Cloning and Detection of DNA from a Nonculturable Plant Pathogenic Mycoplasma-like Organism.

The ability to detect, quantify, and differentiate nonculturable mycoplasma-like organisms (MLOs) would greatly facilitate epidemiological and taxonomical studies of this unique group of plant and insect pathogens. DNA isolated from extracts of insects infected with the Western X-disease MLO was cloned in Escherichia coli. X-disease-specific clones, when labeled and used as probes, readily detected X-disease MLOs in infected plants and insects but did not hybridize with DNA from healthy plants or insects, or from several other plant pathogenic MLOs or spiroplasmas. These methods provide both a sensitive diagnostic tool and a basis for genetically differentiating MLOs.

Interactions between viral coat protein and a specific binding region on turnip crinkle virus RNA.

The turnip crinkle virus coat protein binding sites in the ribonucleoprotein complex resulting from virion dissociation have been identified previously. In this study, RNA binding characteristics of viral coat protein to a region encompassing the protected RNA fragments Fa, Ff, and Fc (Fafc) have been investigated further using an RNA transcript (the Fafc fragment). These experiments have shown that coat protein requires no additional viral RNA elements to bind to this region. Such binding was shown to be specific for turnip crinkle virus coat protein using an ultra-violet light cross-linking assay. Gel mobility shift analyses demonstrated that the protein-RNA interactions produced two complexes: a homogeneous small ribonucleoprotein complex, and larger complexes which failed to migrate into gels. High salt and limiting protein concentrations favored the formation of the small ribonucleoprotein complex, whereas low salt and excess protein concentrations favored the larger complexes. RNA competition experiments demonstrated that small ribonucleoprotein complex formation coincided with specific RNA binding of the coat protein to the Fafc fragment. In addition, the coat protein possessed a poly(U)-binding site(s), which enabled it to interact with single-stranded RNA in a sequence non-specific manner to form large complexes. The results suggest that the coat protein contains both specific and non-specific RNA binding activities located at physically distinct sites. These results are consistent with the proposed assembly model for turnip crinkle virus.

Structure and assembly of turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA.

Structural studies of turnip crinkle virus have been extended to include the identification of high-affinity coat protein binding sites on the RNA genome. Virus was dissociated at elevated pH and ionic strength, and a ribonucleoprotein complex (rp-complex) was isolated by chromatography on Sephacryl S-200. Genomic RNA fragments in the rp-complex, resistant to RNase A and RNase T1 digestion and associated with tightly bound coat protein subunits, were isolated using coat-protein-specific antibodies. The identity of the protected fragments was determined by direct RNA sequencing. These approaches allowed us to study the specific RNA-protein interactions in the rp-complex obtained from dissociated virus particles. The location of one protected fragment downstream from the amber terminator codon in the first and largest of the three viral open reading frames suggests that the coat protein may play a role in the regulation of the expression of the polymerase gene. We have also identified an additional cluster of T1-protected fragments in the region of the coat protein gene that may represent further high-affinity sites involved in assembly recognition.

Structure and assembly of turnip crinkle virus. IV. Analysis of the coat protein gene and implications of the subunit primary structure.

The structure of the turnip crinkle virus (TCV) coat protein and coat protein gene has been examined by cDNA cloning, nucleotide sequencing and high-resolution mRNA mapping. We have cloned a 1450-nucleotide cDNA fragment, representing the 3' end of the TCV genome, using genomic RNA polyadenylated in vitro as the reverse transcriptional template. Nucleic acid sequence analysis reveals the presence of a 1053 nucleotide open reading frame capable of encoding a protein of 38,131 Mr, identified as the coat protein subunit. The 1446 base subgenomic mRNA for the coat protein, mapped using high-resolution primer extension techniques, contains a 137 nucleotide leader sequence upstream from the initiation codon. We have characterized a second subgenomic RNA of approximately 1700 bases, roughly 250 nucleotides longer than the 1446 base species in the 5' direction. No TCV-related RNAs are polyadenylated in vivo. The derived amino acid sequence of the TCV coat protein has been built into the 3.2 A resolution electron density map of TCV reported in paper I of this series. We describe here some of the important features of the structure. Alignment of the three-dimensional structures of tomato bushy stunt virus and southern bean mosaic virus shows significant sequence relationships in the arms and S domains, although the conserved residues do not appear to have any special role in stabilizing the beta-barrel fold or in mediating subunit interactions. The sequences of TCV and carnation mottle virus can be aligned. Comparisons among the four are discussed in terms of the organization of the S domain.

