Overexpression of a DEAD box protein (DDX1) in neuroblastoma and retinoblastoma cell lines.

The DEAD box gene, DDX1, is a putative RNA helicase that is co-amplified with MYCN in a subset of retinoblastoma (RB) and neuroblastoma (NB) tumors and cell lines. Although gene amplification usually involves hundreds to thousands of kilobase pairs of DNA, a number of studies suggest that co-amplified genes are only overexpressed if they provide a selective advantage to the cells in which they are amplified. Here, we further characterize DDX1 by identifying its putative transcription and translation initiation sites. We analyze DDX1 protein levels in MYCN/DDX1-amplified NB and RB cell lines using polyclonal antibodies specific to DDX1 and show that there is a good correlation with DDX1 gene copy number, DDX1 transcript levels, and DDX1 protein levels in all cell lines studied. DDX1 protein is found in both the nucleus and cytoplasm of DDX1-amplified lines but is localized primarily to the nucleus of nonamplified cells. Our results indicate that DDX1 may be involved in either the formation or progression of a subset of NB and RB tumors and suggest that DDX1 normally plays a role in the metabolism of RNAs located in the nucleus of the cell.

Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes.

In investigating the composition of stored (maternal) mRNP particles in Xenopus oocytes, attention has focussed primarily on the phosphoproteins pp60/56, which are Y-box proteins involved in a general packaging of mRNA. We now identify a third, abundant, integral component of stored mRNP particles, Xp54, which belongs to the family of DEAD-box RNA helicases. Xp54 was first detected by its ability to photocrosslink ATP. Subsequent sequence analysis identifies Xp54 as a member of a helicase subfamily which includes: human p54, encoded at a chromosomal breakpoint in the B-cell lymphoma cell line, RC-K8; Drosophila ME31B, encoded by a maternally-expressed gene, and Saccharomyces pombe Ste13, cloned by complementation of the sterility mutant ste13. Expression studies reveal that the gene encoding Xp54 is transcribed maximally at early oogenesis: no transcripts are detected in adult tissues, other than ovary. Using a monospecific antibody raised against native Xp54, its presence in mRNP particles is confirmed by immunoblotting fractions bound to oligo(dT)-cellulose and separated by rate sedimentation and buoyant density. On isolating Xp54 from mRNP particles, it is shown to possess an ATP-dependent RNA helicase activity. Possible functions of Xp54 are discussed in relation to the assembly and utilization of mRNP particles.

A DEAD-box RNA helicase in the Escherichia coli RNA degradosome.

The Escherichia coli RNA degradosome is a multi-enzyme complex that contains the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. Both enzymes are important in RNA processing and messenger RNA degradation. Here we report that enolase and RhlB are two other major components of the degradosome. Enolase is a glycolytic enzyme with an unknown role in RNA metabolism. RhlB is a member of the DEAD-box family of ATP-dependent RNA helicases, which are found in both prokaryotes and eukaryotes. We show that the degradosome has an ATP-dependent activity that aids the degradation of structured RNA by PNPase. Incubation of the degradosome with affinity-purified antibody against RhlB inhibited the ATP-stimulated RNA degradation. These results suggest that RhlB acts by unwinding RNA structures that impede the processive activity of PNPase. RhlB is thus an important enzyme in mRNA turnover.

A nucleolar RNA helicase recognized by autoimmune antibodies from a patient with watermelon stomach disease.

Watermelon stomach is characterized by prominent stripes of ectatic vascular tissue in the stomach similar to stripes on a watermelon; in patients with this disorder chronic gastrointestinal bleeding occurs and approximately half of these patients have associated autoimmune disorders. In the serum of one patient, an antinucleolar antibody titer of 1:25 600 was found; the antibodies specifically recognized an approximately 100 kDa nucleolar protein, which we referred to as the 'Gu' protein. Its cDNA was cloned and sequenced. The Gu protein is a member of a new subgroup of RNA helicases, the DEXD box family. Gu protein fused with glutathione S-transferase contains ATP-dependent RNA helicase activity which preferably translocates in the 5'-->3' direction. Its RNA folding activity, RNA-dependent ATPase and dATPase activities, and its translocation direction are similar to those of RNA helicase II [Flores-Rozas, H. and Hurwitz, J. (1993) J. Biol. Chem. 268, 21372-21383]. Sequencing of 209 amino acids of RNA helicase II peptides showed 96.7% identity with the cDNA-derived amino acid sequence of the Gu protein. The precise biological roles of this RNA helicase in the biogenesis of ribosomal RNA and the pathogenesis of watermelon disease and autoimmune disorder require further study.

