Identification and nucleotide sequence analysis of an open reading frame involved in high-frequency conversion of turbid to clear plaque mutants of filamentous phage Cf1t

The gene catIII, encoding a type III enterobacterial chloramphenicol acetyltransferase, was cloned from the transmissible plasmid R387 into pBR322 and bacteriophage M13 mp8. Nucleotide sequence analysis of 1160 bp of DNA identified an open reading frame encoding a protein of 213 amino acid residues and a calculated molecular mass of 24965 Da. The predicted N-terminal sequence is identical with that determined by Edman degradation of chloramphenicol acetyltransferase purified from Escherichia coli harbouring R387. Sequences equivalent to the consensus motifs for initiation and rho-factor-independent termination of transcription in E. coli occur 5′ and 3′ to the catIII open reading frame. In contrast with the catI gene, present on transposon Tn9 and many enterobacterial plasmids, expression of catIII is not subject to cyclic AMP-mediated catabolite repression in vivo and there is no sequence in the 5′ non-coding DNA that resembles that deduced as the consensus for the binding of cyclic AMP receptor protein. Unique restriction-endonuclease cleavage sites were introduced adjacent to the catIII reading frame by using oligonucleotide-directed mutagenesis to facilitate insertion into E. coli expression vectors. Fully active chloramphenicol acetyltransferase represents 30-50% of the soluble protein component of cell-free extracts of E. coli containing the appropriate plasmids.

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Development of a Self-Cloning System for Actinomadura verrucosospora and Identification of Polyketide Synthase Genes Essential for Production of the Angucyclic Antibiotic Pradimicin

Applied and Environmental Microbiology ◽

10.1128/aem.65.6.2703-2709.1999 ◽

1999 ◽

Vol 65 (6) ◽

pp. 2703-2709 ◽

Cited By ~ 10

Author(s):

Tohru Dairi ◽

Yoshimitsu Hamano ◽

Tamotsu Furumai ◽

Toshikazu Oki

Keyword(s):

Nucleotide Sequence ◽

Polyketide Synthase ◽

Selectable Marker ◽

Plasmid Vector ◽

Open Reading Frame ◽

Antibiotic Biosynthesis ◽

Nucleotide Sequence Analysis ◽

Reading Frame ◽

Cloning System ◽

Open Reading Frame 1

ABSTRACT A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadurastrain. The system has an efficiency of more than 105CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.

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Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1570-1575.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1570-1575 ◽

Cited By ~ 4

Author(s):

Dae Heoun Baek ◽

Jae Jun Song ◽

Seok-Joon Kwon ◽

Chung Park ◽

Chang-Min Jung ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Open Reading Frame ◽

Nucleotide Sequence Analysis ◽

Reading Frame ◽

E Coli ◽

Specificity Constant ◽

Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 Ã¯Â¿Â½ 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 Ã¯Â¿Â½ 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55Ã¯Â¿Â½C. The enzyme was stable up to 55Ã¯Â¿Â½C, and the optimal pH and temperature were 8.5 and 65Ã¯Â¿Â½C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

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The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2221 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2221-2230 ◽

Cited By ~ 69

Author(s):

W D Burke ◽

C C Calalang ◽

T H Eickbush

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Transposable Elements ◽

Reverse Transcriptase ◽

Bombyx Mori ◽

Open Reading Frame ◽

Nucleotide Sequence Analysis ◽

Type Ii ◽

Site Specific ◽

Reading Frame

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.

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The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2221-2230.1987 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2221-2230

Author(s):

W D Burke ◽

C C Calalang ◽

T H Eickbush

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Transposable Elements ◽

Reverse Transcriptase ◽

Bombyx Mori ◽

Open Reading Frame ◽

Nucleotide Sequence Analysis ◽

Type Ii ◽

Site Specific ◽

Reading Frame

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.

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CHL12, a gene essential for the fidelity of chromosome transmission in the yeast Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/138.4.1067 ◽

1994 ◽

Vol 138 (4) ◽

pp. 1067-1079 ◽

Cited By ~ 5

Author(s):

N Kouprina ◽

E Kroll ◽

A Kirillov ◽

V Bannikov ◽

V Zakharyev ◽

...

Keyword(s):

Cell Cycle ◽

Sequence Analysis ◽

Critical Role ◽

Open Reading Frame ◽

Phase Cell ◽

Nucleotide Sequence Analysis ◽

Protein Sequence Analysis ◽

Dna Metabolism ◽

Reading Frame ◽

Factor C

Abstract We have analyzed the CHL12 gene, earlier identified in a screen for yeast mutants with increased rates of mitotic loss of chromosome III and circular centromeric plasmids. A genomic clone of CHL12 was isolated and used to map its physical position on the right arm of chromosome XIII near the ADH3 locus. Nucleotide sequence analysis of CHL12 revealed a 2.2-kb open reading frame with a 84-kD predicted protein sequence. Analysis of the sequence upstream of the CHL12 open reading frame revealed the presence of two imperfect copies of MluI motif, ACGCGT, a sequence associated with many DNA metabolism genes in yeast. Analysis of the amino acid sequence revealed that the protein contains a NTP-binding domain and shares a low degree of homology with subunits of replication factor C (RF-C). A strain containing a null allele of CHL12 was viable under standard growth conditions, and as well as original mutants exhibited an increase in the level of spontaneous mitotic recombination, slow growth and cold-sensitive phenotypes. Most of cells carrying the null chl12 mutation arrested as large budded cells with the nucleus in the neck at nonpermissive temperature that typical for cell division cycle (cdc) mutants that arrest in the cell cycle at a point either immediately preceding M phase or during S phase. Cell cycle arrest of the chl12 mutant requires the RAD9 gene. We conclude that the CHL12 gene product has critical role in DNA metabolism.

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