Cloning, characterization and transient expression of the gene encoding a rice U3 small nuclear RNA.

A rice U3 small nuclear RNA (snRNA)-encoding gene has been isolated. The coding region of this gene contains all five conserved sequence boxes common to plant U3 snRNAs. The upstream and downstream regions of the gene harbour characteristic sequence elements required for transcription with RNA polymerase III (pol III), as well as three monocot-specific promoter (MSP) elements [Connelly et al., Mol. Cell Biol.14 (1994) 5910-5919], two of them comprising a palindromic G+C-rich segment. The sequence (TTTAAAA) of the TATA-box in this gene does not fit the established consensus [Marshallsay et al., Plant Mol. Biol. 19(1992) 973-983], making this gene unique among reported snRNA-encoding genes of plants. An RNase protection assay showed that the gene is expressed properly in cucumber protoplasts. We thus suggest that, with other promoter elements present, the TATA-box for RNA pol III-specific snRNA-encoding genes may not be as well conserved as that for RNA pol II-specific genes.

Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector.

Production of structural proteins from foot-and-mouth disease virus (FMDV) and bovine herpes virus (BHV-1) in Nicotiana benthamiana through the use of a tobacco mosaic virus-based vector (TMV-30B) has been reported previously. The development of the TMV-30B-HISc vector, a new version that adds a C-terminal histidine (His) sequence to the foreign protein expressed is described. Coding sequences from the FMDV VPl protein and the core protein, p24, from a clade C HIV-1 isolate from Zambia were cloned into the new vector and infective RNAs were generated for each construct to inoculate N. benthamiana plants. His-tagged proteins were purified from inoculated leaves using immobilized metal affinity chromatography (IMAC) as detected by Coomassie blue staining and proteins were further characterized in Western blot assays using a commercial anti-6xHis mAb and specific polyclonal antisera for each protein. While yields obtained for the VPl-His protein after purification were similar to those in crude extracts obtained with the previous TMV-VPl vector, p24-His yields were 10-15 times higher than those of VPl-His. Twenty-five grams of TMV-p24-HISc inoculated leaves were processed to obtain 2.5 mg of isolated p24-His and the recombinant protein was inoculated in rabbits to test immunogenicity and antigenic integrity of the plant-produced p24-His. Animals developed a strong and specific humoral response to the p24-His after the first booster and immune sera was able to recognize the native p24 from a different clade expressed on the surface of the HIV-1 chronically infected HUT78/ARV T-cell line. Importantly, the recombinant p24-His proved its efficiency by confirming the serology of 117 samples previously tested by two rapid HIV-1 tests, thus representing an excellent alternative for production of highly specific diagnostic reagents for HIV endemic regions in the developing world.

Maximum shortening velocity and myosin heavy-chain isoform expression in human masseter muscle fibers.

While human masseter muscle is known to have unusual co-expression of myosin heavy-chain proteins, cellular kinetics of individual fibers has not yet been tested. Here we examine if myosin heavy-chain protein content is closely correlated to fiber-shortening speed, as previously reported in other human muscles, or if these proteins do not correlate well to shortening speeds, as has been demonstrated previously in rat muscle. Slack-test recordings of single, skinned human masseter fibers at 15 degrees C revealed maximum shortening velocities generally slower and much more variable than those recorded in human limb muscle. The slowest fiber recorded had a maximum shortening velocity (V0) value of 0.027 muscle lengths x s(-1), several times slower than the slowest type I fibers previously measured in humans. By contrast, human limb muscle controls produced V0 measurements comparable with previously published results. Analysis by gel electrophoresis found 63% of masseter fibers to contain pure type I MyHC and the remainder to co-express mostly type I in various combinations with IIA and IIX isoforms. V0 in masseter fibers forms a continuum in which no clear relationship to MyHC isoform content is apparent.

