the important enzymes regulating tryptophan—niacin metabolism. In the present study, we purified the enzyme from rat liver and kidney, and cloned the cDNA encoding rat ACMSD. The molecular masses of rat ACMSDs purified from the liver and kidney were both estimated to be 39kDa by SDS/PAGE. Analysis of N-terminal amino acid sequences showed that these two ACMSDs share the same sequence. An expressed sequence tag (EST) of the mouse cited from the DNA database was found to be identical with this N-terminal sequence. Reverse transcription-PCR (RT-PCR) was performed using synthetic oligonucleotide primers having the partial sequences of the EST, and then cDNAs encoding rat ACMSDs were isolated by using subsequent 3′-rapid amplification of cDNA ends and RT-PCR methods. ACMSD cDNAs isolated from liver and kidney were shown to be identical, consisting of a 1008bp open reading frame (ORF) encoding 336 amino acid residues with a molecular mass of 38091Da. The rat ACMSD ORF was inserted into a mammalian expression vector, before transfection into human hepatoma HepG2 cells. The transfected cells expressed ACMSD activity, whereas the enzyme activity was not detected in uninfected parental HepG2 cells. The distribution of ACMSD mRNA expression in various tissues was investigated in the rat by RT-PCR. ACMSD was expressed in the liver and kidney, but not in the other principal organs examined.

scholarly journals A New Method for Extraction and Analysis of Ricin Samples through MALDI-TOF-MS/MS

Toxins ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 201 ◽  
Author(s):  
Roberto B. Sousa ◽  
Keila S. C. Lima ◽  
Caleb G. M. Santos ◽  
Tanos C. C. França ◽  
Eugenie Nepovimova ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Intact Protein ◽  
Toxic Activity ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Sds Page ◽  
Tof Ms

We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.

scholarly journals Evidence for the Presence of an F-Type ATP Synthase Involved in Sulfate Respiration in Desulfovibrio vulgaris

2000 ◽  
Vol 182 (8) ◽  
pp. 2200-2206 ◽  
Author(s):  
Kiyoshi Ozawa ◽  
Takanori Meikari ◽  
Ken Motohashi ◽  
Masasuke Yoshida ◽  
Hideo Akutsu
Keyword(s):  
Amino Acid ◽  
Atp Synthase ◽  
Cytoplasmic Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Level ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Desulfovibrio Vulgaris ◽  
F Cells

ABSTRACT Using a library of genomic DNA from Desulfovibrio vulgaris Miyazaki F, a strict anaerobe, and two synthetic deoxyoligonucleotide probes designed for F-type ATPases, the genes for open reading frames (ORFs) 1 to 5 were cloned and sequenced. The predicted protein sequences of the gene products indicate that they are composed of 172, 488, 294, 471, and 134 amino acids, respectively, and that they share considerable identity at the amino acid level with δ, α, γ, β, and ɛ subunits found in other F-type ATPases, respectively. Furthermore, a component carrying ATPase activity was partially purified from the cytoplasmic membrane fraction of theD. vulgaris Miyazaki F cells. The N-terminal amino acid sequences of three major polypeptides separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis were identical to those of the products predicted by the sequences of ORF-2, ORF-3, and ORF-4, suggesting that an F-type ATPase is functioning in the D. vulgaris Miyazaki F cytoplasmic membrane. The amount of the F-type ATPase produced in the D. vulgaris Miyazaki F cells is similar to that in the Escherichia coli cells cultured aerobically. It indicates that the enzyme works as an ATP synthase in the D. vulgaris Miyazaki F cells in connection with sulfate respiration.

scholarly journals Molecular Analysis of the Enterococcus faecalis Serotype 2 Polysaccharide Determinant

2003 ◽  
Vol 185 (15) ◽  
pp. 4393-4401 ◽  
Author(s):  
Lynn E. Hancock ◽  
Brett D. Shepard ◽  
Michael S. Gilmore
Keyword(s):  
Enterococcus Faecalis ◽  
Transcription Initiation ◽  
Capsular Polysaccharide ◽  
Open Reading Frames ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Promoter Fusion ◽  
Specific Polysaccharide ◽  
Linkage Of Genes

