Sequence of DNA complementary to a small RNA segment of influenza virus A/NT/60/68.

A small RNA segment from the influenza virus strain A/NT/60/68 (H3N2) was converted to cDNA and then to double-stranded DNA using synthetic oligodeoxynucleotide primers. The double-stranded form was cloned into the bacteriophage M1 3mp7. Clones yielding single-strand recombinant templates in opposite orientation were sequenced by the Sanger dideoxynucleotide chain termination technique. The small viral RNA was 422 nucleotides long and the evidence indicated that it was formed by internal deletion of segment 3. It also contained sequences homologous to segment 1.     

The sequence of RNA segment 1 of influenza virus A/NT/60/68 and its comparison with the corresponding segment of strains A/PR/8/34 and A/WSN/33.

The complete nucleotide sequence of RNA segment 1 of influenza virus A/NT/60/68, corresponding to the PB2 protein, has been determined. It is 2341 nucleotides long, encoding a predicted product of 759 amino acids with a net charge of +27 1/2 at neutral pH. The predicted amino acid sequence has been compared to the equivalent sequences in influenza viruses A/PR/8/34 and A/WSN/33. Evolutionary divergence, assuming a direct lineage from A/PR/8/34 and allowing for "laboratory drift", is 0.08% per year. The alignment of RNA segment 10 of A/NT/60/68 with segments 1 and 3 is completed, confirming that it is a mosaic of regions from these two segments.     

The sequence of the nucleoprotein gene of human influenza A virus, strain A/NT/60/68.

The nucleotide sequence of the nucleoprotein gene of influenza A/NT/60/68 was established after using improved cloning methods to obtain full length cDNA clones in pBr322. The gene is 1565 residues long and codes for a basic protein of 498 amino acids. There are only 30 amino acid differences between it and the homologous sequence in A/PR/8/35, all occurring as point mutations. Assuming a common lineage, the evolutionary rate of divergence of the two strains is 0.18% amino acid per year. This confirms there is a slow but significant rate of evolution.     

Sequence of the N2 neuraminidase from influenza virus A/NT/60/68.

The complete sequence of the neuraminidase gene of influenza virus A/NT/60/68 (N2 subtype) was determined following cloning of full length complementary DNA into pBR322. Comparison of the predicted amino acid sequence with a closely related neuraminidase from A/Udorn/72 suggests that point mutations over an extensive region of the primary sequence can contribute to antigenic drift, although the region between amino acid residues 308 and 371 may be particularly significant.     

Nucleotide sequence of human influenza A/PR/8/34 segment 2.

The nucleotide sequence of RNA segment 2 of human influenza strain A/PR/8/34 has been determined. Segment 2 in 2341 nucleotides long and encodes a protein of 757 amino acids (86,500 daltons molecular weight) which is involved in RNA synthesis. Although segment 2 is identical in size to segment 1, which encodes a protein of related function, neither the nucleotide sequences of these two RNA segments nor the amino acid sequences of the encoded proteins appear to be homologous. The sequence of segment 2 completes the sequence of the virus (total 13,588 nucleotides).     

The complete amino acid sequence of the rabbit P2 protein

P2 protein is a small basic protein (Mr = 14,820) found in peripheral nerve myelin and spinal cord myelin. There is now overwhelming evidence that P2 protein is the crucial antigen involved in the induction of experimental allergic neuritis, an autoimmune disease of the peripheral nervous system. The complete amino acid sequence of rabbit P2 protein was derived by sequence analysis of cyanogen bromide peptides and peptides obtained by proteolysis using Staphylococcus aureus V8 enzyme, trypsin, or clostripain. There are 131 amino acids and an excess of the basic amino acids lysine and arginine; histidine is absent. There are 3 highly hydrophobic regions in the P2 molecule. Probability analysis of the sequence predicts a high degree of beta structure, essentially in agreement with CD data.     