A putative mammalian RNA helicase with an arginine-serine-rich domain colocalizes with a splicing factor.

We have cloned a rat cDNA whose deduced primary structure yields a protein of 117.4 kDa. Because this protein contains RNA helicase consensus motifs, among them a "DEAD" box, we have termed it HEL117 (for helicase of 117.4 kDa). Besides the helicase consensus motifs, HEL117 contains an arginine-serine (RS)-rich domain, which occurs in some proteins involved in RNA splicing. Moreover, the COOH-terminal region of 78 residues of HEL117 is 38.5% identical and 59% similar to the COOH-terminal region of a yeast PRP5 protein that is involved in RNA splicing. Rabbit antibodies generated against a synthetic peptide of HEL117 identified a single polypeptide not only in rat cells but also in cells of other mammals as well as chicken. The antibodies revealed a finely punctate and speckled intranuclear staining in immunofluorescence microscopy. A monoclonal antibody against a human splicing factor containing an RS domain (SC35) showed, in double immunofluorescence microscopy, largely overlapping staining consistent with HEL117 being involved in RNA splicing.

A 130 kDa polypeptide immunologically related to the 180 kDa catalytic subunit of DNA polymerase alpha-primase complex is detected in early embryos of Drosophila.

An immunocytochemical method using specific antibodies was employed to detect DNA polymerase alpha-primase complex in Drosophila melanogaster embryos during the first 13 nuclear division cycles. A monoclonal antibody specific to the 72 kDa polypeptide stained interphase nuclei, but not metaphase chromosome, while at late anaphase and thereafter staining of the chromosome was regained. On the other hand, a polyclonal antibody specific to the 180 kDa polypeptide stained not only the interphase nuclei but also the cytoplasmic regions surrounding interphase nuclei. These results suggest that the distributions of the 180 kDa and the 72 kDa polypeptides of DNA polymerase alpha-primase complex are different. We detected the 180 kDa and the 72 kDa polypeptides in the extract prepared from a single Drosophila embryo by Western blotting, and a 130 kDa polypeptide immunologically related to the 180 kDa polypeptide was also detected in the extract. These polypeptides (180, 130, and 72 kDa) in the embryos were detected at similar levels at interphase and at the mitotic phase. These three polypeptides were also detected in unfertilized eggs, showing that they were maternally stored. The 130 kDa polypeptide was detected till cycle 10, then began to decrease, and finally disappeared at cycle 14, whereas the 180 kDa and the 72 kDa polypeptides were present without marked fluctuation in quantity throughout the developmental stages. Even in unfertilized eggs, the level of the 130 kDa polypeptide decreased gradually with a similar time course to that in fertilized ones, but the levels of the 180 kDa and the 72 kDa polypeptides remained unchanged. This is the first report suggesting the existence of the 130 kDa polypeptide in vivo in the early embryos of Drosophila. The significance of the 130 kDa polypeptide is discussed.

Localization and functions of Japanese encephalitis virus nonstructural proteins NS3 and NS5 for viral RNA synthesis in the infected cells.

Recently it has been reported that Japanese encephalitis virus (JEV)-specific RNAs can be synthesized in vitro in the subcellular fraction including outer-nuclear membrane (Takegami and Hotta, 1989). The results of Western blot analysis and indirect immunofluorescence test using two kinds of monospecific antisera against JEV nonstructural proteins NS3 and NS5 showed that NS3 and NS5 were membrane-associated proteins and formed the complex at the perinuclear site in the infected cells. Both antisera against NS3 and NS5 inhibited in vitro RNA synthesis. These results suggest that NS5 and NS3 play important role(s) in flavivirus RNA replication.

