The heterogeneous nuclear ribonucleoprotein (hnRNP) type I, a modulator of alternative splicing, localizes in the nucleoplasm of mammalian cells and in a discrete perinucleolar structure. HnRNP I contains a novel type of bipartite nuclear localization signal (NLS) at the N-terminus of the protein that we have previously named nuclear determinant localization type I (NLD-I). Recently, a neural counterpart of hnRNP I has been identified that contains a putative NLS with two strings of basic amino acids separated by a spacer of 30 residues. In the present study we show that the neural hnRNP I NLS is necessary and sufficient for nuclear localization and represents a variant of the novel bipartite NLS present in the NLD-I domain. Furthermore, we demonstrate that the NLD-I is transported into the nucleus by cytoplasmic factor(s) with active transport modality. Binding assays using recombinant importin a show an interaction with NLD-I similar to that of SV40 large T antigen NLS. Deletion analysis indicates that both stretches of basic residues are necessary for binding to importin a. The above experimental results lead to the conclusion that importin a acts as cytoplasmic receptor for proteins characterized by a bipartite NLS signal that extends up to 37 residues.Keywords: heterogeneous ribonucleoprotein-I; polypyrimidine tract-binding protein; PTB; nuclear localization signal; importin a.Transport of proteins and RNA into and out of the nucleus occurs through nuclear pore complexes (NPCs), which are plugged through the double membrane of the nuclear envelope [1,2]. Small molecules and ions may pass the NPC passively, while macromolecules larger than 40-45 kDa are actively transported through the NPC. The active nuclear import and export of proteins is mediated by specific aminoacid sequences that are referred as nuclear localization signals (NLSs) [3,4] and nuclear export signal (NESs) [5]. At least two different types of ÔclassicalÕ NLSs have been defined: a short stretch of basic amino acids, exemplified by the SV40 large T antigen NLS (T-ag PKKKRKV) [6] and a bipartite NLS composed of two stretches of basic amino acids separated by a spacer of 10-12 amino acids, exemplified by nucleoplasmin (KRPAATKKAGQAKKKK). The two sets of basic residues of bipartite-type NLS are required for sufficient nuclear localization, while the spacer is mutant-tolerant in sequence [7]. NLSs are usually recognized by the heterodimeric import receptor complex comprising importins a and b, also named karyopherins [8,9]. Importins a contain the NLS-binding site and importins b are responsible for the docking of the importin-substrate complex to the cytoplasmic side of the NPC and its subsequent translocation through the pore. Transfer through the pore of importin-NLS protein complex requires two additional soluble proteins, RanGTPase and nuclear transport factor-2 (NTF2) [1]. Once inside the nucleus, Ran-GTP binding to importin b causes the dissociation of the import complex and release of the cargo [10,11]. The directionality of the nuclear...
Edited by Ulrike KutayKeywords: RBM20 DCM Alternative splicing Nuclear localization Ribonucleoprotein SR a b s t r a c t RBM20 is a nuclear protein which regulates alternative splicing of expressed genes that have a key role in cardiac function. By cloning the human and mouse RBM20 cDNA, producing expressing vectors for truncated proteins, and comparing their sub-cellular distribution in transfected cells, we have identified the sequences necessary for RBM20 full nuclear retention. The region overlaps an RNA binding motif and a serine-arginine domain. The sequence is conserved in many species but belongs only to RBM20 orthologs. The RMB20 tissue specificity, together with the properties of its nuclear localization determinant, demonstrates a specific evolutionary selection of post-transcriptional regulation factors.
We explored APC gene mutations and chromosome 5q21 allelic losses (5qLOH) in 18 neoplasms of the papilla of Vater, including 6 early-stage tumours (3 adenomas, 3 carcinomas) and I 2 advanced-stage cancers. Eleven PCR-amplified polymorphic sequences were used to analyse 5qLOH. AfK mutations were investigated both by an In vitro APC-protein truncation test and by single-strand conformation polymorphism analysis. Mutations in the Ki-ras, N-ras and p53 genes were also assessed. We found: 5qLOH in 8 of 16 cases (50%), including I adenoma. 3 early-and 4 advanced-stage cancers; APC mutations in 2 adenomas and I advanced-stage carcinoma; Ki-or N-ras mutations in 3 adenomas and 3 advanced-stage cancers; pS3 mutations in 2 early-stage and 7 advanced-stage adenocarcinomas. Our results suggest that SqLOH, APC mutations and ras mutations are present at early stages, whereas p53 inactivation is associated with progression of malignancy in a large proportion of cases. These data indicate that sporadic ampullary tumours differ from those occurring in familial adenomatous polyposis in the frequency (I 7% vs. 64Oh) as well as in the site of APC somatic mutations, suggesting a different molecular pathogenesis in the 2 conditions. cri 1906 Wilq-Liss, Inc.Ampullary epithelial neoplasms include benign (5%) and malignant tumours (95%) centered in the region of the papilla of Vater. They represent 5% of all sporadic gastrointestinal tumours, but account for up to 36% of the surgically operable pancreatoduodenal tumours, whereas sporadic duodenal neoplasms not originating from ampullary structures arc exccedingly rare. Ampullary and duodenal non-ampullary neoplasms occur at a high frequency in patients affected by familial adenomatous polyposis (FAP) (reviewed in Gallinger et a/.,
In the present paper we report the exclusive microbial preparation of polyhydroxyalkanoates (PHA) containing 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB) as comonomers through the use of unexpensive carbon sources such as whey from dairy industry. Polymers were produced by growing H. pseudoflava DSM 1034 in minimal medium supplemented with sucrose, lactose or whey without any co-substrate added. The chemical and physical properties of the polymers were fully characterized by GPC, DSC, TGA analyses and the composition by GC and (1)H NMR examinations to especially confirm the content of different monomeric units. The presence of 4HB units into PHA samples is particularly aimed in thermoplastic applications where greater flexibility is required and conventional rigid PHAs tend to fail. Usually the insertion of 4HB into chain backbone consisting of 3-hydroxyalkanoates requires expensive carbon sources mostly of petrochemical origin. According to our study the production of P(3HB-co-3HV-co-4HB) terpolymer can be obtained directly by the use of lactose or waste raw materials such as cheese whey as carbon sources. Although the amount of 4HB in the produced terpolymers was usually low and not exceeding 10% of the total molar composition, a PHA containing 18.4% of 4HB units was produced in 1 step fermentation process from this structurally unrelated carbon sources. The crystallinity of the terpolymer is basically to be markedly affected with respect to that of conventional PHAs, thus obtaining a comparatively less rigid material and easier to be processed.
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