Cloning of influenza cDNA ino M13: the sequence of the RNA segment encoding the A/PR/8/34 matrix protein

A strategy has been developed for sequencing the single-stranded RNA genes of influenza virus. Restriction fragments derived from double-stranded cDNA copies of total influenza RNA were cloned into the bacteriophage M13mp2 and sequenced by the dideoxy technique. Sequences were extended and overlapped by using the virion RNA as template and priming with small restriction fragments. In the course of this strategy, the nucleotide sequence of segment 7 (1027 nucleotides) was completed and provides the primary structure of the matrix protein (27, 861 daltons). In addition, there is a second long reading frame which partly overlaps the reading frame of the matrix protein.     

The complete sequence of a chicken-muscle cDNA encoding the acidic ribosomal protein P1

We have isolated and sequenced a complete cDNA encoding the acidic phosphoprotein P1 from chicken. The analysis of the deduced protein sequence and its comparison with the known sequence of P proteins from human, rat and Artemia salina indicates that the central, alanine-rich region of these proteins was probably generated by internal duplications of the gene followed by modifications within each repeat. This observation explains the length heterogeneity and sequence divergence of this particular region when compared with the highly conserved structure of the remaining segments of the protein.     

The complete sequence of genome segment 8 of bluetongue virus, serotype 1, which encodes the nonstructural protein, NS2.

Bluetongue virus has a ten-segment double-stranded RNA genome, of which segment 8 encodes a nonstructural protein NS2. This protein is the only bluetongue viral protein to be phosphorylated and also has the ability to bind single-stranded RNA. At present, the function of NS2 is unknown and in order to analyse its characteristics in more detail, it was first necessary to obtain a full-length cDNA clone of the genome segment.     

Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus.

The biochemical properties of a second protein (CM2) encoded by RNA segment 6 of influenza C virus were investigated. Three forms of CM2 with different electrophoretic mobilities (CM2(0), CM2a, and CM2b) were detected in infected cells by immunoprecipitation with antiserum to the glutathione S-transferase (GST)-CM2 fusion protein. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with endoglycosidase H or peptide-N-glycosidase F suggested that a mannose-rich oligosaccharide core is added to unglycosylated CM2(0) (Mr, approximately 16,000) to form CM2a (Mr, approximately 18,000) and that the processing of the carbohydrate chain from the high-mannose type to the complex type converts CM2a into CM2b, which is heterogeneous in electrophoretic mobility (Mr, approximately 22,000 to 30,000). Labeling of infected cells with [3H]palmitic acid showed that CM2 is fatty acylated. The fatty acid bond was sensitive to treatment with hydroxylamine and mercaptoethanol, which indicates a labile thioester-type linkage. The CM2 protein was also found to form disulfide-linked dimers and tetramers on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Trypsin treatment of infected cell surfaces as well as of microsome vesicles from infected cells followed by immunoprecipitation with antiserum to the GST fusion protein containing the 56 C-terminal amino acid residues of CM2 suggested that this C-terminal domain is intracellular and exposed to the cytoplasms of microsomes. Furthermore, evidence that a small amount of CM2 is incorporated into progeny virus particles was obtained by Western blot analysis. These results, altogether, suggest that CM2 is an integral membrane protein with biochemical properties similar to those of influenza A virus M2 and influenza B virus NB proteins.     

The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA

The p53-induced Wig-1 gene encodes a double stranded RNA-binding zinc finger protein. We generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. Induction of Wig-1 expression by doxycycline inhibited cell growth in a long-term assay but did not cause any changes in cell cycle distribution nor increased fraction of apoptotic cells. Using co-immunoprecipitation and mass spectrometry, we identified two Wig-1-binding proteins, hnRNP A2/B1 and RNA Helicase A, both of which are involved in RNA processing. The binding was dependent on the presence of RNA. Our results establish a link between the p53 tumor suppressor and RNA processing via hnRNPA2/B1 and RNA Helicase A.     

Interference is controlled by segment 2 and possibly by segment 8 of the nondefective interfering influenza virus variant A/FM/1/47-MA.