Abundant expression of myosin heavy-chain IIB RNA in a subset of human masseter muscle fibres.

Type IIB fast fibres are typically demonstrated in human skeletal muscle by histochemical staining for the ATPase activity of myosin heavy-chain (MyHC) isoforms. However, the monoclonal antibody specific for the mammalian IIB isoform does not detect MyHC IIB protein in man and MyHC IIX RNA is found in histochemically identified IIB fibres, suggesting that the IIB protein isoform may not be present in man; if this is not so, jaw-closing muscles, which express a diversity of isoforms, are likely candidates for their presence. ATPase histochemistry, immunohistochemistry polyacrylamide gel electrophoresis and in situ hybridization, which included a MyHC IIB-specific mRNA riboprobe, were used to compare the composition and RNA expression of MyHC isoforms in a human jaw-closing muscle, the masseter, an upper limb muscle, the triceps, an abdominal muscle, the external oblique, and a lower limb muscle, the gastrocnemius. The external oblique contained a mixture of histochemically defined type I, IIA and IIB fibres distributed in a mosaic pattern, while the triceps and gastrocnemius contained only type I and IIA fibres. Typical of limb muscle fibres, the MyHC I-specific mRNA probes hybridized with histochemically defined type I fibres, the IIA-specific probes with type IIA fibres and the IIX-specific probes with type IIB fibres. The MyHC IIB mRNA probe hybridized only with a few histochemically defined type I fibres in the sample from the external oblique; in addition to this IIB message, these fibres also expressed RNAs for MyHC I, IIA and IIX. MyHC IIB RNA was abundantly expressed in histochemical and immunohistochemical type IIA fibres of the masseter, together with transcripts for IIA and in some cases IIX. No MyHC IIB protein was detected in fibres and extracts of either the external oblique or masseter by immunohistochemistry, immunoblotting and electrophoresis. Thus, IIB RNA, but not protein, was found in the fibres of two different human skeletal muscles. It is believed this is the first report of the substantial expression of IIB mRNA in man as demonstrated in a subset of masseter fibres, but rarely in limb muscle, and in only a few fibres of the external oblique. These findings provide further evidence for the complexity of myosin gene expression, especially in jaw-closing muscles.

Doxycycline-induced esophageal ulceration in the U.S. Military service.

U.S. military forces are frequently deployed with little warning to regions of the world where chloroquine-resistant malaria is endemic. Doxycycline is often used for malaria chemoprophylaxis in these environments. The use of doxycycline can be complicated by esophageal injury. Two cases of esophageal ulceration will be discussed, followed by a review of the literature. Doxycycline causes esophageal injury through a combination of drug-specific factors, the circumstances of drug administration, and individual patient conditions. Patients with dysphagia attributable to esophageal ulceration are managed by intravenous fluid support and control of gastric acid reflux until their symptoms resolve over 5 to 7 days. The risk of esophageal injury can be minimized by use of fresh capsules, drug administration in the upright position well before lying down to sleep, and drinking at least 100 ml of water after swallowing the medication.

What is SNOMED CT and why should the ISSHP care?

SNOMED CT (Systematized NOmenclature of MEDicine Clinical Terms) is a standardized multilingual healthcare terminology. It was developed to meet the needs of our electronic world so that care can be documented and clinicians can retrieve and transmit data in electronic format. It is anticipated that SNOMED CT will provide the core general terminology for electronic health records and, as such, replace existing classification systems such as the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10). At present, there is no special interest group for the hypertensive disorders of pregnancy (HDP) within the SNOMED CT initiative. We believe that members of the ISSHP, and others interested in the HDP, should take a leadership role in this regard for a number of reasons.

Optimizing Rayleigh laser guide star range-gate depth during initial loop closing.

The use of a Rayleigh laser guide star (LGS) as a wavefront reference for adaptive optics (AO) allows complete control over the distance to, and depth of, the slice of the Rayleigh plume that is selected. By altering the LGS range-gate depth (RGD) and distance at three defined stages during closed-loop operations, the LGS performance can be optimized. For the given example of a 20 km LGS on a 4.2 m telescope the application of this technique would increase the permissible RGD from 200to930 m after optimization. This can allow either an increase in LGS altitude or an increase in wavefront sensor frame rate, thereby increasing AO system performance.

