scholarly journals The Cloning by Complementation of the pawn-A Gene in Paramecium

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 947-957 ◽  
Author(s):  
W John Haynes ◽  
Brian Vaillant ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Dna Sequences ◽  
Avoidance Reaction ◽  
Cdna Clones ◽  
Genetic Dissection ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rnase Protection ◽  
Reading Frame ◽  
Voltage Dependent ◽  
Mutant Phenotypes

Abstract The genetic dissection of a simple avoidance reaction behavior in Paramecium tetraurelia has shown that ion channels are a critical molecular element in signal transduction. Pawn mutants, for example, were originally selected for their inability to swim backward, a trait that has since been shown to result from the loss of a voltage-dependent calcium current. The several genes defined by this phenotype were anticipated to be difficult to clone since the 800-ploid somatic macronucleus of P. tetraurelia is a formidable obstacle to cloning by complementation. Nonetheless, when the macronucleus of a pawn mutant (pwA/pwA) was injected with total wild-type DNA or a fractional library of DNA, its clonal descendants all responded to stimuli like the wild type. By sorting a fractional library, we cloned and sequenced a 2.3-kb fragment that restores the Ca2+ current and excitability missing in pawn-A. Data from RNase protection assays, followed by the sequencing of mutant alleles and cDNA clones, established an open reading frame. The conceptually translated product suggests a novel protein that may be glycophosphatidylinositol anchored. We also discuss the general usefulness of this method in cloning other unknown DNA sequences from Paramecium that are functionally responsible for various mutant phenotypes.

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.

scholarly journals Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

1991 ◽  
Vol 11 (2) ◽  
pp. 1133-1137 ◽  
Author(s):  
Y You ◽  
K Aufderheide ◽  
J Morand ◽  
K Rodkey ◽  
J Forney
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Mutant Cell ◽  
Restriction Pattern ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Coding Region ◽  
Cytoplasmic Factor ◽  
In The Wild ◽  
Dna Restriction

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

scholarly journals Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia

1991 ◽  
Vol 11 (2) ◽  
pp. 1133-1137
Author(s):  
Y You ◽  
K Aufderheide ◽  
J Morand ◽  
K Rodkey ◽  
J Forney
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Mutant Cell ◽  
Restriction Pattern ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Coding Region ◽  
Cytoplasmic Factor ◽  
In The Wild ◽  
Dna Restriction

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

scholarly journals CFP, the Putative Cercosporin Transporter of Cercospora kikuchii, Is Required for Wild Type Cercosporin Production, Resistance, and Virulence on Soybean

1999 ◽  
Vol 12 (10) ◽  
pp. 901-910 ◽  
Author(s):  
Terrence M. Callahan ◽  
Mark S. Rose ◽  
Maura J. Meade ◽  
Marilyn Ehrenshaft ◽  
Robert G. Upchurch
Keyword(s):  
Major Facilitator Superfamily ◽  
Cdna Clones ◽  
Membrane Transporter ◽  
Wild Type ◽  
Drastic Reduction ◽  
Targeted Disruption ◽  
Reading Frame ◽  
Mrna Transcripts ◽  
Increased Sensitivity ◽  
Major Facilitator

Many species of the fungal genus Cercospora, including the soybean pathogen C. kikuchii, produce the phytotoxic polyketide cercosporin. Cercosporin production is induced by light. Previously, we identified several cDNA clones of mRNA transcripts that exhibited light-enhanced accumulation in C. kikuchii. Targeted disruption of the genomic copy of one of these, now designated CFP (cercosporin facilitator protein), results in a drastic reduction in cercosporin production, greatly reduced virulence of the fungus to soybean, and increased sensitivity to exogenous cercosporin. Sequence analysis of CFP reveals an 1,821-bp open reading frame encoding a 65.4-kDa protein similar to several members of the major facilitator superfamily (MFS) of integral membrane transporter proteins known to confer resistance to various antibiotics and toxins in fungi and bacteria. We propose that CFP encodes a cercosporin transporter that contributes resistance to cercosporin by actively exporting cercosporin, thus maintaining low cellular concentrations of the toxin.

scholarly journals Identification of a Fourth Formate Dehydrogenase in Methylobacterium extorquens AM1 and Confirmation of the Essential Role of Formate Oxidation in Methylotrophy

