Filtering "genic" open reading frames from genomic DNA samples for advanced annotation

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescensQ2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipientPseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlEand phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, andphlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.

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Divergent overlapping transcripts at the PET122 locus in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3027-3035.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3027-3035

Author(s):

J D Ohmen ◽

K A Burke ◽

J E McEwen

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Sequence ◽

Cytochrome C Oxidase ◽

Genomic Dna ◽

Transcription Unit ◽

Open Reading Frames ◽

Base Pairs ◽

Opposite Strand ◽

Overlapping Transcripts ◽

Reading Frames

PET122 is one of three nuclear genes specifically required for translation of the mitochondrial mRNA for cytochrome c oxidase subunit III in Saccharomyces cerevisiae. The nucleotide sequence of 2,862 base pairs (bp) of yeast genomic DNA encompassing the PET122 locus shows very close spacing between the PET122 gene (254 codons) and two unidentified open reading frames, termed ORF2 and ORF3. ORF2 is encoded by the same strand of DNA as PET122 and is located 53 bp downstream of PET122, while ORF3 is encoded on the opposite strand and is located 215 bp upstream of PET122. Five transcripts, with sizes of 2.9, 2.3, 2.1, 1.5, and 1.4 kilobases (kb), are produced from this locus. The 2.1- and 1.4-kb transcripts encode ORF3, the 1.5-kb transcript encodes ORF2, and the 2.9- and 2.3-kb transcripts encode PET122. A particularly interesting feature of the ORF3-PET122-ORF2 transcription unit is a 535-base overlap between the 2.3-kb PET122 transcript produced from one strand and a 2.1-kb ORF3 transcript produced from the opposite strand. Similarly, the 2.9-kb PET122 transcript overlaps the 2.1-kb ORF3 transcript by more than 900 bases and the 1.5-kb ORF3 transcript by at least 200 bases. Hence, these pairs of transcripts are antisense to one another and have the potential to regulate, in an interdependent fashion, the posttranscriptional expression of ORF3 and PET122.

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Identification and Characterization of Phytoplasmal Genes, Employing a Novel Method of Isolating Phytoplasmal Genomic DNA

Journal of Bacteriology ◽

10.1128/jb.185.22.6513-6521.2003 ◽

2003 ◽

Vol 185 (22) ◽

pp. 6513-6521 ◽

Cited By ~ 8

Author(s):

Sharon Melamed ◽

Edna Tanne ◽

Raz Ben-Haim ◽

Orit Edelbaum ◽

David Yogev ◽

...

Keyword(s):

Genomic Dna ◽

Plant Pathogens ◽

Open Reading Frames ◽

Rrna Genes ◽

Unique Method ◽

Novel Method ◽

Identification And Characterization ◽

Reading Frames ◽

Large Background

ABSTRACT Phytoplasmas are unculturable, insect-transmissible plant pathogens belonging to the class Mollicutes. To be transmitted, the phytoplasmas replicate in the insect body and are delivered to the insect's salivary glands, from where they are injected into the recipient plant. Because phytoplasmas cannot be cultured, any attempt to recover phytoplasmal DNA from infected plants or insects has resulted in preparations with a large background of host DNA. Thus, studies of the phytoplasmal genome have been greatly hampered, and aside from the rRNA genes, only a few genes have hitherto been isolated and characterized. We developed a unique method to obtain host-free phytoplasmal genomic DNA from the insect vector's saliva, and we demonstrated the feasibility of this method by isolating and characterizing 78 new putative phytoplasmal open reading frames and their deduced proteins. Based on the newly accumulated information on phytoplasmal genes, preliminary characteristics of the phytoplasmal genome are discussed.

