Variation in nucleotide sequences coding for the N-terminal regions of the matrix and nonstructural proteins of influenza A viruses.

Nucleotide sequences have been determined for complementary DNA transcribed from the 3' ends of RNA segments 7 (matrix gene) and 8 (nonstructural gene) from a number of human influenza A viruses isolated over a period of 43 years and representing H0N1, H1N1, H2N2, and H3N2 subtypes. The pattern of nucleotide variation in both genes suggests that RNA segments 7 and 8 were conserved during the reassortment events which were responsible for the antigenic shifts H1N1 leads to H2N2 and H2N2 leads to H3N2. During the 23-year period between the isolation of A/PR/8/34(H0N1) and A/RI/5-/57(H2N2), substitutions have occurred at 7 of 230 nucleotides in RNA segment 7 and 13 of 220 nucleotides in RNA segment 8, and in 20 years A/RI/5-/57(H2N2) to A/Canberra Grammar/77(H3N2) substitutions have occurred at 5 of 230 nucleotides in RNA segment 7 and 12 of 220 nucleotides in RNA segment 8. These give rise to 2 of 67, 5 of 64, 1 of 67, and 5 of 64 amino acid changes, respectively. The number of nucleotide and amino acid changes observed is of the same order of magnitude as that which occurs over a comparable period of drift in RNA segments 4 and 6, which code for the variable antigenic determinants hemagglutinin and neuraminidase.     

The M segment of the 2009 new pandemic H1N1 influenza virus is critical for its high transmission efficiency in the guinea pig model

A remarkable feature of the 2009 pandemic H1N1 influenza virus is its efficient transmissibility in humans compared to that of precursor strains from the triple-reassortant swine influenza virus lineage, which cause only sporadic infections in humans. The viral components essential for this phenotype have not been fully elucidated. In this study, we aimed to determine the viral factors critical for aerosol transmission of the 2009 pandemic virus. Single or multiple segment reassortments were made between the pandemic A/California/04/09 (H1N1) (Cal/09) virus and another H1N1 strain, A/Puerto Rico/8/34 (H1N1) (PR8). These viruses were then tested in the guinea pig model to understand which segment of Cal/09 virus conferred transmissibility to the poorly transmissible PR8 virus. We confirmed our findings by generating recombinant A/swine/Texas/1998 (H3N2) (sw/Tx/98) virus, a representative triple-reassortant swine virus, containing segments of the Cal/09 virus. The data showed that the M segment of the Cal/09 virus promoted aerosol transmissibility to recombinant viruses with PR8 and sw/Tx/98 virus backgrounds, suggesting that the M segment is a critical factor supporting the transmission of the 2009 pandemic virus.     

Interaction between the C8 alpha-gamma and C8 beta subunits of human complement C8: role of the C8 beta N-terminal thrombospondin type 1 module and membrane attack complex/perforin domain

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex (MAC). It is an oligomeric protein composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous; both contain an N-terminal thrombospondin type 1 (TSP1) module, a low-density lipoprotein receptor class A (LDLRA) module, an extended central segment referred to as the membrane attack/perforin (MACPF) domain, an epidermal growth factor (EGF) module, and a second TSP1 module at the C-terminus. In this study, the segment of C8beta that confers binding specificity toward C8alpha-gamma was identified using recombinant C8beta constructs in which the N- and/or C-terminal modules were deleted or exchanged with those from C8alpha. Constructs were tested for their ability to bind C8alpha-gamma in solution and express C8 hemolytic activity. Binding to C8alpha-gamma was found to be dependent on the TSP1 + LDLRA + MACPF segment of C8beta. Within this segment, the TSP1 module and MACPF domain are principally involved and act cooperatively to mediate binding. Results from activity assays suggest that residues within this segment also mediate binding and incorporation of C8 into the MAC.     

Synthesis, cloning and determination of the primary structure of a full-size DNA copy of the NP protein gene from influenza virus type A

Complete nucleotide sequence of the cloned full-length DNA copy of the influenza virus A (H1N1) RNA segment 5 has been determined. The degree of nucleotide difference between two variants of A/PR/8/34 strain is estimated. The possible secondary structure of the segment 5 is deduced from the nucleotide sequence of some clones.     

Mapping of the influenza virus genome. III. Identification of genes coding for nucleoprotein, membrane protein, and nonstructural protein.