2007 ◽  
Vol 189 (24) ◽  
pp. 9076-9081 ◽  
Author(s):  
Ludmila Chistoserdova ◽  
Gregory J. Crowther ◽  
Julia A. Vorholt ◽  
Elizabeth Skovran ◽  
Jean-Charles Portais ◽  
...  
Keyword(s):  
Formate Dehydrogenase ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Methanol Metabolism ◽  
Reading Frame ◽  
Formate Oxidation ◽  
In The Wild ◽  
Mutant Phenotypes ◽  
Small Open Reading Frame

ABSTRACT A mutant of Methylobacterium extorquens AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [13C]formate, as well as 14C-labeling experiments, demonstrated production of labeled CO2 in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (fdh4A) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO2 formation in growing cultures. Mutation of a small open reading frame (fdh4B) downstream of fdh4A resulted in mutant phenotypes similar to the phenotypes of fdh4A mutants, suggesting that fdh4B is also involved in formate oxidation.

Evidence for alternative splicing of the chicken vasoactive intestinal polypeptide gene transcript

1995 ◽  
Vol 15 (1) ◽  
pp. 81-91 ◽  
Author(s):  
R T Talbot ◽  
I C Dunn ◽  
P W Wilson ◽  
H M Sang ◽  
P J Sharp
Keyword(s):  
Vasoactive Intestinal Polypeptide ◽  
Cdna Sequence ◽  
Rnase Protection Assay ◽  
Protein Product ◽  
Cdna Clones ◽  
Gene Transcript ◽  
Rt Pcr ◽  
Rnase Protection ◽  
Reading Frame ◽  
Intestinal Polypeptide

ABSTRACT Two forms of chicken vasoactive intestinal polypeptide (VIP) mRNA have been identified by reverse transcription (RT)-PCR and RNase protection assay. The shorter form of chicken VIP mRNA encodes a protein that does not contain an analogue of rat peptide histidine isoleucine (PHI) 1–27 or human peptide histidine methionine 1–27. The larger form encodes both VIP and a chicken analogue of PHI 1–27 in the same protein product. Three VIP cDNAs isolated from a chicken hypothalamic cDNA library were derived from the shorter mRNA. Sequence analysis of the longest clone identified an open reading frame that codes for a 165 amino acid preproVIP protein and contains two polyadenylation signals. In situ hybridisation with an oligonucleotide probe from the VIP cDNA sequence showed that VIP-encoding mRNA occurs in cells in the basal hypothalamus, an area of the brain known to contain VIP neurosecretory neurones. RT-PCR of total RNA from liver, kidney, gut, pancreas, pituitary, cerebellum, forebrain and hypothalamus, using primers derived from the VIP cDNA sequence, showed that the shorter form of VIP mRNA is present in all of these tissues. The sequence of the longer form of VIP mRNA was obtained by sequencing a portion of the VIP gene from genomic DNA. This revealed a potential exon that was not represented in the VIP cDNA clones analysed. RT-PCR with primers from this sequence showed that it was expressed in the gut and hypothalamus. RNase protection assays confirmed the presence of the two forms of mRNA in gut and hypothalamus. The relative proportions of the two mRNA forms were: 97·8% VIP only, 2·2% PHI/VIP in the hypothalamus and 98·5% VIP only, 1·5% PHI/VIP in the gut. In conclusion, chicken VIP mRNA is alternatively spliced. The shortest form, which encodes a preproprotein containing only the VIP peptide, is the most abundant. The longer form of chicken VIP mRNA encodes a preproprotein containing sequences for both VIP and a chicken form of PHI.

scholarly journals Genetic Dissection Defines the Roles of Elsinochrome Phytotoxin for Fungal Pathogenesis and Conidiation of the Citrus Pathogen Elsinoë fawcettii

2008 ◽  
Vol 21 (4) ◽  
pp. 469-479 ◽  
Author(s):  
Hui-Ling Liao ◽  
Kuang-Ren Chung
Keyword(s):  
Polyketide Synthase ◽  
Functional Characterization ◽  
Chemical Properties ◽  
Fungal Pathogenesis ◽  
Genetic Dissection ◽  
Wild Type ◽  
Rough Lemon ◽  
Mutant Phenotypes ◽  
Axenic Conditions

