Genome reconstitution and nucleic acid hybridization as methods of identifying particle-deficient isolates of tobacco rattle virus in potato plants with stem-mottle disease

NM isolates of tobacco rattle virus (TRV) are perpetuated by the larger genome nucleic acid (RNA-1) but do not produce nucleoprotein particles; their identification therefore presents special problems. Assays of the infectivity of nucleic acid extracts, and of previously frozen sap, suggested that such isolates occur in potato plants with stem-mottle disease. These isolates were identified by two tests. In the first, TRV nucleoprotein particles were produced in plants inoculated with a mixture of nucleic acid from diseased potato leaves and non-infective RNA-2 of an authentic culture of TRV, showing that the TRV genome had been reconstituted. In the second test, TRV RNA was identified in nucleic acid from diseased potato leaves by hybridization to 3H-labelled DNA complementary in nucleotide sequence to RNA-1 of the PRN strain of TRV. This cDNA test consistently identified TRV in stem-mottle affected potato plants grown in the glasshouse or in field crops. Although particle-producing isolates of TRV have been reported in potato, stem-mottle disease is apparently usually caused by infection with NM isolates.     

Introduction. The investigation of lexical semantic representation in Alzheimer''s disease

In northern blots, cDNA probes prepared to soil-borne wheat mosaic virus (SBWMV) RNA-1 and RNA-2 hybridized to RNA-1 and RNA-2, respectively, from a UK isolate of oat golden stripe virus (OGSV), as well as to their homologous RNAs. RT-PCR was used to amplify, clone and sequence a region of about 750 nucleotides spanning the capsid protein gene and part of the readthrough protein on RNA-2 from OGSV, a French isolate of SBWMV and two stable deletion mutants (Lab1 and Okl-7) of SBWMV isolates from Nebraska and Oklahoma respectively. There was very high (96.7-99.1%) nucleotide homology between all these sequences and the wild-type SBWMV sequences from Nebraska and Oklahoma. OGSV was more similar to SBWMV from France and Nebraska than were any of the isolates to SBWMV from Oklahoma. Of the few differences in the deduced amino acid sequences of the capsid proteins from the different isolates, OGSV differed from all SBWMV isolates only in one amino acid (isoleucine for valine at position 88). The high degree of similarity suggests that OGSV may best be classified as an oat strain of SBWMV.     

Diurnal and ultradian rhythmicity of plasma leptin: effects of gender and adiposity

Cloning and sequencing of the circular, single-stranded DNA of one isolate of psittacine beak and feather disease virus (BFDV) demonstrate a genome composed of a circular molecule of 1993 nucleotide bases. An analysis of the assembled replicative form demonstrated seven open reading frames (ORFs) (three in the virion strand and four in the complementary strand), potentially encoding seven viral proteins of >8.7 kDa. High amino acid sequence similarity was demonstrated between a potential 33.3-kDa protein product of ORF1 of BFDV and the replicase-associated protein of porcine circovirus (PCV), subterranean clover stunt virus, and faba bean necrotic yellows virus. However, significant similarity in nucleotide or amino acid sequences was not present between BFDV and chicken anaemia virus. A potential stem-loop structure similar to that found in PCV and plant circoviruses was present in the putative encapsidated strand of the BFDV genome. At the top of this structure, a nonanucleotide motif (TAGTATTAC) similar to that of PCV, plant circoviruses, and geminiviruses also was recognised. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1% identity, and in both viruses, ORF2 is located on the complementary strand, beginning close to or within the hairpin stem. Our findings provide further evidence of a close relationship among BFDV, PCV, and plant circoviruses but not chicken anaemia virus.     

