Cloning of the ATP sulphurylase gene of Schizosaccharomyces pombe by functional complementation

2008 ◽  
Vol 54 (1) ◽  
pp. 71-74 ◽  
Author(s):  
Tibor Simonics ◽  
Anna Maráz
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Genomic Library ◽  
Resistant Mutant ◽  
Selection Marker ◽  
Wild Type ◽  
Comparable Amount ◽  
Reading Frame ◽  
Negative Selection Marker ◽  
Efficient Selection ◽  
Atp Sulphurylase

The ATP sulphurylase gene of Schizosaccharomyces pombe has been cloned by complementation of cysteine auxotrophy of a selenate-resistant mutant, which supposedly had a defect in ATP sulphurylase. A sulphate nonutilizing (cysteine auxotrophic) and selenate-resistant mutant of S. pombe was transformed with a wild-type S. pombe genomic library and sulphate-utilizing clones were isolated. The open reading frame encoding the ATP sulphurylase enzyme was found to be responsible for the restoration of sulphate assimilation. Transformants became as sensitive for selenate as the wild-type strain and produced a comparable amount of ATP sulphurylase as the prototrophic strains. The cloned ATP sulphurylase gene (sua1) proved to be an efficient selection marker in an ARS vector, when different isogenic or nonisogenic S. pombe selenate-resistant mutants were used as cloning hosts. Complementation of sua1– mutations by sua1-bearing multicopy vectors functions as a useful dual positive and negative selection marker. The cloned sua1 gene also complemented the met3 (ATP sulphurylase deficient) mutation in Saccharomyces cerevisiae .

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

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
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.

Characterization of a gene locus from Erwinia amylovora with regulatory functions in exopolysaccharide synthesis of Erwinia spp.

1998 ◽  
Vol 44 (7) ◽  
pp. 657-666 ◽  
Author(s):  
Phillip Aldridge ◽  
Frank Bernhard ◽  
Peter Bugert ◽  
David L Coplin ◽  
Klaus Geider
Keyword(s):  
Structural Gene ◽  
Erwinia Amylovora ◽  
Genomic Library ◽  
Expression Vectors ◽  
External Signal ◽  
Wild Type ◽  
Reading Frame ◽  
Gus Reporter ◽  
Acid Protein ◽  
Erwinia Stewartii

In a genomic library of Erwinia amylovora, a locus has been identified that can suppress an Erwinia stewartii rcsA mutant. In addition, the locus induced a mucoid sticky phenotype of colonies in a wild-type strain of Erwinia stewartii and increased exopolysaccharide synthesis in several species of bacteria belonging to the genus Erwinia. An open reading frame was identified at this locus encoding a 225 amino acid protein that contained a helix-turn-helix motif typical of transcriptional regulators. The corresponding gene was subsequently named rcsV (regulator of capsular synthesis affecting viscosity). A mutant of rcsV in wild-type Erwinia amylovora had no detectable phenotype and produced typical levels of amylovoran under laboratory conditions. The rcsV gene on a high copy number plasmid under the control of its own promoter did not alter amylovoran production, in contrast to in-frame fusions of the structural gene in expression vectors. Since even the lac promoter was inert in the expression of rcsV, a DNA-binding protein could inhibit transcription of the gene in Erwinia amylovora. On the other hand, an Erwinia amylovora rcsA mutant was suppressed by rcsV when its promoter was replaced and the structural gene fused in-frame with lacZ' or malE. Northern blots, with total RNA from Erwinia amylovora, or promoter analysis using the GUS reporter gene did not show expression of rcsV in Erwinia amylovora, although primer extension analysis did. RcsV could be a component involved in the regulation of amylovoran synthesis, and gene expression may require an unknown external signal during the life cycle or pathogenesis of Erwinia amylovora. Key words: amylovoran, fire blight, rcsA-like activator, fusion protein.

SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis

1993 ◽  
Vol 13 (7) ◽  
pp. 4039-4048
Author(s):  
T Roemer ◽  
S Delaney ◽  
H Bussey
Keyword(s):  
Genomic Library ◽  
Killer Toxin ◽  
Resistant Mutant ◽  
Null Alleles ◽  
Dependent Manner ◽  
Wild Type ◽  
Beta Glucan ◽  
Glucan Synthesis ◽  
Dose Dependent

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.