Elsinochrome pigments produced by many phytopathogenic Elsinoë spp. are nonhost-selective toxins which react with oxygen molecules after light activation to produce highly toxic reactive oxygen species. The structures and chemical properties of four derivatives are well known. However, the biological roles of elsinochromes in fungal pathogenesis are poorly understood. Many isolates of Elsinoë fawcettii causing citrus scab are able to produce elsinochromes under axenic conditions. In this article, we report the cloning, expression, and functional characterization of the polyketide synthase-encoding gene, EfPKS1, which we show is required for the production of elsinochromes and fungal pathogenesis. Targeted disruption of EfPKS1 in E.fawcettii completely abrogated elsinochrome production, drastically reduced conidiation, and significantly decreased lesion formation on rough lemon leaves. All mutant phenotypes were restored to the wild type in fungal strains expressing a functional copy of EfPKS1. Accumulation of the EfPKS1 transcript and elsinochromes by a wild-type strain appears to be coordinately regulated by light, nutrients, and pH. The results indicate that the product of EfPKS1 is involved in the biosynthesis of elsinochromes via a fungal polyketide pathway, and that elsinochromes play an important role in fungal pathogenesis.

scholarly journals A Pathogenicity Gene Isolated from the pPATH Plasmid of Erwinia herbicola pv. gypsophilae Determines Host Specificity

1998 ◽  
Vol 11 (8) ◽  
pp. 753-762 ◽  
Author(s):  
Lea Valinsky ◽  
Shulamit Manulis ◽  
Roni Nizan ◽  
David Ezra ◽  
Isaac Barash
Keyword(s):  
Dna Sequences ◽  
Wild Type ◽  
Erwinia Herbicola ◽  
Putative Gene ◽  
Reading Frame ◽  
Ribosome Binding ◽  
Significant Homology ◽  
In Trans ◽  
Dna Fragment ◽  
Specific Virulence

The host range of the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) is restricted to the gypsophila plant whereas E. herbicola pv. betae (Ehb) incites galls on beet as well as gypsophila. The pathogenicity of Ehg and Ehb was previously shown to be dependent on a plasmid (pPATH). Transposon mutagenesis was used to generate mutants on the cosmid pLA150 of the pPATH from Ehg824-1. A cluster of nonpathogenic mutations flanked by two IS1327 elements was identified on a 3.2-kb NdeI DNA fragment. All mutants were restored to pathogenicity by complementation in trans with the wild-type Ehg DNA. DNA sequence analysis of the 3.2-kb NdeI fragment revealed a single open reading frame (ORF) of 2 kb as well as a potential ribosome binding site and a putative hrp box upstream to the ORF. The ORF had no significant homology to known genes. Southern analysis also revealed the presence of DNA sequences that hybridized to the ORF in the beet pathovar Ehb4188. This gene was isolated and sequenced. Marker exchange mutants generated in the ORF of Ehb eliminated the pathogenicity of Ehb on gypsophila but fully retained its pathogenicity on beet. Since the putative gene appeared to encode a host-specific virulence factor for gypsophila it was designated as hsvG.

scholarly journals Permanent rescue of a non-Mendelian mutation of Paramecium by microinjection of specific DNA sequences.

Genetics ◽  
1991 ◽  
Vol 129 (3) ◽  
pp. 727-734
Author(s):  
H Jessop-Murray ◽  
L D Martin ◽  
D Gilley ◽  
J R Preer ◽  
B Polisky
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Large Deletion ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Cytoplasmic Factor ◽  
Gene Copies ◽  
Unusual Phenomenon ◽  
Small Stable Rna

Abstract The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its surface. Although the A gene is intact in the micronuclei of d48, the A gene copies in the macronucleus contain a large deletion eliminating virtually the entire coding sequence. Previous studies showed that microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently restored A expression after autogamy. Together with other data, this result suggests that in wild type cells the A gene in the old macronucleus ensures the presence of a cytoplasmic factor that prevents A gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular mechanisms involved in this unusual phenomenon, we attempted to define the region(s) of the A gene necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments of the A gene have the ability to rescue d48 to different extents, some being more efficient than others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might act as or encode a cytoplasmic factor. Molecular mechanisms that may be involved in the rescue of d48 are discussed.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

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