DNA polymerase alpha--primase complex of Physarum polycephalum.

DNA polymerase alpha and DNA polymerase alpha--primase complex of Physarum polycephalum were purified by rapid methods, and antibodies were raised against the complex. In crude extracts, immune-reactive polypeptides of 220 kDa, 180 kDa, 150 kDa, 140 kDa, 110 kDa, 86 kDa, 57 kDa and 52 kDa were identified. The structural relationships between the 220 kDa, 110 kDa and 140 kDa (the most abundant form) was investigated by peptide mapping. The 140 kDa form was active DNA polymerase alpha. The 57 kDa and the 52 kDa polypeptides were identified as primase subunits by auto-catalytic labelling. In amoebae, the immune-reactive 140 kDa polypeptide was replaced by a 135 kDa active DNA polymerase alpha.

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.

Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone recognized dengue virus types 1, 2, and 3. Four dengue virus serotype-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4. One flavivirus-cross-reactive clone recognized dengue virus types 1, 2, 3, and 4 and West Nile virus (WNV), but did not recognize yellow fever virus (YFV), whereas three flavivirus-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4, WNV, and YFV. HLA restriction in the lysis by these T-cell clones was also heterogeneous. HLA-DP, HLA-DQ, and HLA-DR were used as restriction elements by various T-cell clones. We also examined the recognition of viral nonstructural protein NS3, purified from cells infected with dengue virus type 3 or WNV, by these T-cell clones. One serotype-specific clone, two dengue virus subcomplex-specific clones, and three dengue virus serotype-cross-reactive clones recognized NS3 of dengue virus type 3. One flavivirus-cross-reactive clone recognized NS3 of dengue virus type 3 and WNV. These results indicate that heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes.

Immunoaffinity purification and properties of Drosophila melanogaster DNA polymerase alpha-primase complex.

Two hybrid cell lines (DM88-5E12 and DM88-4C9) secreting monoclonal antibodies against DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells were established by immunizing mice with the complex partially purified by a conventional method. The IgG subclasses of both antibodies were IgG1. Both antibodies immunoprecipitated the DNA polymerase alpha-primase complex from D. melanogaster Kc cells. The DNA-polymerizing activity was neutralized by 4C9 antibody, but not by 5E12 antibody. The DNA priming activity was not neutralized by either antibody. These antibodies did not cross-react to HeLa DNA polymerase alpha-primase complex. A rapid, two-step purification of DNA polymerase alpha-primase complex from D. melanogaster Kc cell was carried out by 5E12 antibody column chromatography followed by single-stranded DNA cellulose column chromatography. The immunoaffinity-purified enzyme had both DNA-polymerizing and DNA-priming activities with the specific activities of 50,000 and 2,000 units/mg, respectively. The effects of aphidicolin, NEM, ddTTP, BuPdGTP, and DMSO on the enzyme activity showed that the purified enzyme was DNA polymerase alpha, but not DNA polymerase beta, gamma, or delta. The purified enzyme consisted of polypeptides with apparent molecular weights of 180 (and 145, 140, 130 kDa), 72, 63, 51, and 49 kDa. The 5E12 antibody was shown to bind to all the high-molecular-weight polypeptides, 180, 145, 140, and 130 kDa, by immuno-Western blotting analysis.

SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.

Purified SV40 large T antigen and purified DNA polymerase alpha-primase form a complex detectable by ELISA and by a modified immunoblotting technique. The interaction is specific for the large catalytic subunit of polymerase alpha. The amino terminal 83 amino acids of T antigen are both necessary and sufficient for binding to the polymerase. However, antibody epitopes located in the carboxy terminal ATPase domain of T antigen are masked in the polymerase-T antigen complex, and complex formation is inhibited by an antibody directed against the carboxy terminus of T antigen, suggesting that this region of T antigen, though not required for binding, is in close proximity to the bound polymerase. The affinity of human DNA polymerase alpha for T antigen is approximately 10-fold greater than that of polymerase alpha from calf thymus, consistent with the interpretation that polymerase alpha is at least in part responsible for the primate-specific replication of SV40 DNA in vivo and in vitro. The results suggest that specific protein-protein interaction between DNA polymerase alpha and T antigen plays an important role in viral DNA replication.

