[PubMed] [Google Scholar] 57. clone (NS1-associated protein 1 [NSAP1]) that interacts with NS11C276 and NS11C638. An additional sequence was predicted from human EST (expressed sequence tag) data, and the cDNA was estimated to be at least 2,221 bp long, potentially encoding a 562-amino-acid protein product. A polyclonal antibody raised to a synthetic peptide within NSAP1 recognizes an 65-kDa cellular protein. This NSAP1 cDNA has not previously been characterized, but the predicted protein Rabbit polyclonal to SP3 sequence is usually 80% identical to the recently recognized heterogeneous nuclear ribonucleoprotein (hnRNP) R (W. Hassfeld et al., Nucleic Acids Res. 26:439C445, 1998). NSAP1 contains four ribonucleoprotein domains, as well as a highly repetitive C-terminal region. A closely related mouse cDNA (deduced from murine EST data) GSK2838232A encodes a protein with only a single amino acid residue change from the human protein. NSAP1 is predicted to be a 65-kDa polynucleotide binding protein, and it likely functions in the regulation of splicing and/or transport of mRNAs from your nucleus. Minute computer virus of mice (MVM) is an autonomously replicating parvovirus. MVM has a small, single-stranded, negative-sense DNA genome of 5,149 nucleotides (nt) with nonidentical terminal palindromic hairpins. Replication is dependent on its major GSK2838232A nonstructural protein, NS1, a multifunctional 83-kDa nuclear phosphoprotein. NS1 supplied in allows replication of MVM minigenomes which include only the viral hairpins and an essential DH5 (Life Technologies) or SURE (Stratagene) strains. Bacteria were transformed by electroporation (Bio-Rad). Plasmids for the transcription transactivation test have been explained previously (47). pSG424 encodes the GAL4 DNA-binding domain name (GAL4DB) from your SV40 early promoter. This vector was used to produce GAL4DB-NS1 constructs. pG5BCAT contains five tandem GAL4 specific 17-mers upstream of a TATA box directing the expression of a CAT gene. Yeast plasmids GSK2838232A required for the two-hybrid selection method were generously provided by the Brent laboratory (21), and detailed information is available from your Massachusetts General Hospital Molecular Biology Internet Gopher server (http://xanadu.mgh.harvard.edu/brentlabweb/). pLexA (pEG202) baits were constructed from pEG202 by cloning the desired sequence in frame with LexA (202 aa, including the DNA binding and dimerization domains). LexA-NS1 junctions were sequenced by using the Sequenase kit (USB). Fusions are expressed from your strong constitutive promoter. The reporter pLexAop-lacZ (pSH18-34) was created by inserting four (reporter gene with glucose- and galactose-responsive elements deleted. pLexA-GAL4TA (pSH17-4) is usually a positive control encoding the DNA binding LexA1C87 fused to the activator GAL474C881 expressed from your promoter. pJG4-5 is usually a vector utilized for the HeLa-acid cDNA library of fusion proteins. The HeLa cDNA clones are fused to a sequence encoding the acidic B42 activator, the SV40 nuclear localization signal, and the hemagglutinin epitope. The HeLa-acid fusion proteins are expressed from your galactose-induced promoter. This promoter is usually repressed by glucose. pGAL4TA-NS1 (pPCNS1) is usually a plasmid that encodes a GAL4TA-NS1 fusion expressed from your constitutive promoter. The plasmid was created by cloning the entire coding sequence of NS1 in frame into pPC86 (7). Screening of baits. Before the baits can be used in the GSK2838232A two-hybrid system, they must be tested to ensure that they are expressed, are transcriptionally inert, and can bind to the in the yeast nucleus. A Western blot was performed to confirm that each bait plasmid expressed a LexA-NS1 fusion. A mouse -LexA monoclonal antibody (MAb; Clontech) was used to detect each hybrid. LexA fusions of approximately the expected size.