In previous communications we reported that the eight RNA segments of influenza A/PR/8/34 (HON1) virus could be distinguished from corresponding segments of influenza A/Hong Kong/8/68 (H3N2) virus by migration on polyacrylamide-urea gels. Examination of the RNA patterns of the two parent viruses and recombinants derived from them in concert with serological identification of surface proteins and analysis of the other proteins on sodium dodecyl sulfate gradient gels permitted the identification of the genes coding for hemagglutinin, neuraminidase, and the P1, P2, and P3 proteins (Palese and Schulman, 1976; P. Palese et al., Virology, in press). In the present report we have extended these observations using similar techniques to examine other recombinants and have identified the genes coding for the remaining virus-specific moving RNA segment as 1) and segment 6 of Hong Kong virus coding for the respective nucleoproteins, and that segment 7 of both viruses codes for the membtane protein and RNA segment 8 codes for the nonstructural protein. This completes the mapping of the influenza A virus genome.     

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.     

A new species of the genus Lightiella: the first record of Cephalocarida (Crustacea) in Europe

A new species of Cephalocarida belonging to the genus Lightiella is described. Like all known species of Lightiella , the new species is characterized by reduction of trunk segment 8, which also lacks both pleura and thoracopods. The diagnostic characters of the species are: (1) one seta on the inner distal corner of the penultimate endopodal segment of second maxilla and thoracopods 1–5; (2) only one claw on the distal segment of the endopod of thoracopod 6. A cladistic analysis of 27 morphological characters was used to estimate the phylogeny of all species of Lightiella , with all other cephalocarid species used as outgroups. The discovery of this species in the Mediterranean fills a gap in the distribution of the genus and of the entire class.  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 148 , 209–220.     

Obervations on the Induction of Position Effect Variegation of Euchromatic Genes in Drosophila Melanogaster

In the T(1;2)dor(var7) translocation, the 1A-2B7-8 segment of the X chromosome is brought to the vicinity of 2R-chromosome heterochromatin resulting in position effect variegation of dor, BR-C and more distal genes, as well as compaction of chromatin in this segment. By irradiation of T(1;2)dor(var7), nine reversions (rev) to a normal phenotype were recovered. In two cases (rev27, rev226), the 1A-2B7-8 section is relocated to the 19A region of the X chromosome, forming free duplications (1A-2B7-8/19A-20F-X-het). Modifiers of position effect do not change the normal expression of the BR-C and dor genes in these duplications. In five reversions (rev3, rev40, rev60, rev167, rev175), free duplications have formed from the 1A-2B7-8 fragment and X chromosome heterochromatin. In these rearrangements, modifiers of position effect (low temperature, removal of Y and 2R-chromosome heterochromatin and a genetic enhancer (E-var(3)201) induce position-effect again. Two reversions (rev45 and rev110) are associated with additional inversions in the original dor(var7) chromosomes. The inversions relocate part of the heterochromatin adjacent to the 1A-2B7-8 section into new positions. In T(1;2)dor(rev45), position-effect is seen in the 2B7-8-7A element as compaction spreading from 2B7-8 proximally in some cases as far as the 5D region. Thus, in rev45 the pattern of euchromatin compaction is reciprocal to that of the initial dor(var7) strain. Apparently, it is due to the same variegation-evoking center near the 2R centromere in both cases. In all nine revertants, weakening or complete disappearance of the position-effect is observed despite retention of the 20- kb heterochromatic segment adjacent to the 1A-2B7-8 region. Thus, a 20-kb heterochromatic sequence does not inactivate euchromatin joined to it.     

Magnesium secretion by the distal segment of the Malpighian tubules of the black field cricket Teleogryllus oceanicus

The short distal segment of unstimulated Teleogryllus Malpighian tubules secreted hyperosmotic fluid containing primarily Mg (125mmoll(-1)), Cl (242mmoll(-1)) and Na (43mmoll(-1)). Remarkably, the volume secreted by the distal segment in unit time was independent of segment length, i.e. the volume was constant regardless of the length of the segment. Magnesium was secreted at a rate of 75.5pmolmin(-1)mm(-1); the highest rate recorded for any epithelium. Low concentrations of K (20mmoll(-1)) were present but almost no P or S. Ca (2.5mmoll(-1)) concentration was higher than in the main segment. The short distal segment secreted 100% of the Mg, 54% of the Cl and 23% of the Na secreted by the whole tubule. The main segment secreted fluid containing primarily K (199mmoll(-1)), Cl (149mmoll(-1)), Na (104mmoll(-1)) and P (48mmoll(-1)) with very low concentrations of Ca (1mmoll(-1)) and S. The main segment appeared to reabsorb a small fraction of the Mg secreted by the distal segment. The fluid secreted by the whole tubule was isosmotic and alkaline, approximately pH8.     