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

2003 ◽  
Vol 69 (2) ◽  
pp. 1263-1269 ◽  
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.

scholarly journals SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis.

1993 ◽  
Vol 13 (7) ◽  
pp. 4039-4048 ◽  
Author(s):  
T Roemer ◽  
S Delaney ◽  
H Bussey
Keyword(s):  
Genomic Library ◽  
Killer Toxin ◽  
Resistant Mutant ◽  
Null Alleles ◽  
Dependent Manner ◽  
Wild Type ◽  
Beta Glucan ◽  
Glucan Synthesis ◽  
Dose Dependent

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.

scholarly journals Saccharomyces cerevisiae Σ1278b Has Novel Genes of the N-Acetyltransferase Gene Superfamily Required for l-Proline Analogue Resistance

2000 ◽  
Vol 182 (15) ◽  
pp. 4249-4256 ◽  
Author(s):  
Hiroshi Takagi ◽  
Mika Shichiri ◽  
Miho Takemura ◽  
Miho Mohri ◽  
Shigeru Nakamori
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carboxylic Acid ◽  
Genomic Library ◽  
Acid Resistance ◽  
Resistant Mutant ◽  
Genome Project ◽  
The Novel ◽  
Novel Genes ◽  
Global Function ◽  
Reading Frame

ABSTRACT We discovered on the chromosome of Saccharomyces cerevisiae Σ1278b novel genes involved in l-proline analogue l-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of thel-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Σ1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable forl-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of theN-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required forl-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Σ1278b background strains. When this novelMPR1 or MPR2 gene (sigma 1278b gene forl-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to l-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae.

scholarly journals Role of rpoS in Stress Survival and Virulence of Vibrio cholerae

1998 ◽  
Vol 180 (4) ◽  
pp. 773-784 ◽  
Author(s):  
Fitnat H. Yildiz ◽  
Gary K. Schoolnik
Keyword(s):  
Vibrio Cholerae ◽  
Genomic Library ◽  
Bacterial Species ◽  
Wild Type ◽  
Reading Frame ◽  
Infant Mouse ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded byrpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivatedrpoS by homologous recombination. V. choleraestrains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoSmutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.

scholarly journals Intrinsic Resistance of Mycobacteriumsmegmatis to Fluoroquinolones May Be Influenced by New Pentapeptide Protein MfpA

2001 ◽  
Vol 45 (12) ◽  
pp. 3387-3392 ◽  
Author(s):  
Clemente Montero ◽  
Guaniri Mateu ◽  
Rosalva Rodriguez ◽  
Howard Takiff
Keyword(s):  
Genomic Library ◽  
Type Ii ◽  
Wild Type ◽  
Intrinsic Resistance ◽  
Reading Frame ◽  
Putative Protein ◽  
Development Of Resistance ◽  
The Family ◽  
Multistep Process ◽  
Level Resistance

ABSTRACT The fluoroquinolones (FQ) are used in the treatment ofMycobacterium tuberculosis, but the development of resistance could limit their effectiveness. FQ resistance (FQR) is a multistep process involving alterations in the type II topoisomerases and perhaps in the regulation of efflux pumps, but several of the steps remain unidentified. Recombinant plasmid pGADIV was selected from a genomic library of wild-type (WT), FQ-sensitive M. smegmatisby its ability to confer low-level resistance to sparfloxacin (SPX). In WT M. smegmatis, pGADIV increased the MICs of ciprofloxacin (CIP) by fourfold and of SPX by eightfold, and inM. bovis BCG it increased the MICs of both CIP and SPX by fourfold. It had no effect on the accumulation of14C-labeled CIP or SPX. The open reading frame responsible for the increase in FQR, mfpA, encodes a putative protein belonging to the family of pentapeptides, in which almost every fifth amino acid is either leucine or phenylalanine. Very similar proteins are also present in M.tuberculosis and M. avium. The MICs of CIP and SPX were lower for an M.smegmatis mutant strain lacking an intactmfpA gene than for the WT strain, suggesting that, by some unknown mechanism, the gene product plays a role in determining the innate level of FQR in M.smegmatis.

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