Abstract of related literatures |
1. The Y-box factors interact specifically with both DNA and RNA. Biologically they have roles in both transcriptional and translational regulation. Conserved through evolution from prokaryotic to eukaryotic organisms they represent a new family of nucleic acid binding proteins. PMID: [1622927]
2. We have isolated a cDNA that encodes the murine CCAAT-binding protein mYB-1. The deduced amino acid sequence shows 95% identity with its presumed human homologue (hYB-1A) which was originally isolated as a protein that binds to the Y box of MHC class II genes. In vitro translated mYB-1 binds to CCAAT boxes of the MHCIIE alpha, HSVTK and mouse PCNA promoters but not to alpha-globin or human thymidine kinase CCAAT boxes. Interestingly, complexes formed between the in vitro translated protein and the various CCAAT boxes display the property of being competed more efficiently with self competitor DNA, regardless of the CCAAT box initially used as a probe. A similar phenomenon was observed in a cell extract of Con-A stimulated murine splenocytes when the same competition assays were performed. These results may reflect the generation of multiple forms of a particular CCAAT-binding protein, such as mYB-1, that display distinct, yet overlapping, DNA binding specificities. PMID: [1741293]
3. We have isolated a mouse cDNA clone, which encodes the protein MSY1 (mouse Y box protein 1), a new member of the Y box family of proteins. Northern analysis indicates that MSY1 mRNA accumulates over 100-fold more in testis than in other tissues. Moreover, MSY1 mRNA is developmentally regulated, initially appearing at the pachytene stage of spermatogenesis. This is the stage of maximal transcription and translation in the spermatocyte. In Xenopus laevis, homologous Y box proteins, FRGY1 and FRGY2, positively regulate transcription from promoters containing a Y box (CTGATTGGCCAA). In addition, the germ cell-specific Y box protein FRGY2 binds maternal mRNA within 60-80 S mRNP storage particles and in doing so regulates translation in the developing oocyte and embryo (Smith, L. D., Richter, J. D., and Taylor, M. A. (1984) in Molecular Biology of Development (Davidson, E. R., and Firtel, R. A., eds) pp. 129-141, Alan R. Liss, New York). The MSY1 protein can be isolated from a 60-80 S mRNP fraction of testis which like the frog oocyte contains stored, untranslated mRNAs. Furthermore, cross-linking experiments demonstrate that MSY1 is bound to mRNAs of this fraction. Finally, mobility shift analysis performed using the isolated protein indicates that MSY1 has nucleic acid binding properties similar to those of the FRGY proteins. These data suggest that the mouse Y box protein, MSY1, functions similarly to the FRGY2 protein in regulating the storage and translation of germ cell RNAs. PMID: [8505341]
4. The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID: [15489334]
5. Transcriptional activity of the mouse vascular smooth muscle alpha-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Puralpha and Purbeta (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997) J. Biol. Chem. 272, 26727-26733). In this study, we demonstrate that Puralpha and Purbeta interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Puralpha, Purbeta, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle alpha-actin MCAT enhancer in fibroblasts. PMID: [10318844]
6. A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation. PMID: [17203969]
7. Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a "dipolar" motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation. PMID: [17242355]
8. Kinases play a prominent role in tumor development, pointing to the presence of specific phosphorylation patterns in tumor tissues. Here, we investigate whether recently developed high resolution mass spectrometric (MS) methods for proteome and phosphoproteome analysis can also be applied to solid tumors. As tumor model, we used TG3 mutant mice carrying skin melanomas. At total of 100 microg of solid tumor lysate yielded a melanoma proteome of 4443 identified proteins, including at least 88 putative melanoma markers previously found by cDNA microarray technology. Analysis of 2 mg of lysate from dissected melanoma with titansphere chromatography and 8 mg with strong cation exchange together resulted in the identification of more than 5600 phosphorylation sites on 2250 proteins. The phosphoproteome included many hits from pathways important in melanoma. One-month storage at -80 degrees C did not significantly decrease the number of identified phosphorylation sites. Thus, solid tumor can be analyzed by MS-based proteomics with similar efficiency as cell culture models and in amounts compatible with biopsies. PMID: [19367708]
9. Cleavage of histone pre-mRNAs at the 3' end is guided by the U7 snRNP, which is a component of a larger 3'-end processing complex. To identify other components of this complex, we isolated proteins that stably associate with a fragment of histone pre-mRNA containing all necessary processing elements and a biotin affinity tag at the 5' end. Among the isolated proteins, we identified three well-characterized processing factors: the stem-loop binding protein (SLBP), which interacts with the stem-loop structure upstream of the cleavage site, and both Lsm11 and SmB, which are components of the U7-specific Sm ring. We also identified 3'hExo/Eri-1, a multifunctional 3' exonuclease that is known to trim the 3' end of 5.8S rRNA. 3'hExo primarily binds to the downstream portion of the stem-loop structure in mature histone mRNA, with the upstream portion being occupied by SLBP. The two proteins bind their respective RNA sites in a cooperative manner, and 3'hExo can recruit SLBP to a mutant stem-loop that itself does not interact with SLBP. UV-cross-linking studies used to characterize interactions within the processing complex demonstrated that 3'hExo also interacts in a U7-dependent manner with unprocessed histone pre-mRNA. However, this interaction is not required for the cleavage reaction. The region between the cleavage site and the U7-binding site interacts with three low-molecular-weight proteins, which were identified as components of the U7-specific Sm core: SmB, SmD3, and Lsm10. These proteins likely rigidify the substrate and function as the molecular ruler in determining the site of cleavage. PMID: [19470752]
10. We used on-line electron capture dissociation (ECD) for the large scale identification and localization of sites of phosphorylation. Each FT-ICR ECD event was paired with a linear ion trap collision-induced dissociation (CID) event, allowing a direct comparison of the relative merits of ECD and CID for phosphopeptide identification and site localization. Linear ion trap CID was shown to be most efficient for phosphopeptide identification, whereas FT-ICR ECD was superior for localization of sites of phosphorylation. The combination of confident CID and ECD identification and confident CID and ECD localization is particularly valuable in cases where a phosphopeptide is identified just once within a phosphoproteomics experiment. PMID: [19131326]
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