The probable significance of tracheal tufts in the 8th abdominal segment of Heliothis virescens (F.) on the development of its parasitoid, Toxoneuron nigriceps (Viereck)

The 8th abdominal segment of Heliothis virescens (Fabricius) larvae contains aerating trachea and tracheole tufts that end in the hemocoel of the 8th segment, unlike the tracheae that invade tissues in other segments. These tracheal tufts from the 8th abdominal segment extend to the tokus region, which along with the telson cavity is known to act as a “lung” for hemocytes in Calpodes ethlius and a few other lepidopteran larvae. The goal of this research was to study the effects of these tracheal tufts in the 8th abdominal segment on parasitoid development inside the host larvae, H. virescens. The first objective was to determine if the eggs of the parasitoid, Toxoneuron nigriceps, are predominantly located among the tracheal tufts of the 8th abdominal segment compared to other body cavity regions irrespective of their oviposition site or the position of the host larvae. The results showed that several hours after oviposition most of the eggs are found in the 8th abdominal segment irrespective of the oviposition site or the position of the host larvae. The second objective was to study the effect of varying oxygen concentrations in vitro on various developmental stages of the egg. The results showed that decreasing oxygen concentrations adversely affects the parasitoid egg development in vitro. A third objective was to determine the oxygen concentration in 8th abdominal segment of the host larvae and compare it to other regions of the body using an oxygen sensor placed in vivo. The results suggested relatively high concentration of oxygen in the 8th abdominal segment compared to other regions of the host, thus supporting our hypothesis that the increased oxygen level in the 8th abdominal segment is important to the development of the parasitoid eggs.     

Structure and localization of sympathetic neurons in the cat spinal cord

By the method of retrograde axonal transport of horseradish peroxidase (HP) structure and localization of sympathetic neurons sending axons to the cranial cervical ganglion (CCG) have been revealed ipsilaterally in the ventral horns and in 4 nuclei of the spinal cord: nucl ILp, nucl. ILf. nucl. IC, nucl. ICpe. Orientation of the neurons, their number, structure of the nuclei formed by them, degree of the CCG efferentation by the preganglionic fibres, which run from various nuclei, are different. In nucl. ILf two types of neurons have been revealed-triangle and spindle-shaped, they always orienting by their long axis in mediolateral direction. The greatest amount of HP-positive neurons are found in nucl. ILp. They form a well distinquished compact nucleus in the lateral horns. HP-labelled neurons in nucl. ILp are found at the level of segments T1-T8 with their maximal amount at the level of segments T1-T3. HP-positive neurons are detected in nucl. ILf beginning from the segment C8 up to the middle of T4, in nucl. IC-from the segment C8 up to T6, in nucl. ICpe-from the segment C8 up to T5, in the ventral horns-from the segment T1 up to T5. In rostocaudal direction from the segment C8 up to T8 the number of HP-positive neurons is decreasing, but the part of nucl. ILp neurons in the CCG efferentation, comparing to the neurons in other sympathetic structures of the spinal cord, is increasing.     

Identification of the trapped calcium in the gelsolin segment 1—actincomplex: Implications for the role of calcium in the control of gelsolin activity

The X-ray structure of the complex of actin with gelsolin segment 1 revealed the presence of two calcium ions, one bound at an intramolecular site within segment 1 and the other bridging the segment directly to actin. Although earlier calcium binding studies at pH 8.0 revealed only a single calcium trapped in the complex (and also in the binary gelsolin-actin complex), it is here shown that two calcium ions are bound under the conditions of crystallization at physiological pH. Mutation of acidic residues in either actin or segment 1 involved in ligation of the intermolecular calcium ion resulted in loss of one of the bound calcium ions at pH <7, but not at pH 8. Thus the calcium ion trapped in the segment 1-actin complex is that located at the intramolecular site. The implications of this for gelsolin function are discussed.     

单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析

本文利用T4 RNA连接酶将5'－磷酸,3'-氨基修饰的引物1连接到马尾松毛虫质型多角体病毒第8片段dsRNA的3'-OH端,经逆转录,退火,补齐形成全长双链cDNA。使用单一的互补引物2进行PCR 增,扩增产物克隆在pMD18-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长330bp,S'端具有CPV－1型末端保守序列AGTAAA'端具有保守序列GTTAGCC。起始密码子从ATG位于38－40残基,终止密码子TAA位于1208－1210残基。推测S8片段编码390年氨基酸多肽,分子量为44kDa。与舞毒蛾质多角体病（LdCPV)第8片段相比较,核苷酸和氨基酸同源性分别为97％和98％。与家蚕质型多角体病毒（BmCPV）第8片段相比较,核苷酸和氨基酸的同源性分别为83％和85％。与人的呼肠孤病毒第8片段比较没有明显的同源性。     

Attenuated influenza virus construct with enhanced hemagglutinin protein expression

Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein expressed lower levels of hemagglutinin (HA) protein in infected cells than did wild-type viruses. This reduction in HA protein expression correlated with a reduction in HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not that of the nucleoprotein (NP). This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated recombinant influenza A/Puerto Rico/8/34 (rPR8) virus carrying the G3A C8U "superpromoter" mutations in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and carrying the G3A C8U mutations in the HA segment demonstrated enhanced protection from wild-type virus challenge over that for mice vaccinated with an rPR8 virus encoding the truncated NS1 protein alone.     