Evidence for a single infectious species of potato spindle tuber viroid.

Potato spindle tuber viroid (PSTV) has been separated into two electrophoretic species on polyacrylamide gels containing 8 M urea at 60 degrees. Only the slower-migrating electrophoretic form was infectious and is presumably the circular form of the molecule. Both forms had similar base compositions with a 55-58% GC content, similar to the citrus exocortis viroid. Labeling studies in vivo suggest that the viroid is not methylated.

Skeletal muscle function and fibre types: the relationship between occlusal function and the phenotype of jaw-closing muscles in human.

Mammalian skeletal muscle cells are composed of repeated sarcomeric units containing thick and thin filaments of myosin and actin, respectively. Excitation of the myosin ATPase enzyme is possible only with presence of Mg-ATP and Ca(2+). Skeletal muscle fibres may be classified into several types according to the isoform of myosin they contain. Nine isoforms of myosin heavy chain are known to exist in mammalian skeletal muscle including type I, IIA, IIB, IIX, IIM, alpha, neonatal, embryonic, and extra-ocular. Healthy adult human limb skeletal muscle contains type I, IIA, IIB, and IIX myosin heavy chains. The jaw-closing muscles of most carnivores and primates have tissue-specific expression of the type IIM or 'type II masticatory' myosin heavy chain. Adult human jaw-closing muscles, however, do not contain IIM myosin. Rather, they express type I, IIA, IIX (as in human limb muscle), and myosins typically expressed in developing or cardiac muscle. The morphology of human jaw-closing muscle fibres is also unusual in that the type II fibres are of smaller diameter that type I fibres, except in cases of increased function and hypertrophy. This paper describes the relationship of fibre types and motor unit function to changes in human occlusion and masticatory activity. Refereed Scientific Paper

RNA determinants of junction site selection in RNA virus recombinants and defective interfering RNAs.

RNA recombination plays an important role in the diversification and evolution of RNA viruses. Most of these events are believed to be mediated by an actively copying viral replicase switching from a donor template to an acceptor template, where it resumes synthesis. In addition, intramolecular replicase-mediated events (i.e., rearrangements) can lead to the generation of replicable deleted forms of a viral genome, termed defective interfering (DI) RNAs. To gain further insight into the recombination process, the effect of various primary and secondary structures on recombination site selection in vivo was examined using plant RNA tombusviruses. The effect of sequence identity and complementarity on deletion events that generate DI RNAs was also investigated. Our results suggest that (1) 5' termini and strong hairpin structures in donor templates represent preferred sites for recombinations, (2) junction sites in acceptor templates do not occur in double-stranded regions, (3) nucleotide homology can shift donor and acceptor recombination sites closer to regions of identity and, (4) both sequence identity and complementarity can direct deletion sites in DI RNAs. These results further define RNA determinants of tombusvirus RNA recombination and rearrangement.

Quantitative analysis of the binding of turnip crinkle virus coat protein to RNA fails to demonstrate binding specificity but reveals a highly cooperative assembly interaction.

An element(s) within a 386-nucleotide segment (TCV-386 RNA) of the turnip crinkle virus (TCV) genome was previously implicated in virus assembly nucleation. To localize the proposed high-affinity binding determinants, we analyzed the ability of the coat protein to bind the full-length and truncated derivatives of the TCV-386 RNA using gel retardation and nitrocellulose filter retention assays. Quantitation of the binding data indicated that the coat protein did not preferentially recognize a particular region of the RNA. Moreover, the affinity of the coat protein for the TCV-386 RNA [apparent dissociation constant (Kd) approximately 0.5 microM) did not appreciably differ from its affinity for other comparably sized RNAs tested, including nonviral RNAs. However, the quantitative studies also suggested that the coat protein binds RNA in a cooperative manner and this was supported by evidence that all of the RNAs examined were bound by multiple copies of the coat protein. Based on the number of binding intermediates which could be detected in titrations involving RNAs of different chain length, it appeared that each coat protein binding unit occupies 35-40 nucleotides. Our results demonstrate that encapsidation of TCV RNA results from highly cooperative binding of the coat protein on the large viral genome. However, we were not able to confirm that assembly is mediated by initiation at a high-affinity binding site on the viral RNA.

