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

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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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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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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Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains ofPorphyromonas gingivalis

Infection and Immunity ◽

10.1128/iai.67.11.5621-5625.1999 ◽

1999 ◽

Vol 67 (11) ◽

pp. 5621-5625 ◽

Cited By ~ 27

Author(s):

Koichi Sawada ◽

Susumu Kokeguchi ◽

Hiroshi Hongyo ◽

Satoko Sawada ◽

Manabu Miyamoto ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Subtractive Hybridization ◽

Open Reading Frame ◽

Specific Marker ◽

Avirulent Strain ◽

Target Sequence ◽

Dna Fragments ◽

Reading Frame

ABSTRACT Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected inP. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalisstrains.

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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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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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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 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4169-4177 ◽

Cited By ~ 126

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

Applied and Environmental Microbiology ◽

10.1128/aem.64.3.836-842.1998 ◽

1998 ◽

Vol 64 (3) ◽

pp. 836-842 ◽

Cited By ~ 45

Author(s):

Seiji Nishikawa ◽

Tomonori Sonoki ◽

Tatsuhide Kasahara ◽

Takahiro Obi ◽

Shoko Kubota ◽

...

Keyword(s):

Open Reading Frame ◽

Cosmid Library ◽

Sequencing Analysis ◽

Cloning And Sequencing ◽

Significant Similarity ◽

Reading Frame ◽

Formyltetrahydrofolate Synthetase ◽

Dna Fragment ◽

Pseudomonas Paucimobilis ◽

Dimeric Model

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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SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.202 ◽

1991 ◽

Vol 11 (1) ◽

pp. 202-212 ◽

Cited By ~ 54

Author(s):

F Damak ◽

E Boy-Marcotte ◽

D Le-Roscouet ◽

R Guilbaud ◽

M Jacquet

Keyword(s):

Saccharomyces Cerevisiae ◽

Open Reading Frame ◽

Gene Products ◽

Terminal Part ◽

Yeast Saccharomyces Cerevisiae ◽

Reading Frame ◽

Dna Fragment ◽

Degree Of Similarity ◽

Entire Gene

In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.

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