ATP-Dependent Remodeling of the Spliceosome: Intragenic Suppressors of Release-Defective Mutants of Saccharomyces cerevisiae Prp22

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 407-415 ◽  
Author(s):  
Eva Campodonico ◽  
Beate Schwer
Keyword(s):  
Atp Hydrolysis ◽  
Splicing Factor ◽  
Nonpermissive Temperature ◽  
Mutant Proteins ◽  
Growth Defects ◽  
Isolation And Characterization ◽  
Cold Sensitive ◽  
Rna Unwinding

Abstract The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.

Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae

1988 ◽  
Vol 8 (10) ◽  
pp. 3997-4008
Author(s):  
M Wittekind ◽  
J Dodd ◽  
L Vu ◽  
J M Kolb ◽  
J M Buhler ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Polymerase I

The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon.

scholarly journals Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (10) ◽  
pp. 3997-4008 ◽  
Author(s):  
M Wittekind ◽  
J Dodd ◽  
L Vu ◽  
J M Kolb ◽  
J M Buhler ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Polymerase I

The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon.

scholarly journals Isolation and Characterization of a Second Subunit of Molecular Chaperonin from Pyrococcus kodakaraensis KOD1: Analysis of an ATPase-Deficient Mutant Enzyme

1999 ◽  
Vol 65 (4) ◽  
pp. 1801-1805 ◽  
Author(s):  
Michi Izumi ◽  
Shinsuke Fujiwara ◽  
Masahiro Takagi ◽  
Shigenori Kanaya ◽  
Tadayuki Imanaka
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Deficient Mutant ◽  
Α Subunit ◽  
Mutant Proteins ◽  
E Coli ◽  
Isolation And Characterization ◽  
Insoluble Form ◽  
Cobyric Acid

ABSTRACT The cpkA gene encoding a second (α) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (β subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.

PENGEMBANGAN TEKNOLOGI SELEKSI IN VITRO BERULANG (REPEAT CYCLING–IN VITRO SELECTION) TOLERAN STRES KEKERINGAN UNTUK PERBAIKAN MUTU GENETIK DAN PRODUKSI TANAMAN KEDELAI BERMIKORIZA

2017 ◽  
Vol 1 (1) ◽  
pp. 74-84
Author(s):  
Ahmad Riduan ◽  
Rainiyati Rainiyati ◽  
Yulia Alia
Keyword(s):  
Arbuscular Mycorrhizae ◽  
In Vitro Selection ◽  
Soil Samples ◽  
Single Spore ◽  
Isolation And Characterization ◽  
Single Spore Culture ◽  
Spore Culture ◽  
Glomus Sp

Every plant rhizospheres in any ecosystem there are various living microorganisms including Arbuscular Mycorrhizae Fungi (AMF).  An isolation and characterization is required to investigate the species or type of the AMF. This research was aimed at studying the isolation and characterization of AMF sporulation in soybean rhizospheres in Jambi Province. The results of evaluation on soil samples before trapping showed that there are spores from three genus of AMF twelve types Glomus , two types Acaulospora and one type of Enthrophospora.  Following single spore culture in soybean rhizosphere, 5 spore types were obtained:  Glomus sp-1, Glomus sp-4, Glomus sp-7, Glomus sp-8 Glomus sp-10.

Arginine synthesis by hepatomas in vitro. II. Isolation and characterization of Morris hepatoma variants unable to convert ornithine to arginine, and modulation of urea-cycle enzymes by dexamethasone and cyclic-AMP

1984 ◽  
Vol 68 (1) ◽  
pp. 305-319
Author(s):  
S.J. Goss
Keyword(s):  
Cyclic Amp ◽  
Liver Cells ◽  
Isolation And Characterization ◽  
Rat Liver Cells ◽  
Morris Hepatoma ◽  
Ornithine Transcarbamoylase ◽  
Cellular Incorporation

‘77orn’, a derivative of the Morris rat hepatoma 7777, stably expresses high levels of ornithine transcarbamoylase (OTC) and carbamoylphosphate synthetase I (CPS-I), and is able to grow indefinitely in ornithine-medium (medium with ornithine in place of arginine). Variants that have lost this ability are isolated from 77orn by a ‘suicide’ selective technique dependent on the cellular incorporation of [3H]ornithine. These variants, which have reduced levels of CPS-I, or of both CPS-I and OTC, are shown to have developed multiple hormonal requirements; their enzyme deficiencies can be reversed by use of an appropriately supplemented medium. In particular, CPS-I is inducible by dexamethasone and dibutyryl-cyclic-AMP in combination. Cholera toxin can be used instead of cyclic-AMP, but then butyrate is additionally required if the induction is to be maintained in the long term. The use of these agents in excess can depress OTC. Several other hepatomas, and alos explanted foetal rat liver cells, have similar requirements for CPS-I expression. It is argued that multiple hormonal requirements for CPS-I production are normal in liver cells in vitro, and that hormone-independent hepatomas should be regarded as abnormal. The implications of this for the somatic cell genetic investigation of differentiation are briefly discussed.

