Fibronectin-replating experiment: procedure and analysis.

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.

Real time RT-PCR with a newly designed set of primers confirmed the presence of 2b and 2x/d myosin heavy chain mRNAs in the rat slow soleus muscle.

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.

Mass spectrometry analyses of rat 2b myosin heavy chain isoform.

We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDS-PAGE 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 (MALDI-TOF 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.

Cyclophilins of a novel subfamily interact with SNW/SKIP coregulator in Dictyostelium discoideum and Schizosaccharomyces pombe.

We screened the Dictyostelium discoideum two-hybrid cDNA library with the SNW/SKIP transcription coregulator SnwA and identified a novel cyclophilin CypE. Independently, the Schizosaccharomyces pombe cDNA library was screened with the SnwA ortholog Snw1 and the ortholog of CypE (named Cyp2) was found. Both cyclophilins bind the respective SNW protein in their autologous systems. The interaction was localized to the N-terminal part of SnwA as well as of Snw1. CypE was confirmed in vitro to be a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase. Remarkably, both SNW proteins bind the cyclophilins in a cyclosporin A independent manner, possibly serving as adaptors for these novel isomerases. These results are the first characterization of the members of a novel cyclophilin subfamily, which includes the human CGI-124/PPIL1 protein.

The homolog of chromatin binding protein Bx42 identified in Dictyostelium.

We identified in Dictyostelium a gene snwA containing a region of similarity to SH2 domains of higher eukaryotes. snwA is homologous to a novel human gene SNW1 and to Bx42 from Drosophila melanogaster, a gene coding for a chromatin binding protein responsive to 20-OH-ecdysone. snwA has one mRNA transcript of an approximate size of 2.5 kb.

Molecular characterization of a calmodulin-like dictyostelium protein CalB.

A gene named calB was cloned and characterized in Dictyostelium. A relationship to calmodulin (CaM) is suggested by sequence identity (50%), similar exon-intron structure and cross-reactivity with anti-CaM sera. The level of calB mRNA is developmentally regulated with maxima during aggregation and in spores. CalB null cells grow normally, develop and produce viable spores. We demonstrated the capacity of tagged CalB to bind Ca(2+) using the (45)Ca(2+) overlay assay and showed that its mobility on SDS-PAGE is dependent on Ca(2+)/EGTA pretreatment.

Blasticidin resistance cassette in symmetrical polylinkers for insertional inactivation of genes in Dictyostelium.

In Dictyostelium discoideum inactivation of developmentally regulated genes via homologous recombination has become an important tool in studying systematically the entire developmental program of this model organism. The Dictyostelium genome is very A/T-rich,which presents obstacles to the preparation of knockout constructs. The coding regions offer few suitable restriction sites and the low complexity intergenic regions do not guarantee specificity of recombination. We present here the preparation of plasmids pBsR479, pBsR503, and pBsR519, in which a blasticidin resistance-cassette is positioned in the center of various symmetrical polylinkers. This design simplifies the cloning process and gives more flexibility in positioning the selectable marker within the coding regions.

Expression of protein tyrosine kinase pp60v-src variants in Dictyostelium discoideum.

We achieved production of v-Src of the low-oncogenic PRC and its variant proviral structure H19 in Dictyostelium discoideum, an emerging host system suitable for synthesis of heterologous proteins. To accomplish their expression, the first six codons of the N-terminus of v-src had to be changed according to the D. discoideum codon preference. Alternatively, N-terminal fusions of 6xHis-tag or GFP were sufficient to overcome the incompatibility in codon usage. D. discoideum-expressed v-Src kinases of the expected molecular weight were recognized by Src-specific antibodies; GFP-PRC was distributed uniformly in the cytosol. In contrast to other lower eukaryotes, where the accumulation of v-Src leads to growth inhibition, D. discoideum cells silenced the kinase activity of PRC-derived v-Src and showed no developmental or growth defects.

Construction of a new Escherichia coli-Saccharomyces cerevisiae shuttle plasmid cloning vector allowing positive selection for cloned fragments.

A new E. coli-S. cerevisiae shuttle plasmid cloning vector (pPW263) with a positive type of selection, was constructed. The selection system, based on the regulatory region of lambda phage controlling the expression of tetracycline resistance, was derived from the cloning vector pUN121 (Nilsson et al. 1983). There are three cloning sites in the cI gene, EcoRI, HindIII and BglII, and, in addition, two unique sites in the neighborhood, BamHI and SalI. The size of the vector is 7.8 kb. The maintenance of the vector and the selection in yeast was ensured by the replication region of the 2 mu plasmid and by the URA3 marker gene, respectively.

Direct selection shuttle plasmid vector, pPW264, used for cloning the alpha-amylase gene of Schwanniomyces occidentalis.

We constructed a novel cloning system with positive selection for inserted fragments. The gene for tetracycline resistance (tetR) originally used in plasmid pTR262 was replaced with the gene for chloramphenicol acetyltransferase (cat) and terminator sequences were introduced downstream of the cat gene. The terminator sequences stop transcription originating on strong PR promoter that would otherwise proceed through the region of replication origin and interfere with plasmid replication. Thus the copy number of recombinant plasmid molecules is stabilized. The cloning system has been constructed in a new YEp type shuttle vector, pPW264. The 8.1 kb-vector carries two unique cloning sites, BglII and HindIII. The maintenance of the vector and selection in yeast is ensured by URA3 Saccharomyces cerevisiae gene. The vector was employed in cloning of the gene for alpha-amylase from Schwanniomyces occidentalis.

Transcriptional coregulator SNW/SKIP: the concealed tie of dissimilar pathways.

Eukaryotic gene expression requires that all the steps of messenger RNA production are regulated in concert to integrate the diverse inputs cells receive. We discuss the functioning of SNW/SKIP, an essential spliceosomal component and transcriptional coregulator, which may provide regulatory coupling of transcription initiation and splicing. SNW/SKIP potentiates the activity of important transcription factors, such as vitamin D receptor, CBF1 (RBP-Jkappa), Smad2/3, and MyoD. It synergizes with Ski in overcoming pRb-mediated cell cycle arrest, and it is targeted by the viral transactivators EBNA2 and E7. SNW/SKIP may aid in conformational transition of the gene expression machine through its avidity to nuclear matrix fractions or by recruiting foldases such as the prolyl isomerase PPIL1. The extensive list of SNW/SKIP partners, its unique primary structure, conserved from yeast to humans, and its essential character suggest a distinct function of general importance.

The fission yeast ortholog of the coregulator SKIP interacts with the small subunit of U2AF.

The mode of action of transcriptional coregulators may involve the recruitment of spliceosome components. Using the two-hybrid screen, we examined the interaction partners of spSNW1, the S. pombe ortholog of the human coregulator SNW1/SKIP/NCoA-62, and found it to interact with the small subunit of the splicing factor U2AF (spU2AF23). The interaction involves the C-terminal parts of spU2AF23 and spSNW1. Tagged variants of both proteins were expressed in S. pombe and the interaction was proved by coprecipitation in nuclear extracts. This interaction would explain the finding of SKIP in nuclear speckles (Mintz, P. J., et al., EMBO J. 18, 4308-4320, 1999) and in reconstituted spliceosomes (Neubauer, G., et al., Nat. Genet. 20, 46-50, 1998). We deleted the spSNW1 gene in the diploid strain and demonstrated that spSNW1 is an essential gene in S. pombe.