Cloning and Sequencing of theSphingomonas (Pseudomonas)paucimobilis Gene Essential for the O Demethylation of Vanillate and Syringate

ABSTRACT Sphingomonas (Pseudomonas)paucimobilis SYK-6 is able to grow on 5,5′-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2′,3′-trihydroxy-3-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase ofClostridium thermoaceticum.

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Coexistence of Two Different O Demethylation Systems in Lignin Metabolism by Sphingomonas paucimobilis SYK-6: Cloning and Sequencing of the Lignin Biphenyl-Specific O-Demethylase (LigX) Gene

Applied and Environmental Microbiology ◽

10.1128/aem.66.5.2125-2132.2000 ◽

2000 ◽

Vol 66 (5) ◽

pp. 2125-2132 ◽

Cited By ~ 33

Author(s):

Tomonori Sonoki ◽

Takahiro Obi ◽

Sachiko Kubota ◽

Motoo Higashi ◽

Eiji Masai ◽

...

Keyword(s):

Polyclonal Antiserum ◽

Sphingomonas Paucimobilis ◽

Sequencing Analysis ◽

Cloning And Sequencing ◽

Reading Frame ◽

Cell Extracts ◽

Essential Enzyme ◽

Lignin Metabolism ◽

Dimeric Model ◽

Demethylation Activity

ABSTRACT Sphingomonas paucimobilis SYK-6 can grow on several dimeric model compounds of lignin as a carbon and energy source. It has O demethylation systems on three kinds of substrates: 5,5′-dehydrodivanillic acid (DDVA), syringate, and vanillate. We previously reported the cloning of a gene involved in the tetrahydrofolate-dependent O demethylation of syringate and vanillate. In the study reported here, we cloned the gene responsible for DDVA O demethylation. Using nitrosoguanidine mutagenesis, a mutant strain, NT-1, which could not degrade DDVA but could degrade syringate and vanillate, was isolated and was used to clone the gene responsible for the O demethylation of DDVA by complementation. Sequencing analysis showed an open reading frame (designatedligX) of 1,266 bp in this fragment. The deduced amino acid sequence of LigX had similarity to class I type oxygenases. LigX was involved in O demethylation activity on DDVA but not on vanillate and syringate. DDVA O demethylation activity in S. paucimobilisSYK-6 cell extracts was inhibited by addition of the LigX polyclonal antiserum. Thus, LigX is an essential enzyme for DDVA O demethylation in SYK-6. S. paucimobilis SYK-6 has two O demethylation systems: one is an oxygenative demethylase system, and the other is a tetrahydrofolate-dependent methyltransferase system.

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Phylogeny and Characterization of Three nifH-Homologous Genes from Paenibacillus azotofixans

Applied and Environmental Microbiology ◽

10.1128/aem.69.6.3658-3662.2003 ◽

2003 ◽

Vol 69 (6) ◽

pp. 3658-3662 ◽

Cited By ~ 24

Author(s):

Quok-Cheong Choo ◽

Mohd-Razip Samian ◽

Nazalan Najimudin

Keyword(s):

Phylogenetic Analysis ◽

Methanogenic Archaea ◽

Gene Organization ◽

Open Reading Frame ◽

The Other ◽

Sequencing Analysis ◽

Reading Frame ◽

Homologous Genes ◽

Dna Fragment

ABSTRACT In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.

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The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽

10.1093/genetics/155.3.1105 ◽

2000 ◽

Vol 155 (3) ◽

pp. 1105-1117 ◽

Cited By ~ 4

Author(s):

W John Haynes ◽

Kit-Yin Ling ◽

Robin R Preston ◽

Yoshiro Saimi ◽

Ching Kung

Keyword(s):

Genomic Library ◽

Open Reading Frame ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Rt Pcr ◽

Reading Frame ◽

Close Proximity ◽

In The Wild ◽

Dna Fragment ◽

Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

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Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

Applied and Environmental Microbiology ◽

10.1128/aem.68.12.6237-6245.2002 ◽

2002 ◽

Vol 68 (12) ◽

pp. 6237-6245 ◽

Cited By ~ 36

Author(s):

Tara D. Sutherland ◽

Irene Horne ◽

Robyn J. Russell ◽

John G. Oakeshott

Keyword(s):

Shuttle Vector ◽

Open Reading Frame ◽

Cosmid Library ◽

Flavin Mononucleotide ◽

Flavin Reductase ◽

Reading Frame ◽

Reductase Gene ◽

Degradation Activity ◽

Layer Chromatography

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

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One of the tightly clustered genes of the mouse surfeit locus is a highly expressed member of a multigene family whose other members are predominantly processed pseudogenes.