Cloning and characterization of mycovirus double-stranded RNA from the plant pathogenic fungus, Fusarium solani f. sp. robiniae

A mycovirus (named FusoV) from the phytopathogenic fungus, Fusarium solani f. sp. robiniae SUF704, has two kinds of double-stranded (ds) RNA genomes, designated M1 and M2. The cDNAs were constructed from FusoV genomic dsRNAs. The sequences of M1 and M2 cDNAs comprised 1645 and 1445bp, respectively. Sequence analysis showed that each dsRNA had a single long open reading frame (ORF) on only one of the strands. M1 ORF encodes a 519-amino acid residue polypeptide with a predicted molecular mass of 60 kDa. RNA-dependent RNA polymerase-conserved motifs were identified in the predicted amino acid sequence, and the polymerase synthesized dsRNA in vitro. The M2 ORF encodes a polypeptide of 413 amino acid residues with a predicted molecular mass of 44 kDa. The predicted amino acid sequence contained the sequence corresponding to those found in the purified 44-kDa capsid protein of FusoV.     

Cloning and sequence analysis of cDNA encoding a putative juvenile hormone esterase from the Colorado potato beetle

In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5'RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequence of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs.     

Isolation of an ovine genomic sequence containing the full-length angiotensin II type-1 receptor

We have isolated from a genomic library using PCR amplification an 1171 base sequence containing a putative ovine AT1-R protein coding sequence of 1080 bases. As expected the protein coding sequence is of greater than 99% homology to the partial protein coding sequence reported by Robillard et al, with only one base difference. Relative to other species, highest homology at the level of the cDNA protein coding sequence is to bovine (97.6%) and lowest homology to rat Type 1a (83.3%). The predicted protein amino acid sequence in turn encodes a protein with the properties of a seven alpha-helix transmembrane receptor (by TMPred) sharing closest homology (98.6%) to the bovine receptor and lowest to the rat Type 1a (90.2%). As expected from such a high degree of interspecies homology, amino acids identified by site-directed mutagenesis of the human or rat AT1A-R as involved in binding and action of AII are very highly conserved in the ovine sequence. In addition, both bovine and ovine AT1-R are known to exhibit lower affinity for DuP753 than human AT1-R, and in bovine AT1-R this has been suggested to coincide with the amino acid substitutions Ala->Thr (163) and Leu->Met (262) relative to the human sequence. Our ovine AT1-R cDNA sequence shares these same bovine substitutions.     

Expression and regulation of protein inhibitor of neuronal nitric oxide synthase in ventilatory muscles

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.     

The somatostatin analogue octreotide protects against ethanol-induced microcirculatory stasis and elevated vascular permeability in rat gastric mucosa

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)- RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)- RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

Banana bunchy top virus DNA-3 encodes the viral coat protein

Banana bunchy top virus (BBTV) has a multicomponent genome consisting of at least six circular single-stranded DNAs each with a single large open reading frame (ORF) in the virion sense. A protein of approximately 20 kDa has been associated with purified virions and is assumed to be the viral coat protein. The N-terminus of this protein was sequenced and compared to the predicted amino acid sequence of the large ORF of BBTV DNA-1 to 6. This comparison indicated that the ORF of BBTV DNA-3, which potentially encodes a protein of 19.3 kDa, was the coat protein gene of BBTV. The ORF sequence of BBTV DNA-3 was cloned into a prokaryotic expression vector, pMAL-c2, and the resulting maltose binding-BBTV coat protein fusion product was purified and used for the production of polyclonal antiserum in a rabbit. The resultant antiserum was able to detect the presence of BBTV in infected leaf tissue confirming that the large virion sense ORF of BBTV DNA-3 encodes the viral coat protein.     

Characteristic distribution of bases and codons around the initiation and termination codons in whole reading frames in bacteria and yeast genomes

Recently the complete nucleotide sequence of the entire genome was determined for yeast and a few kinds of bacterium. To see characteristic features of base sequence in the cistron (actually the open reading frame, ORF) and in the regions around a cistron (ORF), the biases of appearance frequency of bases from the base ratio were studied statistically. In the regions before the base biases were observed. The characteristic base distribution patterns were similar to all the cases of bacteria, but different from yeast. The base biases are reflected on the appearance frequency of amino acids near the N-termini and C-termini of proteins. The characteristic biases found in the amino acid sequence of the N-terminal part of bacteria proteins are different from that in yeast proteins.     