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Divergent overlapping transcripts at the PET122 locus in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3027 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3027-3035 ◽

Cited By ~ 8

Author(s):

J D Ohmen ◽

K A Burke ◽

J E McEwen

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Sequence ◽

Cytochrome C Oxidase ◽

Genomic Dna ◽

Transcription Unit ◽

Open Reading Frames ◽

Base Pairs ◽

Opposite Strand ◽

Overlapping Transcripts ◽

Reading Frames

PET122 is one of three nuclear genes specifically required for translation of the mitochondrial mRNA for cytochrome c oxidase subunit III in Saccharomyces cerevisiae. The nucleotide sequence of 2,862 base pairs (bp) of yeast genomic DNA encompassing the PET122 locus shows very close spacing between the PET122 gene (254 codons) and two unidentified open reading frames, termed ORF2 and ORF3. ORF2 is encoded by the same strand of DNA as PET122 and is located 53 bp downstream of PET122, while ORF3 is encoded on the opposite strand and is located 215 bp upstream of PET122. Five transcripts, with sizes of 2.9, 2.3, 2.1, 1.5, and 1.4 kilobases (kb), are produced from this locus. The 2.1- and 1.4-kb transcripts encode ORF3, the 1.5-kb transcript encodes ORF2, and the 2.9- and 2.3-kb transcripts encode PET122. A particularly interesting feature of the ORF3-PET122-ORF2 transcription unit is a 535-base overlap between the 2.3-kb PET122 transcript produced from one strand and a 2.1-kb ORF3 transcript produced from the opposite strand. Similarly, the 2.9-kb PET122 transcript overlaps the 2.1-kb ORF3 transcript by more than 900 bases and the 1.5-kb ORF3 transcript by at least 200 bases. Hence, these pairs of transcripts are antisense to one another and have the potential to regulate, in an interdependent fashion, the posttranscriptional expression of ORF3 and PET122.

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Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.1.230-240.2005 ◽

2005 ◽

Vol 49 (1) ◽

pp. 230-240 ◽

Cited By ~ 65

Author(s):

Joshua A. V. Blodgett ◽

Jun Kai Zhang ◽

William W. Metcalf

Keyword(s):

Sequence Analysis ◽

Heterologous Expression ◽

Gene Cluster ◽

Genomic Dna ◽

Biosynthetic Gene Cluster ◽

Fosmid Library ◽

Open Reading Frames ◽

Biosynthetic Gene ◽

Heterologous Host ◽

Reading Frames

ABSTRACT A fosmid library from genomic DNA of Streptomyces viridochromogenes DSM 40736 was constructed and screened for the presence of genes known to be involved in the biosynthesis of phosphinothricin tripeptide (PTT). Eight positives were identified, one of which was able to confer PTT biosynthetic capability upon Streptomyces lividans after integration of the fosmid into the chromosome of this heterologous host. Sequence analysis of the 40,241-bp fosmid insert revealed 29 complete open reading frames (ORFs). Deletion analysis demonstrated that a minimum set of 24 ORFs were required for PTT production in the heterologous host. Sequence analysis revealed that most of these PTT genes have been previously identified in either S. viridochromogenes or S. hygroscopicus (or both), although only 11 out of 24 of these ORFs have experimentally defined functions. Three previously unknown genes within the cluster were identified and are likely to have roles in the stepwise production of phosphonoformate from phosphonoacetaldehyde. This is the first report detailing the entire PTT gene cluster from any producing streptomycete.

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Identification of Campylobacter jejuni ATCC 43431-Specific Genes by Whole Microbial Genome Comparisons

Journal of Bacteriology ◽

10.1128/jb.186.14.4781-4795.2004 ◽

2004 ◽

Vol 186 (14) ◽

pp. 4781-4795 ◽

Cited By ~ 47

Author(s):

Frédéric Poly ◽

Deborah Threadgill ◽

Alain Stintzi

Keyword(s):

Dna Sequences ◽

Genomic Dna ◽

Genomic Library ◽

Open Reading Frames ◽

Dna Fragments ◽

Helicobacter Hepaticus ◽

Type Iv ◽

Novel Approach ◽

The Mean ◽

Reading Frames

ABSTRACT This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.