Immunochemical detection of a primase activity related subunit of DNA polymerase alpha from human and mouse cells using the monoclonal antibody.

A hybrid cell line (HDR-854-E4) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase alpha was established by immunizing mice with DNA replicase complex (DNA polymerase alpha-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralizes the primase activity as assessed either by the direct primase assay (incorporation of [alpha-32P]AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase alpha activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase alpha. The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDa) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3S DNA polymerase alpha which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase alpha. Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDa polypeptide is associated with DNA polymerase alpha, and not with the primase.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological analysis of the polypeptide structure of calf thymus DNA polymerase-primase complex.

Five major polypeptides are found in immunoaffinity-purified calf thymus DNA polymerase-DNA primase complex: 185, 160, 68, 55, and 48 kDa. Individual polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to produce antibodies in rabbits to aid in identifying the relationships between these polypeptides by immunoblotting and enzyme neutralization procedures. Immunoblot analyses showed that the 160-kDa peptide is derived from the 185-kDa peptide and the 48-kDa peptide is derived from the 68-kDa peptide while antibodies to the 55-kDa peptide do not cross-react with other peptides found in the complex. Direct enzyme neutralization studies demonstrated that antibodies to 185- and 160-kDa peptides inhibit DNA polymerase activity in the complex, confirming earlier suggestions that these peptides are the catalytic peptides for DNA polymerase. DNA primase activity in the complex is inhibited by antibodies to 68-, 55-, and 48-kDa peptides and to a lesser extent by antibodies to the 160-kDa peptide. Free DNA primase isolated from the complex was estimated to have a native molecular weight of about 110,000. The 55- and 48-kDa peptides are found to be associated with the free primase activity. Rabbit antibodies to both 55- and 48-kDa peptides are inhibitory to this primase activity. From these results we suggest that the native calf thymus DNA polymerase-DNA primase complex contains only three unique peptides with the 185-kDa peptide as the catalytic peptide of DNA polymerase and the 55- and 68-kDa peptides constituting the primase peptides. A model illustrating the roles of these peptides in initiation and replication of DNA is presented.

Plasmid RP4 encodes two forms of a DNA primase.

The pri gene locus of the conjugative broad host range plasmid RP4 maps between coordinates 40.3 and 43.5 and encodes two antigenically related forms of a DNA primase with a molecular mass of 118 and 80 kDa (kilodalton). Genesis of these two products has been examined using Pri+-recombinant plasmids. As shown by deletion analysis, the primase polypeptides are tow separate translation products which arise from an in-phase overlapping gene arrangement. It is suggested that transcription of a set of RP4 genes including the pri gene starts at a promoter site within the Tra1 region. In vivo, RP4 mutant primase can apparently substitute for Escherichia coli primase as demonstrated by measuring suppression of the dnaG3 (ts) mutant.

Antibodies against a synthetic peptide of the poliovirus replicase protein: reaction with native, virus-encoded proteins and inhibition of virus-specific polymerase activities in vitro.

A carboxy-terminal peptide of the poliovirus replicase protein (p63) was chemically synthesized, coupled to bovine serum albumin carrier, and injected into rabbits. The resulting antisera reacted with six virus-specific proteins from HeLa cells infected with poliovirus: NCVP 0b, NCVP 1b, NCVP 2, a protein of about 60,000 daltons, p63, and NCVP 6b. The identity of the 60,000-dalton protein is not known, but the other results were consistent with previous experimental approaches which demonstrated that p63 and the other four polypeptides have common coding sequences. An amino-terminal peptide of p63 failed to elicit an immune response in rabbits. Antibodies raised against the p63 carboxy-terminal peptide inhibited poliovirus replicase and polyuridylic acid polymerase activities in vitro, providing strong support for earlier suggestions that these activities are a property of a single virus-specific polypeptide.