Q55168148
Dotaz
Zobrazit nápovědu
141 s. : il.
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- embryologie a teratologie
- biologie
- genetika, lékařská genetika
- NLK Publikační typ
- učebnice vysokých škol
Human transcription co-regulator SNW1/SKIP is implicated in the regulation of both transcription elongation and alternative splicing. Prp45, the SNW/SKIP ortholog in yeast, is assumed to be essential for pre-mRNA processing. Here, we characterize prp45(1-169), a temperature sensitive allele of PRP45, which at permissive temperature elicits cell division defects and hypersensitivity to microtubule inhibitors. Using a synthetic lethality screen, we found that prp45(1-169) genetically interacts with alleles of NTC members SYF1, CLF1/SYF3, NTC20, and CEF1, and 2nd step splicing factors SLU7, PRP17, PRP18, and PRP22. Cwc2-associated spliceosomal complexes purified from prp45(1-169) cells showed decreased stoichiometry of Prp22, suggesting its deranged interaction with the spliceosome. In vivo splicing assays in prp45(1-169) cells revealed that branch point mutants accumulated more pre-mRNA whereas 5' and 3' splice site mutants showed elevated levels of lariat-exon intermediate as compared to wild-type cells. Splicing of canonical intron was unimpeded. Notably, the expression of Prp45(119-379) in prp45(1-169) cells restored Prp22 partition in the Cwc2-pulldowns and rescued temperature sensitivity and splicing phenotype of prp45(1-169) strain. Our data suggest that Prp45 contributes, in part through its interaction with the 2nd step-proofreading helicase Prp22, to splicing efficiency of substrates non-conforming to the consensus. 2008 Wiley-Liss, Inc.
- MeSH
- alely MeSH
- DEAD-box RNA-helikasy genetika metabolismus MeSH
- fenotyp MeSH
- financování organizované MeSH
- introny MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- prekurzory RNA metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sestřih RNA MeSH
- spliceozomy metabolismus MeSH
In this review protocols are described for studying protein tyrosine kinase signalling upon integrin-mediated cell adhesion. We have outlined detailed procedures for fibronectin-replating experiment, biochemical examination of the phosphotyrosine content of cellular proteins by immunoblotting using phosphorylation-specific antibodies or immunoprecipitation and analysis with general phosphotyrosine antibodies. Despite great advances that were made toward optimizing the described procedures, all these methods still remain in many respects an art, given the plentiful of variables and the extent to which the optimum conditions vary from one experimental condition to the other. Examples of performed experiments using the described procedures thus also include notes regarding variability of approaches based on experimental conditions.
In order to re-evaluate the presence and relative quantity of 2b and 2x/d myosin heavy chain (MyHC) transcripts in rat slow soleus muscle by using real time RT-PCR we have compared the available relevant cDNA sequences and designed a new set of primers having similar melting temperatures, matching separate MyHC exons in the regions of maximal differences in MyHC coding sequences, and containing G or C at the 3´-end. These also yielded PCR products of corresponding length, which is an important requirement for real time RT-PCR quantification. The experiments were performed on 8-month-old inbred female Lewis strain rats used in our current study of regenerating transplanted muscles. The real time RT-PCR measurement confirmed the expression of all four MyHC mRNAs (type 1, 2a, 2x/d and 2b) in both fast extensor digitorum longus and slow soleus muscles, although in the soleus muscle of adult rats, only type 1 and 2a protein isoforms can be usually detected.
- Klíčová slova
- Rat slow soleus muscle, Rat fast EDL muscle, Myosin heavy chain isoforms, Real time RT-PCR,
- MeSH
- DNA primery MeSH
- financování organizované MeSH
- kosterní svaly chemie MeSH
- krysa rodu rattus MeSH
- messenger RNA analýza MeSH
- polymerázová řetězová reakce MeSH
- potkani inbrední LEW MeSH
- protein - isoformy MeSH
- svalová vlákna typu I chemie MeSH
- svalová vlákna typu II chemie MeSH
- těžké řetězce myosinu genetika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDSPAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDITOF MS) and sequenced by Liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified.
- MeSH
- financování organizované využití MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody využití MeSH
- interpretace statistických dat MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody využití MeSH
- potkani inbrední LEW fyziologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody využití MeSH
- těžké řetězce myosinu fyziologie izolace a purifikace MeSH
- MeSH
- DNA vazebné proteiny fyziologie MeSH
- finanční podpora výzkumu jako téma MeSH
- genetická transkripce imunologie MeSH
- jaderné proteiny fyziologie MeSH
- lidé MeSH
- regulace genové exprese MeSH
- signální transdukce MeSH
- trans-aktivátory fyziologie genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