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3898 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3898-3905 ◽

Cited By ~ 21

Author(s):

C Huxley ◽

T Williams ◽

M Fried

Keyword(s):

Multigene Family ◽

Open Reading Frame ◽

The Other ◽

Base Pairs ◽

Regulation Of Expression ◽

Reading Frame ◽

Processed Pseudogenes ◽

Dna Fragment ◽

Basic Polypeptide

The mouse surfeit locus is unusual in that it contains a number of closely clustered genes (Surf-1, -2, and -4) that alternate in their direction of transcription (T. Williams, J. Yon, C. Huxley, and M. Fried, Proc. Natl. Acad. Sci. USA 85:3527-3530, 1988). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by 15 to 73 base pairs (bp), and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp (T. Williams and M. Fried, Mol. Cell. Biol. 6:4558-4569, 1986; T. Williams and M. Fried, Nature (London) 322:275-279, 1986). A fourth gene in this locus, Surf-3, which is a member of a multigene family, has been identified. The poly(A) addition site of Surf-3 lies only 70 bp from the poly(A) addition site of Surf-1. Transcription of Surf-3 has been studied in the absence of the other members of its multigene family after transfection of a cloned genomic mouse DNA fragment, containing the Surf-3 gene, into heterologous monkey cells. Surf-3 specifies a highly expressed 1.0-kilobase mRNA that contains a long open reading frame of 266 amino acids, which would encode a highly basic polypeptide (23% Arg plus Lys). The other members of the Surf-3 multigene family are predominantly, if not entirely, intronless pseudogenes with the hallmarks of being generated by reverse transcription. The role of the very tight clustering on regulation of expression of the genes in the surfeit locus is discussed.

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Quorum-Sensing Signals and Quorum-Sensing Genes in Burkholderia vietnamiensis

Journal of Bacteriology ◽

10.1128/jb.184.4.1187-1191.2002 ◽

2002 ◽

Vol 184 (4) ◽

pp. 1187-1191 ◽

Cited By ~ 37

Author(s):

Barbara-Ann Conway ◽

E. P. Greenberg

Keyword(s):

Quorum Sensing ◽

Burkholderia Cepacia ◽

Homoserine Lactone ◽

Abundant Species ◽

Open Reading Frame ◽

Reading Frame ◽

Burkholderia Vietnamiensis ◽

Dna Fragment ◽

Low Levels ◽

Quorum Sensing Signals

ABSTRACT Acyl-homoserine lactone (acyl-HSL) quorum sensing is common to many Proteobacteria including a clinical isolate of Burkholderia cepacia. The B. cepacia isolate produces low levels of octanoyl-HSL. We have examined an environmental isolate of Burkholderia vietnamiensis. This isolate produced several acyl-HSLs. The most abundant species was decanoyl-HSL. Decanoyl-HSL in B. vietnamiensis cultures reached concentrations in excess of 20 μM. We isolated a B. vietnamiensis DNA fragment containing a gene for the synthesis of decanoyl-HSL (bviI) and an open reading frame that codes for a putative signal receptor (bviR). A B. vietnamiensis bviI mutant did not produce detectable levels of decanoyl-HSL.

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Identifikasi dan isolasi promoter gen pembungaan kakao TcLFY Identification and isolation for promoter of TcLFY cacao flowering gene

E-Journal Menara Perkebunan ◽

10.22302/ppbbi.jur.mp.v75i1.150 ◽

2016 ◽

Vol 75 (1) ◽

Author(s):

Djoko SANTOS ◽

Agustina A. HANDAYAN ◽

Sukarti MOELJOPAWIRO

Keyword(s):

