Biomedical subjects
Publications and source records attributed to V K Parnaik.
The resolution of the standard micrococcal nuclease assay for sensitivity of active chromatin has been enhanced by the inclusion of an additional step of digesting nuclease-digested DNA with a suitable restriction enzyme prior to Southern hybridization. The improved assay has been used to analyze the chromatin structure of the lamin A, albumin and alpha-fetoprotein genes during rat liver development.
We have studied the regulation of expression of the A-type lamins, which are constituents of the nuclear lamina. During rat liver development, high levels of lamin A and C mRNAs were observed in 15-day fetal liver but were barely detectable in the adult. The chromatin conformation of the lamin A gene was sensitive to DNase I in 15-day fetal liver but became mostly insensitive in the adult. Lamin A and C proteins could be detected in fetal liver and persisted in the adult. Our evidence suggests that the lamin A gene is actively transcribed early in liver differentiation and its activity declines considerably in adult liver.
Nucleocytoplasmic transport of proteins occurs through pores embedded in the nuclear envelope. Recent studies have defined the cytoplasmic factors required for signal-mediated import of nuclear proteins. Considerable progress has been made in understanding the mechanism of nuclear export of proteins by the identification of specific signal sequences needed for export. Regulatory molecules have been shown to adopt novel mechanisms to control their entry into the nucleus and thereby regulate their functions.
We have investigated a possible role for protein phosphorylation in nuclear transport in semi-intact cells, prepared by digitonin permeabilization of rat F-111 fibroblasts. Treatment of semi-intact cells with alkaline phosphatase abolished the import of nuclear transport substrates, namely, signal peptide-albumin conjugates, as well as their signal-dependent binding at the nuclear pores, but did not affect the morphology of the cells, in particular their cytoskeletal network. Authentic transport and functional binding of the karyophilic protein at the nuclear envelope could be restored by incubation of phosphatase-treated cells with cytosol enriched in protein kinase C or with purified protein kinase A (catalytic subunit). Restoration of transport was blocked by specific inhibitors of these kinases. Since the protein phosphorylation required for nuclear transport appeared to be a reasonably stable modification, characterization of the phosphorylated proteins was attempted in kinase reactions with radiolabeled ATP. Two proteins of 60-62 kDa were the predominant substrates phosphorylated by both protein kinase C and protein kinase A under conditions wherein nuclear transport was restored. Our results suggest a requirement for phosphorylation of one or more proteins for binding of a karyophilic protein at the nuclear envelope.
Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.
The 5'-most open reading frame of the c.4kb RNA-2 of Indian peanut clump furovirus (IPCV) encodes a protein of 208 amino acids. This protein is thought to be the coat protein of IPCV because its amino acid composition and M(r) closely resemble those reported for IPCV coat protein and because its amino acid sequence is 61% identical to that of the coat protein of peanut clump virus (PCV) from West Africa. The extent of the sequence identity between IPCV and PCV coat proteins confirms previous conclusions that the viruses are distinct rather than strains of one virus. The sequences of the coat proteins of IPCV and PCV were between 18% and 26% identical to those of other furoviruses and those of unrelated tobamoviruses and tobraviruses. In contrast, the coat protein sequences were 37% (IPCV) and 36% (PCV) identical to that of the coat protein of barley stripe mosaic hordeivirus (BSMV). This similarity between the coat proteins of viruses from different groups (= genera) is unusual but is consistent with previous reports of sequence relatedness in various genes between certain furoviruses and BSMV.
The nuclear pore complexes mediate the selective nuclear import of proteins in a signal- and energy-dependent process. We have earlier reported the characterization of a monoclonal antibody, Mab E2, that recognizes a novel class of nuclear pore phosphoproteins involved in signal-binding and protein transport. In the present study, we have analyzed the pattern of immunoreactivity of Mab E2 in cultured rat fibroblasts and have observed significant differences in the expression of epitopes in proliferating and quiescent cells. Furthermore, the common epitope recognized by Mab E2 is conserved across species, consistent with its essential role in nuclear protein import.