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The dar Genes of Pseudomonas chlororaphis PCL1606 Are Crucial for Biocontrol Activity via Production of the Antifungal Compound 2-Hexyl, 5-Propyl Resorcinol

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-13-0012-r ◽

2013 ◽

Vol 26 (5) ◽

pp. 554-565 ◽

Cited By ~ 28

Author(s):

Claudia E. Calderón ◽

Alejandro Pérez-García ◽

Antonio de Vicente ◽

Francisco M. Cazorla

Keyword(s):

Genomic Dna ◽

Genetic Basis ◽

Genomic Library ◽

Open Reading Frames ◽

Antifungal Compound ◽

Pseudomonas Chlororaphis ◽

Phylogenetic Studies ◽

Biocontrol Activity ◽

Reading Frames

To determine the genetic basis by which 2-hexyl, 5-propyl resorcinol (HPR) is produced by the biocontrol rhizobacterium Pseudomonas chlororaphis (formerly known as P. fluorescens) PCL1606, the presence and role of dar genes were investigated. To accomplish this aim, the pCGNOV-1 plasmid was isolated from a PCL1606 genomic library and was shown to hybridize to various dar probes by Southern blot. An analysis of the pCGNOV-1 genomic DNA revealed the presence of five open reading frames that were homologous to dar genes and had an organization that resembled the arrangement of previously described P. chlororaphis strains. Phylogenetic studies resulted in the clustering of PCL1606 with the P. chlororaphis subgroup, which supported the renaming of this strain from P. fluorescens to P. chlororaphis PCL1606. The construction of insertional mutants for each homologous dar gene in P. chlororaphis PCL1606 along with their corresponding complemented derivative strains restored HPR production and confirmed the key role of the dar A and darB genes in HPR production and in the antagonistic phenotype. Finally, biocontrol assays were performed on avocado–Rosellinia and tomato–Fusarium test systems using the HPR-defective and -complemented derivative strains generated here and demonstrated the crucial role of the biosynthetic dar genes in the biocontrol phenotype of P. chlororaphis PCL1606. This biocontrol phenotype is dependent on the dar genes via their production of the HPR antibiotic. Some of the dar genes not directly involved in the biosynthesis of HPR, such as darS or darR, might contribute to regulatory features of HPR production.

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Complete Genome Sequence of Rathayibacter toxicus Phage NCPPB3778

Genome Announcements ◽

10.1128/genomea.00671-17 ◽

2017 ◽

Vol 5 (42) ◽

Cited By ~ 1

Author(s):

William L. Schneider ◽

Aaron J. Sechler ◽

Elizabeth E. Rogers

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Genomic Dna ◽

Open Reading Frames ◽

Content Type ◽

Phage Proteins ◽

Reading Frames

ABSTRACT The phage NCPPB3778 was isolated from Rathayibacter toxicus strain CS14, and the genomic DNA was sequenced. The genome is similar to siphoviruses, consisting of 44,520 bases including 77 predicted open reading frames. Portions of the genome are annotated as typical phage proteins, but much of the genome sequence is unique from other bacteriophages.

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The R-Type Pyocin of Pseudomonas aeruginosa C Is a Bacteriophage Tail-Like Particle That Contains Single-Stranded DNA

Infection and Immunity ◽

10.1128/iai.67.2.717-725.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 717-725 ◽

Cited By ~ 17

Author(s):

Frank K. N. Lee ◽

Kathleen C. Dudas ◽

Julie A. Hanson ◽

M. Bud Nelson ◽

Philip T. LoVerde ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Genomic Dna ◽

Open Reading Frames ◽

Structural Proteins ◽

Chromosomal Dna ◽

Unusual Structure ◽

Single Stranded Dna ◽

Complementary Oligonucleotide ◽

Oligonucleotide Hybridization ◽

Reading Frames

ABSTRACT Pseudomonas aeruginosa R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from P. aeruginosa C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digestedP. aeruginosa C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18 P. aeruginosa strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of P. aeruginosa C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonalprt genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from P. aeruginosa C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of P. aeruginosa C.

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Characterization of IS1272, an insertion sequence-like element from Staphylococcus haemolyticus.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.4.924 ◽

1996 ◽

Vol 40 (4) ◽

pp. 924-929 ◽

Cited By ~ 49

Author(s):

G L Archer ◽

J A Thanassi ◽

D M Niemeyer ◽

M J Pucci

Keyword(s):

Genomic Dna ◽

Insertion Sequence ◽

Methicillin Resistance ◽

Open Reading Frames ◽

Sequence Element ◽

Amino Acid Homology ◽

Methicillin Resistant ◽

Staphylococcus Haemolyticus ◽

Reading Frames

We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.

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