On mouse adaption of A/FM/1/47, a variant, A/FM/1/47-MA (FM-MA), that had acquired the properties of increased virulence and interference was produced. Coinfection of cells with FM-MA and prototype strains of influenza virus yielded > 100-fold more FM-MA virus than prototype virus, whereas coinfection with the same prototype strains and the parental A/FM/1/47 virus produced equivalent yields, indicating that FM-MA had acquired mutations that confer the property of interference during mouse adaption. FM-MA is a nondefective interfering virus that grows to a high titer in vivo and in vitro. It has previously been shown that segments 4, 7, and 8 and possibly segment 5 account for the increased virulence. In this study we show by genetic analysis of FM-MA x A/HK/1/68 reassortants that segment 2, coding for the polymerase-associated protein PB1, and possibly segment 8, encoding the NS1 and NS2 proteins, control the ability of FM-MA to interfere. Interference could not be overcome by increasing the titer of the coinfecting strain, but delaying FM-MA infection by 4 to 6 h did avoid interference. During interference of A/HK/1/68, protein synthesis was inhibited by less than 65% throughout coinfection. Given the kinetics of interference and the small perturbation in protein synthesis, interference appeared to occur at the level of late genome replication or virus assembly. Virulence and interference in FM-MA were not linked. An interfering avirulent FM-MA x A/HK/1/68 reassortant, E07, was capable of protecting mice against lethal pneumonia due to a virulent noninterfering reassortant, H04.     

The use of nuclease P1 in sequence analysis of end group labeled RNA.

A method is described for the direct sequence analysis of 20-25 nucleotides from the termini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequence of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.     

Evolution of the nucleotide sequence of influenza virus RNA segment 7 during drift of the H3N2 subtype

The complete genetic information contained in the influenza virus RNA segment 7 of the A/Bangkok/ $\text{1}\text{79}$ (H3N2) strain has been cloned by in vitro synthesis of the complementary dsDNA and its insertion into plasmid pBR322. The nucleotide sequence of the viral RNA segment has been determined from the cDNA insert. It is 1027 nucleotides long, and contains two open reading frames, as shown for other influenza virus strains. When compared with the previously published sequence for the A/Udorn/72 (H3N2) strain, 15 nucleotide exchanges are observed, most of them silent mutations, and only two causing amino acid changes in each of the M1 and M2 protein sequences.     

Calcium-binding protein of bovine intestine. The complete amino acid sequence.

The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.     

Nucleotide sequence at the 5' extremity of tobacco-mosaic-virus RNA. 1. The noncoding region (nucleotides 1-68)

The sequence of the 5' noncoding region of tobacco mosaic virus RNA has been determined. The noncoding region is 68 nucleotides long and is unusual in that it contains no internal guanosine residues. The long T1 oligonucleotide containing the guanosine-free tract was isolated from a T1 ribonuclease digest of tobacco mosaic virus RNA and sequenced by labelling techniques in vitro using polynucleotide kinase. The guanosine-free tract is terminated by the first potential initiation codon in the RNA molecule and several lines of evidence suggest that this AUG triplet is operational in initiating viral protein synthesis (see following paper). The 5'-noncoding region cannot base-pair extensively with the 3'-terminal sequence of 18-S ribosomal RNA from rabbit reticulocytes.     

人P68核蛋白在小鼠成纤维细胞中过量表达引起恶性转化的研究

P68核蛋白是一个依赖于ATP的RNA解旋酶,同时具有依赖于RNA的ATPase活性,它与SV40大T抗原有交叉免疫反应。随细胞周期不同,其含量和分布均有较大差异。以前的工作表明P68核蛋白在同一细胞系不同生长阶段的表型不同。它在肿瘤细胞（HeLa和TC3H10）与非肿瘤细胞（NIH3T3和NC3H10）之间表型亦有明显差异。因此,在细胞中过量表达P68核蛋白,可能会影响到细胞的生长特性,报道了将重组人P68表达质粒DNA分别传染NIH3T3及NC3H10细胞,使P68核蛋白在细胞中过量表达,引起细胞恶性转化;（1）转染细胞的形态从纤维状趋于梭形;（2）在单层细胞中形成微球细胞转化灶;（3）转染的细胞在软琼脂上生长形成集落;（4）将转染细胞注射裸鼠,3－4个星期后形成肿瘤。