A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids

1986 ◽  
Vol 6 (11) ◽  
pp. 4053-4059
Author(s):  
Y Kikuchi ◽  
A Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Complementation Group ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Initiation Of Replication ◽  
Low Levels ◽  
Stable Maintenance

We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

scholarly journals A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids.

1986 ◽  
Vol 6 (11) ◽  
pp. 4053-4059 ◽  
Author(s):  
Y Kikuchi ◽  
A Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Complementation Group ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Initiation Of Replication ◽  
Low Levels ◽  
Stable Maintenance

We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (4) ◽  
pp. 832-835 ◽  
Author(s):  
Terri S. Rice ◽  
Min Ding ◽  
David S. Pederson ◽  
Nicholas H. Heintz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Origin Recognition Complex ◽  
Nuclear Division ◽  
Permissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
And Migration ◽  
Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

scholarly journals Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

1986 ◽  
Vol 6 (7) ◽  
pp. 2663-2673 ◽  
Author(s):  
M C Strobel ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trna Gene ◽  
Nuclear Extract ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Specific Sequence ◽  
Made In

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

scholarly journals Binding and Partial Denaturing of G-quartet DNA by Cdc13p ofSaccharomyces cerevisiae

2001 ◽  
Vol 276 (50) ◽  
pp. 47671-47674 ◽  
Author(s):  
Yi-Chien Lin ◽  
Jing-Wen Shih ◽  
Chia-Ling Hsu ◽  
Jing-Jer Lin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intermolecular Association ◽  
Telomere Association ◽  
Dna Quadruplex ◽  
Telomere Replication ◽  
Dna Quadruplexes

The protein Cdc13p binds telomeresin vivoand is essential for the maintenance of the telomeres ofSaccharomyces cerevisiae. In addition, Cdc13p is known to bind single-stranded TG1–3DNAin vitro. Here we have shown that Cdc13p also binds DNA quadruplex, G-quartet, formed by TG1–3DNA. Moreover, the binding of Cdc13p causes a partial denaturing of the G-quartet DNA. Formation of DNA quadruplexes may involve the intermolecular association of TG1–3DNA and inhibit the extension of telomeres by telomerase. Thus, our finding suggests that Cdc13p may disrupt telomere association and facilitate telomere replication.

Sign in / Sign up

Export Citation Format

Share Document