ABSTRACT We previously described a 15-kb genetic cluster consisting of 11 open reading frames (cps2A to cps2K) of Enterococcus faecalis FA2-2 that is responsible for the production of the serotype 2 capsular polysaccharide. By using transcriptional fusions to a promoterless lacZ gene, we identified two independent promoters related to the expression of the polysaccharide. Both transcription initiation sites were mapped by primer extension. Reverse transcription-PCR (RT-PCR) demonstrated the transcriptional linkage of genes present in both transcripts. Real-time RT-PCR quantification of transcripts revealed maximum transcription during log phase growth, an observation confirmed by promoter fusion studies. The heterologous expression of this pathway in Escherichia coli caused reactivity with E. faecalis type 2 antiserum, thus demonstrating the essential role of this pathway in the synthesis of the type-specific polysaccharide.

scholarly journals Proteins Associated with Culex nigripalpus Nucleopolyhedrovirus Occluded Virions

2007 ◽  
Vol 81 (9) ◽  
pp. 4585-4590 ◽  
Author(s):  
Omaththage Perera ◽  
Terry B. Green ◽  
Stanley M. Stevens ◽  
Susan White ◽  
James J. Becnel
Keyword(s):  
Mass Spectrometry ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Time Of Flight ◽  
Open Reading Frames ◽  
Structural Proteins ◽  
Ionization Time ◽  
Protein Databases ◽  
Flight Mass Spectrometry ◽  
Culex Nigripalpus

ABSTRACT Occlusion-derived virions (ODVs) of the nucleopolyhedrovirus of Culex nigripalpus (CuniNPV) were purified by Ludox density gradient ultracentrifugation, and the proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were identified by using Edman sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry, nanoelectrospray quadrupole time-of-flight mass spectrometry, or a combination of these methods. Half of the 44 polypeptide sequences identified in this analysis were unique open reading frames (ORFs) encoded by the CuniNPV genome and did not show similarity to any other sequences present in protein databases. Of the 22 polypeptides that showed similarities to other baculovirus-encoded proteins, only 17 sequences have previously been identified as structural proteins. The newly identified CuniNPV structural proteins cun058, cun059, cun087, cun106, and cun109 are homologues of Autographa californica nucleopolyhedrovirus (AcMNPV) ORFs 68, 62, 98, 81, and 2, respectively. The products of four genes, namely, lef-1 (cun045), alkaline exonuclease (cun054), helicase (cun089), and DNA polymerase (cun091), were not detected in the CuniNPV ODV preparations. These four genes are conserved among all annotated baculovirus genomes, and their homologues have been detected in the ODV of AcMNPV.

scholarly journals Characterization of a Novel Potyvirus Isolated from Maize in Israel

2000 ◽  
Vol 90 (5) ◽  
pp. 505-513 ◽  
Author(s):  
D. L. Seifers ◽  
R. Salomon ◽  
V. Marie-Jeanne ◽  
B. Alliot ◽  
P. Signoret ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Pennisetum Glaucum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Maize Dwarf Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Flight Mass Spectrometry ◽  
Sds Page

A potyvirus (proposed name of Zea mosaic virus [ZeMV]) isolated from maize in Israel was analyzed by serology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of capsid proteins, symptomatology, and sequencing. Parts of the nuclear inclusion b, coat protein, and 3′ regions were sequenced; the amino acid sequence of ZeMV capsid was determined by time-of-flight mass spectrometry (TOFMS). The results of these analyses were compared with those of similar analyses of the following potyviruses: Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus strain MDB (SCMV-MDB), Johnsongrass mosaic virus(JGMV), Sorghum mosaic virus (SrMV), and an isolate of MDMV from Israel. Indirect enzyme-linked immunosorbent assay using ZeMV antiserum detected only ZeMV, and reciprocal tests using MDMV, JGMV, or SrMV antisera failed to detect ZeMV. ZeMV cross-reacted weakly when SCMV-MDB antiserum was used. The mass of ZeMV capsid was determined to be 36,810 Da by SDS-PAGE and 34,216 Da by TOFMS. The ZeMV systemically infected johnsongrass (Sorghum halepense), but did not infect oat (Avena sativa), pearl millet (Pennisetum glaucum), barley (Hordeum vulgare), or rye (Secale cereale). Necrosis was caused in 19 sorghum lines by SrMV, in 15 by ZeMV, in 14 by MDMV, and in 5 by JGMV and SCMV-MDB. The nucleic acid and amino acid sequences of ZeMV clearly showed that it is not a strain of JGMV, MDMV, SCMV, or SrMV.

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