Isolation and characterization of phosphate solubilizing bacteria from the rhizospheric soil of Ponthenpuzha forest, Pathanamthitta (District), Kerala

2021 ◽  
Vol 16 (8) ◽  
pp. 110-117
Author(s):  
Kannan Abhirami ◽  
K. Jayakumar
Keyword(s):  
Bacterial Isolate ◽  
Phosphate Solubilizing Bacteria ◽  
Cymbopogon Citratus ◽  
16S Rrna Sequence ◽  
Isolation And Characterization ◽  
16S Rrna Sequence Analysis ◽  
Phosphate Solubilizing ◽  
Level Identification

Phosphorous is considered as a major parameter for crop yield. Its availability to plant is independent of its abundance. For the plants to utilize phosphorous, it is to be converted to absorbable form. Here, the part rendered by phosphate solubilizing bacteria is significant for it plays a crucial role in the formation of plant usable phosphate from organic forms. In the present work, an effort had been made to isolate and identify phosphate solubilising bacterial isolate from the rhizhospheric soils of various plants in Ponthenpuzha forest. One of the isolate from Cymbopogon citrates responded positively to Pikovskaya’s medium by producing a halo zone during in vitro culture. Colony features and 16S rRNA sequence analysis identified the isolate as Burkholderia sps. We have reported the presence of genus Burkholderia in the rhizospheric zone of Cymbopogon citratus. Further studies are warranted for species level identification of the isolate.

scholarly journals Isolation and characterization of arsenic resistant soil bacteria and their effects on germination of rice under arsenic contamination

2015 ◽  
Vol 2 (2) ◽  
pp. 229-237
Author(s):  
Istiaq Ahmed ◽  
Md Tofazzal Islam ◽  
Md Akhter Hossain Chowdhury ◽  
Md Kamruzzaman
Keyword(s):  
Contaminated Soils ◽  
Sodium Arsenite ◽  
Agar Plate ◽  
Rice Seedling ◽  
Resistant Bacteria ◽  
Gram Staining ◽  
Isolation And Characterization ◽  
Resistant Strains

This study was carried out to isolate, screen and characterize arsenic (As) resistant bacteria from As contaminated soils of Dumrakandi and Matlab under Faridpur and Chandpur districts and to evaluate their efficiency in reducing As toxicity against rice seedlings during germination. Thirteen strains were isolated from the soils which showed resistance to different levels of sodium arsenite (viz. 5, 10, 20 and 40 mM) in both agar plate and broth assay using BSMY I media. Among the isolates, BTL0011, BTL0012, BTL0015 and BTL0022 showed highest resistance to 40 mM sodium arsenite. Gram staining and KOH solubility test revealed that five strains were gram positive and rest eight was gram negative. They grew well in the liquid media at pH 5.5 to 8.5. In-vitro rice seedling bioassay with two superior isolates (BTL0011 and BTL0022) revealed that As resistant strains significantly enhanced seed germination of BRRI dhan29 and BRRI dhan47 at 60 ppm As. This study was laid out in CRD with three replications. The performance of BTL 0022 was superior to BTL0011. The overall results suggest that BTL0011 and BTL0022 can be used for bioremediation of As contaminated soils and to increase the germination and seedling growth of rice in As contaminated soils.Res. Agric., Livest. Fish.2(2): 229-237, August 2015

scholarly journals Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore.

1993 ◽  
Vol 121 (3) ◽  
pp. 513-519 ◽  
Author(s):  
W Jiang ◽  
J Lechner ◽  
J Carbon
Keyword(s):  
Molecular Motors ◽  
Cell Biology ◽  
Budding Yeast ◽  
Isolation And Characterization ◽  
Sequence Homologies ◽  
Binding Factor ◽  
Microtubule Motor

We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present. The CBF2 gene is essential for viability of yeast and is identical to NDC10, in which a conditional mutation leads to a defect in chromosome segregation (Goh, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.

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