The use of synthetic oligodeoxynucleotide primers in cloning and sequencing segment 8 of influenza virus (A/PR/8/34)

Winter, Greg; Fields, Stan; Gait, Michael J.; Brownlee, George G.

doi:10.1093/nar/9.2.237

Cited by 100 publications

(42 citation statements)

References 26 publications

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“…However, mutations also appeared in the C-terminal region, proposed to constitute an effector domain (57). This distribution of ts mutations lend support to the hypothesis, first proposed by Winter et al (71), that the ancestral NS1 protein was shorter than that present in present-day viruses and that the extended C terminus might be generated by mutation of the existing termination codon. It is probable that the extended protein was evolutionary conserved, because new roles for virus replication were selected for, as supported by the isolation of ts mutants in this region (Fig.…”

Section: Discussionsupporting

confidence: 83%

Genetic Analysis of Influenza Virus NS1 Gene: a Temperature-Sensitive Mutant Shows Defective Formation of Virus Particles

Garaigorta

Falcón

Ortı́n

2005

J Virol

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To perform a genetic analysis of the influenza A virus NS1 gene, a library of NS1 mutants was generated by PCR-mediated mutagenesis. A collection of mutant ribonucleic proteins containing the nonstructural genes was generated from the library that were rescued for an infectious virus mutant library by a novel RNP competition virus rescue procedure. Several temperature-sensitive (ts) mutant viruses were obtained by screening of the mutant library, and the sequences of their NS1 genes were determined. Most of the mutations identified led to amino acid exchanges and concentrated in the N-terminal region of the protein, but some of them occurred in the C-terminal region. Mutant 11C contained three mutations that led to amino acid exchanges, V18A, R44K, and S195P, all of which were required for the ts phenotype, and was characterized further. Several steps in the infection were slightly altered: (i) M1, M2, NS1, and neuraminidase (NA) accumulations were reduced and (ii) NS1 protein was retained in the nucleus in a temperature-independent manner, but these modifications could not justify the strong virus titer reduction at restrictive temperature. The most dramatic phenotype was the almost complete absence of virus particles in the culture medium, in spite of normal accumulation and nucleocytoplasmic export of virus RNPs. The function affected in the 11C mutant was required late in the infection, as documented by shift-up and shift-down experiments. The defect in virion production was not due to reduced NA expression, as virus yield could not be rescued by exogenous neuraminidase treatment. All together, the analysis of 11C mutant phenotype may indicate a role for NS1 protein in a late event in virus morphogenesis.The influenza A virus genome encodes NS1, a small, nonstructural, and multifunctional protein important for virus-cell interactions (for reviews, see references 22, 33, and 53). It is translated from the colinear transcript of segment 8, which also encodes NS2 protein from a spliced mRNA (31, 34). NS1 accumulates in the nucleus early in the infection (4, 32) and when it is expressed from cDNA (26,35,56), but at late times in the infection it is also found in the cytoplasm (49), in association with polysomes (8,16,32).Although NS1 protein is apparently nonessential, as a recombinant virus has been generated that lacks the gene (23), several virus mutants have been isolated or generated which contain mutations in the NS1 protein and are severely hampered for replication (12,14,17,27,28,40,60,(63)(64)(65). The phenotypes of these mutant viruses indicate that NS1 protein may be involved in several steps in the virus infectious cycle, including transcription and/or replication of virus RNA, late virus protein synthesis, and interference with the cellular gene expression, and that it eventually modulates the virulence of virus infections in vivo (2, 23, 68).NS1 is an RNA-binding protein that has been shown to interact with virion RNA (vRNA) (29, 43), poly(A)-containing RNAs (58), and U6 snRNA (59). The RNA-binding...

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Section: Discussionsupporting

confidence: 83%

Genetic Analysis of Influenza Virus NS1 Gene: a Temperature-Sensitive Mutant Shows Defective Formation of Virus Particles

Garaigorta

Falcón

Ortı́n

2005

J Virol

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“…The restriction fragmnts were fractionated on a 1.5% agarose gel and the fraction fran 300 to 500 nucleotides collected by eluting the DNA fragIents into a trough cut in the agarose gel (24). The fragents subsequently were concentrated by ethanol precipitation and ligated directly into Ml3rp8 (25) *tidh had been Sma I cleaved and phosphatase treated for blunt-end cloning (26).…”

Section: Nucleic Acids Researchmentioning

confidence: 99%

“…Short direct repeats are formed flanking the Alu family mnbers upon their integration into a site in the genare (4,5,8,9,11,12 (Figure 4) has been highly conserved both in evolution, caroaring hamster and human (26), and aiong the individual members of the human Alu family (positions +5 to +20 in ref. 7).…”

Section: Discusicnmentioning

confidence: 99%

Species-specific homogeneity of the primate Alu family of repeated DNA sequences

et al. 1983

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“…2b). TaqI and HpaII digests were cloned into AccI-digested vector Ml 3 mp7, Sau3A digests into BstI-cut mp7 and AluI digests into the HinCII site of mp7 after phosphatase treatment of the cut vector (Winter et al, 1981). Assembly of the data was achieved by the BATIN and BATOVR programs of Staden (1980).…”

Section: Computingmentioning

confidence: 99%

Ribitol dehydrogenase of Klebsiella aerogenes. Sequence of the structural gene

Loviny¹,

Norton²,

Hartley³

1985

Biochemical Journal

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The ribitol dehydrogenase gene was cloned from wild-type Klebsiella aerogenes and also from a transducing phage lambda prbt which expresses the rbt operon constitutively. The coding sequence for 249 amino acids is separated from the following D-ribulokinase gene by 31 base pairs containing three stop codons, one of which overlaps the ribosome binding site for D-ribulokinase. Three residues in the amino acid sequence differ from that predicted from the DNA sequence: Asp-212 for Asn-212 is probably a protein sequencing error, but -Ala-Val- for -Ser-Ser- at 146-147 appears to be a ‘neutral mutation’ that may have arisen during prolonged chemostat selection of a strain that superproduces the enzyme from which the protein sequence was determined.

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The use of synthetic oligodeoxynucleotide primers in cloning and sequencing segment 8 of influenza virus (A/PR/8/34)

Cited by 100 publications

References 26 publications

Genetic Analysis of Influenza Virus NS1 Gene: a Temperature-Sensitive Mutant Shows Defective Formation of Virus Particles

Genetic Analysis of Influenza Virus NS1 Gene: a Temperature-Sensitive Mutant Shows Defective Formation of Virus Particles

Species-specific homogeneity of the primate Alu family of repeated DNA sequences

Ribitol dehydrogenase of Klebsiella aerogenes. Sequence of the structural gene

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