Nucleotide sequences of influenza virus segments 1 and 3 reveal mosaic structure of a small viral RNA segment

Defective interfering RNAs of influenza virus are small segments derived from viral segments 1, 2 and 3. We present here the complete nucleotide sequences of segments 1 and 3 from the human influenza strain A/PR/8/34 and deduce that the sequence of a small RNA segment from A/NT/60/68, apparently a defective interfering RNA, is derived from five separate regions in segment 3 and from one region in segment 1. These regions, which are located near the termini of the two parental segments, are arranged in the small RNA segment in an alternating fashion: thus a region derived from near a 5′ terminus is adjacent to a region derived from near a 3′ terminus. We propose that the small segment is generated during positive strand synthesis as a result of the viral polymerase pausing at uridine-rich sequences in the template and reinitiating synthesis at another site.     

Genetic and molecular characterization of the Pseudomonas plasmid pVS1

A restriction map of the 30-kb nonconjugative Pseudomonas plasmid pVS1 was constructed. Derivatives of pVS1 obtained in vitro by successive deletions were used to localize on the physical map the determinant for resistance to mercuric ions (carried by transposon Tn501), the gene(s) encoding sulfonamide resistance, a 1.6-kb region affecting plasmid stability and establishment in P. fluorescens ATCC 13525, and a segment required for mobilization of pVS1 by plasmid RP1. The sulfonamide resistance determinant of pVS1 appeared to be closely related to that of transposon Tn21. A mini-pVS1 replicon, pME259, consisting of an essential 1.55-kb segment (designated rep and thought to carry the origin of replication) and a mercury resistance determinant was able to replicate P. aeruginosa PAO but selective pressure was needed for plasmid maintenance. The copy number of pVS1 derivatives was estimated to be 6-8 per chromosome equivalent. Plasmids possessing the essential rep segment plus the adjacent stability region could be established in strains of P. aeruginosa, P. putida, P. fluorescens, P. acidovorans, P. cepacia, P. mendocina, P. stutzeri, P. syringae, Agrobacterium tumefaciens, and Rhizobium leguminosarum.     

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).     

Mapping DNA sequences in a human X-chromosome deletion which extends across the region of the Duchenne muscular dystrophy mutation.

A somatic cell hybrid has been constructed and characterized using fibroblasts from a phenotypically normal woman who possesses an X chromosome with an interstitial deletion of the short arm. High-resolution banding indicates that the deleted segment is either Xp22.13-p11.4 or Xp22.11-p11.23. Southern blot hybridization to previously mapped DNA sequences confirms that the missing segment of the X chromosome is a deletion and not an interstitial translocation and supports the cytogenetic interpretation that the deletion extends proximal of Xp11.3 and therefore probably comprises Xp22.11-p11.23. Three further DNA sequences have been localized to the region of the deleted segment. The following order has been assigned to the seven probes used: Xpter-RC8-pXUT22-(OA1,C7,M2C)-L1.28-RD6 -Xcen.     

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.     

Contribution of the T cell receptor BJ gene to recognition of the P91A tumor antigen in DBA/2 mice

 To understand specific immune responses against a tumor, it is important to characterize T cells that recognize the tumor antigen. The mouse P91A antigen is one of the well-defined tumor antigens that is expressed on the P911 cell line, and T cells responding to the antigen in DBA/2 mice were reported to be restricted to BV8S2/S3 families in their T cell receptor (TCR) BV gene usage. We have further characterized the P91A-responding T cells in DBA/2 mice, focusing on TCR BJ gene usage and using the polymerase chain reaction/enzyme-linked immunosorbent assay and DNA sequencing studies of their third complementarity-determining (CDR3) regions. As a result, T cells with cytotoxic activity to the P91A antigen, induced from murine spleen cells both in vivo and in vitro, showed predominant use of the BJ2S1 gene segment in both BV8S2 and BV8S3 T cells compared to unmanipulated murine spleen cells. Sequencing studies of the CDR3 regions in the BV8S3 T cells revealed clonal expansion of T cells with the BV8S3-BJ2S1 combination in two of three DBA/2 mice tested. In the remaining mouse, clonal expansion was not detected despite predominant use of the BJ2S1 segment by these T cells. These data suggest that P91A-recognizing T cells would predominantly use the BV8S2/S3-BJ2S1 combination. Analysis of T cells with these TCR BV-BJ gene combinations may contribute to the evaluation, monitoring and development of a T-cell-mediated immunotherapeutic strategy. Received: 3 July 1997 / Accepted: 17 November 1997