The possibility that interaction of the nuclear localization signal (NLS) with its pore receptor may directly stimulate nuclear envelope-associated ATPase activity and consequently provide energy for protein translocation across the pore has been studied. ATPase activity was assayed after cross-linking of the prototype NLS peptide with its pore receptor, or after preincubation of envelopes with NLS-albumin conjugates. Neither treatment enhanced enzyme activity. A more complex series of events may be required for energy-generation at the nuclear pore.
The SV40 large T antigen transcripts from an SV40-transformed mouse embryo fibroblast cell line (215) have been analyzed by Northern blots and by mapping and sequencing of the corresponding cDNAs. We have observed that T antigen mRNA is highly overexpressed in the 215 cells, and is mostly unspliced, but there are no sequence changes at the splice sites. However, normal amounts of wild-type T antigen are produced, suggesting that the splicing and translation of T antigen RNA is tightly controlled in these cells.
We have compared the organization of the nuclear lamina in adult and fetal mouse liver. Western blot analysis of the expression of lamins with specific antibodies indicates that lamin B is expressed throughout liver development, unlike lamins A and C which are absent in fetal liver. Using [125I]lamin in blot binding assays, we have observed that lamin B binds to at least three membrane proteins (96, 54 and 34 kDa) and to lamins A and C in adult nuclear envelopes, but only to the 54 and 34 kDa proteins and lamin B itself in fetal nuclear envelopes, where lamin B appears to be hyperphosphorylated.
A radioiodinated, photoactivable synthetic nonapeptide corresponding to the nuclear location signal (NLS) of SV40 large T antigen has been used in photolabelling reactions with purified mouse liver nuclei, nuclear envelopes and other cellular fractions, to identify specific NLS-binding proteins which may be involved in selective transport of karyophilic proteins. SDS-polyacrylamide gel analysis of photolabelled products demonstrates that a 60 kDa nuclear protein and four nuclear envelope proteins (67, 60, 53 and 47 kDa) bind specifically to the native NLS and not to a mutant NLS or unrelated sequences. This binding shows saturation kinetics, with highest affinity of the NLS for the 60 and 67 kDa proteins. The nuclear 60 kDa NLS-binding protein is identical to the nuclear envelope 60 kDa NLS-binding protein by two-dimensional gel analysis of labelled proteins. Biochemical fractionation of labelled nuclear envelopes suggests that the 53 and 47 kDa proteins are peripheral membrane proteins whereas the 67 and 60 kDa proteins can be localized to the pore complex. The NLS also binds to solubilized 67, 60, 53 and 47 kDa proteins but with decreased affinity. Our results suggest that one of the early steps in selective nuclear transport of proteins may be the recognition of the NLS by the 60 kDa and/or 67 kDa binding proteins present in the nuclear pore complex.
A sensitive and versatile assay is described for the nuclear transport of 35S-labeled proteins obtained by the in vitro translation of SP6 plasmid-generated mRNAs. A specific nuclear accumulation of greater than 20-fold is observed for the transformation-related nuclear proteins, p53 and E1b, and the nuclear enzyme, thymidine kinase, whereas transport of the nonnuclear proteins, dihydrofolate reductase and simian virus 40 small t antigen, is negligible within 30 min.
A sensitive technique is described for the rapid identification of nuclear-envelope proteins. Mouse liver nuclei (purified on sucrose gradients) were iodinated with Na125I by the immobilized water-insoluble reagent Iodogen. Iodinated nuclei were digested with RNAase A and DNAase I and then salt-extracted to obtain labelled nuclear envelopes. Nuclear envelopes were characterized by morphological and biochemical criteria and by SDS/polyacrylamide-gel electrophoresis. In all, 13 polypeptides of molecular masses 145, 115, 98, 85, 75, 70, 65, 54, 50, 45, 40, 38 and 36 kDa were identified in the labelled nuclear envelopes. The labelled polypeptides were localized to the nuclear envelope by extraction of the envelope with Triton X-100 and different concentrations of salt. Iodination of intact nuclei was shown to be specific for the nuclear envelope by the absence of labelling of histones and cytoplasmic contaminants.