Nuclear medicine approaches for detection of axillary lymph node metastases

Rhopalosiphum padi virus (RhPV) is an aphid virus that has been considered a member of the Picornaviridae based on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, and to a partial sequence from the 3' end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV, Drosophila C virus, Plautia stali intestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects.     

Molecular cloning and sequencing of the coat protein gene of a Nebraskan isolate of tobacco necrosis virus: the deduced coat protein sequence has only moderate homology with those of strain A and strain D

A partial nucleotide sequence spanning the coat protein (CP) gene of a Nebraskan isolate of tobacco necrosis virus (TNV-NE) has been determined. The sequence contains at least four open reading frames (ORFs). The 5'-terminal ORF encodes a protein that has 86% and 38% homology with the polymerases of strains A (TNV-A) and D (TNV-D), respectively. The second and third ORFs probably encode 10.7 kDa and 6.2 kDa proteins (p 10.7 and p 6.2). These are respectively 90% and 96% amino acid homologous encoded by similar ORFs in TNV-A but only 26% and 20% homologous with those in TNV-D. The fourth 3'-proximal ORF encodes the 30.3 kDa CP. The amino acid sequence of TNV-NE CP is only 51% and 44% homologous to those of TNV-A and TNV-D, respectively. Thus, the CP genes of TNV-NE, TNV-A, and TNV-D are quite different. Like the sequences to the 5' side of the CP gene, that of TNV-NE is more closely related to TNV-A than to TNV-D.     

Functional MRI of the lumbar spine in erect position in a superconducting open-configuration MR system: preliminary results

High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 micrograms/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 microgram/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 microgram/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 micrograms/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 micrograms/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 micrograms/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.     

Complete nucleotide sequence of wheat yellow mosaic bymovirus genomic RNAs

The complete sequences of wheat yellow mosaic bymovirus (WYMV) RNA1 and RNA2 were determined. RNA1 is 7636 nucleotides long [excluding the 3'-poly(A)], and codes for a 269 kDa polyprotein of 2,404 amino acids which contains the capsid protein (CP) at the C terminus and seven putative nonstructural proteins. RNA2 is 3,659 nucleotides long and codes for a polyprotein of 904 amino acids which contains a 28 kDa putative proteinase and a 73 kDa polypeptide. These functional proteins are arranged as in RNA1 and RNA2 of barley yellow mosaic bymovirus (BaYMV). Comparisons with the sequence reported for the 3' half of RNA1 of wheat spindle streak mosaic bymovirus (WSSMV) from Southern France show that WYMV and WSSMV have a similar genetic organization. However, WYMV and WSSMV share only 77% amino acid sequence identity in their deduced CPs in spite of their close serological relationship, and 74% nucleotide sequence identity in their 3' non-coding regions. Thus, the sequence data indicate that WYMV and WSSMV are not strains of the same virus, which has long been suggested, but are distinct virus species within the genus Bymovirus of the family Potyviridae.     

Mutations in Bartonella bacilliformis gyrB confer resistance to coumermycin A1

This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of approximately 77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants of B. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124-->Ser), Arg184-->Gln, and Thr214-->Ala or Thr214-->Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E. coli, Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.     

Synthesis and function of Actinomyces naeslundii T14V type 1 fimbriae require the expression of additional fimbria-associated genes

