Shuttle mutagenesis and targeted disruption of a telomere-located essential gene of Leishmania

Leishmania mutants have contributed greatly to extend our knowledge of this parasite's biology. Here we report the use of the mariner in vitro transposition system as a source of reagents for shuttle mutagenesis and targeted disruption of Leishmania genes. The locus-specific integration was achieved by the disruption of the subtelomeric gene encoding a DNA-directed RNA polymerase III subunit (RPC2). Further inactivation of RPC2 alleles required the complementation of the intact gene, which was transfected in an episomal context. However, attempts to generate a RPC2 chromosomal null mutant resulted in genomic rearrangements that maintained copies of the intact locus in the genome. The maintenance of the RPC2 chromosomal locus in complemented mutants was not mediated by an increase in the number of copies and did not involve chromosomal translocations, which are the typical characteristics of the genomic plasticity of this parasite. Unlike the endogenous locus, the selectable marker used to disrupt RPC2 did not display a tendency to remain in its chromosomal location but was targeted into supernumerary episomal molecules.

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Stu2p: A Microtubule-Binding Protein that Is an Essential Component of the Yeast Spindle Pole Body

The Journal of Cell Biology ◽

10.1083/jcb.139.5.1271 ◽

1997 ◽

Vol 139 (5) ◽

pp. 1271-1280 ◽

Cited By ~ 117

Author(s):

Peijing Jeremy Wang ◽

Tim C. Huffaker

Keyword(s):

Essential Gene ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cold Sensitivity ◽

Gene Encoding ◽

Microtubule Binding ◽

Amino Acid Region ◽

Pole Body ◽

Spindle Function

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.

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Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli

Biochemical Journal ◽

10.1042/bj2560741 ◽

1988 ◽

Vol 256 (3) ◽

pp. 741-749 ◽

Cited By ~ 20

Author(s):

N Allison ◽

C H Williams ◽

J R Guest

Keyword(s):

Escherichia Coli ◽

Transcriptional Control ◽

Kinetic Properties ◽

Temperature Sensitive ◽

Gene Encoding ◽

Lambda Repressor ◽

Intact Gene ◽

In The Wild ◽

Lipoamide Dehydrogenase

A ‘split-gene’ technique for the overexpression and mutagenesis of the gene encoding the lipoamide dehydrogenase of Escherichia coli was developed in order to overcome the instability problems encountered when attempting to mutate the intact gene. The lipoamide dehydrogenase gene, lpd, was dissected into two fragments which were separately subcloned into M13 vectors for mutagenesis in vitro followed by reconstitution in the pJLA504 expression vector under the transcriptional control of the lambda PR and lambda PL promoters and a temperature-sensitive lambda repressor. After thermo-induction, E. coli cells transformed with the plasmid carrying the reconstituted lpd gene contained 4-5 times more lipoamide dehydrogenase activity than is normally found in the wild-type organism. The strategy was used to engineer a Glu-188→Asp replacement in lipoamide dehydrogenase, and this generated an enzyme with markedly different kinetic properties.

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τ91, an Essential Subunit of Yeast Transcription Factor IIIC, Cooperates with τ138 in DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.1 ◽

1998 ◽

Vol 18 (1) ◽

pp. 1-9 ◽

Cited By ~ 26

Author(s):

Rosalía Arrebola ◽

Nathalie Manaud ◽

Sophie Rozenfeld ◽

Marie-Claude Marsolier ◽

Olivier Lefebvre ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Essential Gene ◽

Open Reading Frames ◽

Class Iii ◽

Gene Encoding ◽

Promoter Recognition

ABSTRACT Transcription factor IIIC (TFIIIC) (or τ) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, τ91, present in affinity-purified TFIIIC. τ91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of τ91 was established by the following biochemical and genetic evidence. Antibodies to τ91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to τ91; and a point mutation in TFC6 (τ91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349Emutant affected in the B block binding subunit (τ138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that τ91 cooperates with τ138 for DNA binding. Recombinant τ91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

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The Leishmania infantum cytosolic SIR2-related protein 1 (LiSIR2RP1) is an NAD+-dependent deacetylase and ADP-ribosyltransferase

Biochemical Journal ◽

10.1042/bj20080666 ◽

2008 ◽

Vol 415 (3) ◽

pp. 377-386 ◽

Cited By ~ 29

Author(s):

Joana Tavares ◽

Ali Ouaissi ◽

Nuno Santarém ◽

Denis Sereno ◽

Baptiste Vergnes ◽

...