Input Data ◽

Open Reading Frame ◽

Genome Walking ◽

Dna Fragments ◽

Reading Frame ◽

Dna Fragment

SummaryPromoter is a regulator of geneexpression for a phenotype or trait carried bythe gene. In the structure, a promoter locatedbeyond the 5’ end of the open reading frame ofthe gene on which its expression is regulated.This research aimed to isolate the DNAfragment flanking TcLFY at the 5’ end and toanalyze whether the fragment has charac-teristics of the promoter, primarily the coremotifs of promoter. Using Genome Walkingtechnique, DNA fragments flanking the TcLFYgene at its 5’ end was isolated. Analysis of theDNA sequence was done using onlinecomputer software accessible through web sitewww.softberry.com and an entry sequence ofthe flanking DNA fragment along with the 2.5kb TcLFY sequence. The result indicated thatthe flanking fragment has core motifs for apromoter at proper positions, which are TATAbox at position –80, CAT boxes (CCAAT) at -387 and –626, and GC boxes that are known asUAS were found at the -323 and –537positions. To obtain a conclusive result, thispromoter sequence needs to be furtherexamined to confirm its function.RingkasanPromoter merupakan pengendali ekspresigen untuk memunculkan fenotipe atau karakteryang dibawa oleh gen tersebut. Di dalamstrukturnya, promoter umumnya terletak didaerah ujung 5’ gen yang dikendalikanekspresinya. Tujuan penelitian ini adalahmendapatkan fragmen DNA yang mengapitgen pengendali pembungaan kakao (TcLFY)dan menganalisisnya apakah memilikikarakteristik promoter, yaitu mengandungmotif-motif inti (core motifs) dari promoter.Dengan teknik Genome Walking, fragmenDNA pengapit gen TcLFY di ujung 5’ dapatdiisolasi. Analisis sekuen menggunakanperangkat lunak komputer online (www.softberry.com) dengan input data fragmentersebut ditambah gen TcLFY 2,5 kb dibawahnya, mengindikasikan adanya beberapamotif inti promoter pada posisi yang sesuai,yaitu kotak TATA pada lokasi –80, kotak CAT(CCAAT) di posisi -387 dan –626, dan kotakGC yang merupakan UAS dijumpai padalokasi -323 dan –537. Untuk memperoleh hasilyang bersifat konklusif, sekuen promoter inimasih perlu diuji fungsinya.

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Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4169-4177.1987 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4169-4177

Author(s):

C Dietzel ◽

J Kurjan

Keyword(s):

Copy Number ◽

Open Reading Frame ◽

Mating Types ◽

Pheromone Response ◽

Functional Region ◽

Reading Frame ◽

Pheromone Response Pathway ◽

Low Copy Number ◽

Dna Fragment ◽

Beta Galactosidase

Strains of both haploid mating types containing sst2 mutations are altered in response to pheromone; MATa sst2 cells are supersensitive to alpha-factor, and MAT alpha sst2 cells are supersensitive to a-factor. This phenotype suggests that SST2 encodes a component of the pheromone response pathway that is common to both mating types. We have cloned the SST2 gene by isolation of multicopy plasmids that complement the sst2-1 mutation. One such plasmid contained a 4.5-kilobase HindIII fragment that was able to complement the sst2-1 mutation in high or low copy number, integrated at the SST2 locus, and resulted in an sst2 phenotype when disrupted, indicating that this fragment contained the SST2 gene. We identified the functional region of the complementing DNA fragment by transposon mutagenesis. Sequencing of this fragment identified an open reading frame encoding 698 amino acids at a position that correlated well with the functional region. Expression of an Sst2-beta-galactosidase fusion was haploid specific and induced by exposure to pheromone. We discuss a model in which induction of the SST2 product results in inhibition of a component of the pheromone response pathway, resulting in desensitization to pheromone.

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Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various Streptomyces avermitilis Strains

Applied and Environmental Microbiology ◽

10.1128/aem.69.2.1263-1269.2003 ◽

2003 ◽

Vol 69 (2) ◽

pp. 1263-1269 ◽

Cited By ~ 18

Author(s):

Yong-Soon Hwang ◽

Eung-Soo Kim ◽

Sándor Biró ◽

Cha-Yong Choi

Keyword(s):

Genomic Library ◽

Streptomyces Coelicolor ◽

Streptomyces Avermitilis ◽

Wild Type ◽

Significant Similarity ◽

Reading Frame ◽

Strong Homology ◽

Dna Fragment ◽

Minimal Region ◽

Stimulation Of

ABSTRACT To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis.

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