Nonhomologous RNA recombination in tombusviruses: generation and evolution of defective interfering RNAs by stepwise deletions.

We used a protoplast system to study the mechanisms involved in the generation and evolution of defective interfering (DI) RNAs of tomato bushy stunt tombusvirus (TBSV). Synthetic transcripts corresponding to different naturally occurring TBSV DI RNAs, or to various artificially constructed TBSV defective RNAs, were analyzed. The relative levels of competitiveness of different DI RNAs were determined by coinoculating their corresponding transcripts into protoplasts along with helper genomic RNA transcripts and monitoring the level of DI RNA accumulation. Further studies were performed to assess the contribution of naked DI RNA stability and DI RNA encapsidation efficiency to the observed levels of competitiveness. In addition, the ability of various defective RNAs to evolve to alternative forms was tested by serially passaging protoplast infections initiated with transcripts corresponding to helper genomic RNA and a single type of defective RNA. These studies, and the analysis of the sequences of observed recombinants, indicate that (i) replication competence is a major factor dictating DI RNA competitiveness and is likely a primary determinant in DI RNA evolution, (ii) DI RNAs are capable of evolving to both smaller and larger forms, and the rates at which various transitions occur differ, (iii) DI RNA-DI RNA recombination and/or rearrangement is responsible for the formation of the evolved RNA molecules which were examined, and (iv) sequence complementarities between positive- and negative-sense strands in the regions of the junctions suggest that, in some cases, base pairing between an incomplete replicase-associated nascent strand and acceptor template may mediate selection of recombination sites. On the basis of our data, we propose a stepwise deletion model to describe the temporal order of events leading to the formation of tombusvirus DI RNAs.

De novo generation and accumulation of tomato bushy stunt virus defective interfering RNAs without serial host passage.

Studies were initiated to monitor generation and accumulation of defective interfering (DI) RNAs associated with tomato bushy stunt virus (TBSV) in the absence of serial, high multiplicity of infection passage. Infections were initiated in Nicotiana clevelandii host plants and protoplast cell suspensions by inoculation with in vitro-synthesized infectious TBSV RNA transcripts containing a genomic marker. The infections were then assayed for DI-size RNAs by both Northern blot analysis and reverse transcription coupled with PCR amplification. DI-size RNAs could not be detected by Northern blot analysis in either plants or protoplasts after an evident viral infection. However, RT-PCR amplification permitted the isolation of DI-size cDNAs (600-700 nt) from plant, but not protoplast, infections as early as 8 days postinoculation. Sequence analysis of these DI-size cDNA clones revealed that they contained the four conserved regions found in all previously identified competent DI RNAs. Several DI RNA clones contained the genomic marker which confirmed their de novo generation from the input transcript inoculum. A comparison of the nucleotide sequence of these clones to previously sequenced DI RNAs, isolated from plants after multiple passages, showed that differences existed at the junctions between regions. These results demonstrate that a heterogeneous population of DI RNAs accumulated in plants in the absence of serial host passage. In addition, the similarity of these DI RNAs to previously characterized DI RNAs that accumulate upon passage indicates that evolution can occur very rapidly within the initially inoculated plant.

A new iridovirus of two species of terrestrial isopods, Armadillidium vulgare and Porcellio scaber.

A new iridovirus, herein named isopod iridescent virus (IIV), was isolated from two species of terrestrial isopods in Berkeley, Calif. Infected individuals of Armadillidium vulgare and Porcellio scaber (Crustacea:Isopoda) appeared purple in color; in thin sections, hemocytes, nerve cells, and fat body cells contained cytoplasmic aggregations of virions. Isolated particles, 135 nm in diameter in negative stain, contained DNA had included at least 19 polypeptide components. The virus was transmitted per os and by injection to healthy isopods, and by injection to larvae of a coleopteran. IIV from the isopod hosts differed from Tipula iridescent virus (TIV) in buoyant density, molecular weights of major polypeptides, and serological reactions with antisera to IIV and TIV.