The nucleotide sequence of the chromosomal DNA flanking the Actinomyces naeslundii (formerly A. viscosus) T14V type 1 fimbrial structural subunit gene (fimP) was determined. Six open reading frames (ORFs), in the order 5' ORF3, ORF2, ORF1,fimP, ORF4, ORF5, ORF6 3', were identified. ORF1 encoded a protein of 408 amino acid residues (Mr = 39,270) and had significant sequence homology with the A. naeslundii T14V type 1 and A. naeslundii WVU45 type 2 fimbrial structural subunits. An in-frame fusion of ORF1 to the malE gene of the expression vector, pMAL-c2, yielded a protein that was immunostained with antibodies raised against the maltose binding protein and A. naeslundii T14V whole bacteria. Digestion of the fusion protein with factor Xa released a protein (apparent molecular mass of 34 kDa) that was immunostained only with the antibody directed against A. naeslundii T14V whole bacterial cells. Integration plasmids carrying a kanamycin resistance gene (kan) that was used to substitute for ORF1 or for DNA fragments internal to the coding region of the other five ORFs were used to transform A. naeslundii T14V. Neither type 1 fimbriae nor the 65-kDa fimbrial structural subunit was detected in mutants obtained by allelic replacement of ORF1 or ORF2. Mutants obtained by allelic replacement of ORF3 or ORF4 expressed only the 65-kDa fimbrial structural subunit. These mutants did not bind, in vitro, to proline-rich proteins that serve as the receptors for Actinomyces type 1 fimbriae. In contrast, a mutant in which the integration plasmid DNA had been inserted at a site close to the carboxyl terminus of ORF6 expressed type 1 fimbriae and had adherence properties similar to those observed in the wild-type strain. These results demonstrate the existence of additional genes near fimP that are likely to be involved in the synthesis and function of cell surface fimbriae of A. naeslundii T14V.     

KDEL receptor (Erd2p)-mediated retrograde transport of the cholera toxin A subunit from the Golgi involves COPI, p23, and the COOH terminus of Erd2p

To elucidate changes associated with the attenuated virulence in a modified live porcine reproductive and respiratory syndrome (PRRS) vaccine (Boehringer Ingelheim Animal Health, St. Joseph, MO), derived from an American prototype ATCC virus VR-2332, nucleotide sequence of 3' genome covering open reading frames (ORFs) 2 to 7 coding regions from the vaccine virus was determined by RT-PCR with two overlapping fragments. Comparisons showed 98 base changes (94 substitutions, 3 deletions, and 1 addition) out of 3318 nucleotides between the vaccine virus and its parental virus. There were 15, 26, 17, 29, 9, and 6 base substitutions in ORFs 2, 3, 4, 5, 6, and 7, respectively, resulting in 5, 13, 8, 13, 2, and 3 amino acid (a.a.) substitutions in their deduced proteins, respectively. Most of these a.a. substitutions were also present in 17 known virulent/wild type PRRS virus isolates from North America. However, there were 1, 4, 1, and 1 unique a.a. substitutions in the vaccine virus ORFs 2, 3, 4, and 5 deduced proteins, respectively. These unique amino substitutions may be responsible for the attenuated virulence in the vaccine virus.     

Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus

GP4 is a minor structural glycoprotein encoded by ORF4 of Lelystad virus (LV). When it was immunoprecipitated from cell lysates and extracellular virus of CL2621 cells infected with LV, it was shown to have an apparent molecular mass of approximately 28 and 31 kDa, respectively. This difference in size occurred because its core N-glycans were modified to complex type N-glycans during the transport of the protein through the endoplasmic reticulum and Golgi compartment. A panel of 15 neutralizing monoclonal antibodies (MAbs) reacted with the native GP4 protein expressed by LV and the recombinant GP4 protein expressed in a Semliki Forest virus expression system. However, these MAbs did not react with the GP4 protein of U.S. isolate VR2332. To map the binding site of the MAbs, chimeric constructs composed of ORF4 of LV and VR2332 were generated. The reactivity of these constructs indicated that all the MAbs were directed against a region spanning amino acids 40 to 79 of the GP4 protein of LV. Six MAbs reacted with solid-phase synthetic dodecapeptides. The core of this site consists of amino acids 59 to 67 (SAAQEKISF). Comparison of the amino acid sequences of GP4 proteins from various European and North American isolates indicated that the neutralization domain spanning amino acids 40 to 79 is the most variable region of GP4. The neutralization domain of GP4, described here, is the first identified for LV.