We have analyzed the DNAase I sensitivity of the mouse alpha-fetoprotein and albumin structural genes from fetal liver, adult liver and kidney. The albumin gene shows distinct hypersensitive sites in adult liver in addition to an overall DNAase I sensitivity, but is only slightly nuclease-sensitive in fetal liver. The alpha-fetoprotein gene does not show hypersensitive sites but displays an overall DNAase I sensitivity in fetal liver; however, it is nuclease-insensitive in adult liver. Both genes are insensitive to DNAase I in kidney. The presence of DNAase-I-hypersensitive sites in the albumin structural gene correlates with extensive demethylation of the gene in adult liver.
The effect of Z-conformation of DNA on its template activity in DNA synthesis reactions in vitro has been studied. Normal poly(dG-dC) in the B-form, brominated and unbrominated in the Z-form have been compared for their template activity in DNA synthesis reactions mediated by AMV DNA polymerase and E. coli DNA polymerase I. The results indicate that poly(dG-dC) in the Z-form is totally inactive as a template for DNA synthesis and further that it is a strong competitive inhibitor of copying of the B-form DNA.
Dextransucrase was treated with [14C]sucrose, and the product applied to gel-permeation columns. In the absence of the detergents SDS and Triton X-100, poor recovery of enzyme was observed; however, that enzyme which was recovered was labeled. In the presence of detergents, recovery was increased, but the material appeared to be a large aggregate (mol. wt. greater than 5 X 10(6) ). In addition, the ratio of D-glucose to enzyme suggested that a polymer had been formed. Disc-gel electrophoresis in the presence of a mixture of SDS and Triton X-100 showed similar results, and indicated that the aggregate was disrupted upon treatment with dextranase. Native enzyme that had been immobilized on hydroxylapatite could also be labeled with [14C]sucrose, and the labeling followed saturation kinetics. The labeled protein could be released from the gel with 8M urea, but was aggregated. Radioactive sugars, free from protein, could be released by heating the labeled enzyme. The sugars released consisted of a mixture of D-glucose with oligosaccharides having an average chain-length of 17 D-glucosyl residues. The significance of these observations is discussed.
A D-glucosylated form of dextransucrase, whose preparation and characteristics have just been reported in Carbohydr. Res., was employed in a series of studies designed to explore the question of whether the bound sugars participate in the reactions catalyzed by the enzyme. When exposed to maltose, a good acceptor-substrate, monomeric D-glucosyl groups were rapidly transferred to the disaccharide, affording a trisaccharide. In the absence of an acceptor, monomeric D-glucose was released from the enzyme by hydrolysis. In a reaction with D-fructose, the charged enzyme catalyzed the formation of sucrose. Finally, in the presence of unlabeled sucrose, monomeric D-glucosyl groups were chased into enzyme-associated oligomers. Evidence is also presented which indicates that the various pathways for the bound D-glucosyl groups are competitive. The significance of these observations is discussed.
The kinetics of copying of poly (A)--oligo (dT) and poly (C)--oligo (dG) by reverse transcriptase from avian myeloblastosis virus have been studied, and binding affinity of enzyme for template-primer and primer alone have been determined separately. Although the maximal rate of DNA synthesis obtained with poly (C)-oligo (dG) is higher than that for poly (A)-oligo (dT), the binding affinity of the enzyme for poly (C)-oligo (dG) or oligo (dG) is considerably lower than that for poly (A)--oligo (dT) or oligo (dT). Hence, for the more efficient template, poly (C)--oligo (dG), both template-primer and primer bind less tightly to the enzyme.