Keyword(s):

Leishmania Infantum ◽

Catalytic Domain ◽

Insoluble Fraction ◽

Related Protein ◽

Targeted Disruption ◽

Gene Encoding ◽

Adp Ribosyltransferase ◽

First Time

Proteins of the SIR2 (Silent Information Regulator 2) family are characterized by a conserved catalytic domain that exerts unique NAD+-dependent deacetylase activity on histones and various other cellular substrates. Previous reports from us have identified a Leishmania infantum gene encoding a cytosolic protein termed LiSIR2RP1 (Leishmania infantum SIR2-related protein 1) that belongs to the SIR2 family. Targeted disruption of one LiSIR2RP1 gene allele led to decreased amastigote virulence, in vitro as well as in vivo. In the present study, attempts were made for the first time to explore and characterize the enzymatic functions of LiSIR2RP1. The LiSIR2RP1 exhibited robust NAD+-dependent deacetylase and ADP-ribosyltransferase activities. Moreover, LiSIR2RP1 is capable of deacetylating tubulin, either in dimers or, when present, in taxol-stabilized microtubules or in promastigote and amastigote extracts. Furthermore, the immunostaining of parasites revealed a partial co-localization of α-tubulin and LiSIR2RP1 with punctate labelling, seen on the periphery of both promastigote and amastigote stages. Isolated parasite cytoskeleton reacted with antibodies showed that part of LiSIR2RP1 is associated to the cytoskeleton network of both promastigote and amastigote forms. Moreover, the Western blot analysis of the soluble and insoluble fractions of the detergent of promastigote and amastigote forms revealed the presence of α-tubulin in the insoluble fraction, and the LiSIR2RP1 distributed in both soluble and insoluble fractions of promastigotes as well as amastigotes. Collectively, the results of the present study demonstrate that LiSIR2RP1 is an NAD+-dependent deacetylase that also exerts an ADP-ribosyltransferase activity. The fact that tubulin could be among the targets of LiSIR2RP1 may have significant implications during the remodelling of the morphology of the parasite and its interaction with the host cell.

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Maf1p, a Negative Effector of RNA Polymerase III inSaccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.21.15.5031-5040.2001 ◽

2001 ◽

Vol 21 (15) ◽

pp. 5031-5040 ◽

Cited By ~ 109

Author(s):

Krzysztof Pluta ◽

Olivier Lefebvre ◽

Nancy C. Martin ◽

Wieslaw J. Smagowicz ◽

David R. Stanford ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Growth Defect ◽

Class Iii ◽

Gene Encoding ◽

Cell Extracts ◽

Polymerase Iii ◽

Pol Iii

ABSTRACT Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

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Conditional mutants of RPC160, the gene encoding the largest subunit of RNA polymerase C in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/119.3.517 ◽

1988 ◽

Vol 119 (3) ◽

pp. 517-526

Author(s):

R Gudenus ◽

S Mariotte ◽

A Moenne ◽

A Ruet ◽

S Memet ◽

...

Keyword(s):

Rna Polymerase ◽

Essential Gene ◽

Mutant Cell ◽

Yeast Genome ◽

Temperature Sensitive ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Gene Encoding

Abstract A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO3 and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA. An unlinked mutation (pcil-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation.

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Mpe1, a Zinc Knuckle Protein, Is an Essential Component of Yeast Cleavage and Polyadenylation Factor Required for the Cleavage and Polyadenylation of mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8346-8356.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8346-8356 ◽

Cited By ~ 27

Author(s):

Le Thuy Anh Vo ◽

Michèle Minet ◽

Jean-Marie Schmitter ◽

François Lacroute ◽

Françoise Wyers

Keyword(s):

Rna Binding ◽

Essential Gene ◽

Affinity Purification ◽

Protein A ◽

Gene Encoding ◽

Cleavage And Polyadenylation ◽

The Stability ◽

Polyadenylation Factor

ABSTRACT In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene,MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice ofACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3′-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3′ end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3′ end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.

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Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene

Plants ◽

10.3390/plants10061102 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1102

Author(s):

Galina N. Raldugina ◽

Sergey V. Evsukov ◽

Liliya R. Bogoutdinova ◽

Alexander A. Gulevich ◽

Ekaterina N. Baranova

Keyword(s):

Choline Oxidase ◽

Culture Methods ◽

Gene Encoding ◽

Oxidase Gene ◽

Bacterial Enzyme ◽

Transgenic Lines ◽

High Regeneration ◽

Whole Plants ◽

Implicit Response

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

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