3D average human corneal models.

In this paper we propose a method of building an average model or atlas of the cornea based on topographic data. Specific models can be constructed for the left or right eye, gender, age, or ametropia, to assess differences and similarities. An application of this atlas construction methodology to the study of corneal shape evolution with age is presented. Results show significant differences between age groups. This numerical atlas could also be helpful in the design of algorithms targeting the detection of corneal shape abnormalities, such as keratoconus or previous laser surgery.

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity.

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m(7)GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m(7)GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.

Identification of a 37 kDa plant protein that interacts with the turnip mosaic potyvirus capsid protein using anti-idiotypic-antibodies.

Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV). The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies. A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies. The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced. These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa. Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein. The 37 kDa protein was localized in chloroplasts and was detected in other plant species.

Characterization of the expression and immunogenicity of the ns4b protein of human coronavirus 229E.

Sequencing of complementary DNAs prepared from various coronaviruses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression and immunogenicity of the ns4b gene product from strain 229E of human coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used to demonstrate the expression of ns4b in HCV-229E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice to evaluate whether myelin-viral protein cross-reactive antibody responses could be generated. Each immunogen induced specific but not cross-reactive antibodies. We conclude that ns4b is expressed in infected cells and is immunogenic, although this does not involve amino acids shared with a self protein, at least in the experimental conditions used.

Variability among turnip mosaic potyvirus isolates.

ABSTRACT Eight turnip mosaic potyvirus (TuMV) isolates from the Campania region of Italy were characterized. Experiments based on host range and symptomatology indicated that the isolates were biologically different. In addition, the isolates, with the exception of ITA1 and ITA3, were distinguished from each other by using a combination of monoclonal antibodies recognizing the coat protein. Single-strand conformation polymorphism (SSCP) analysis of the coat protein gene revealed that each isolate produced a specific SSCP profile, except for isolates ITA1 and ITA3. This study indicates that (i) even in a small geographical region, there is a great deal of variation in TuMV isolates; (ii) the use of a set of four differential hosts does not always specify the same pathotype in different environments; (iii) the TuMV isolates with the same pathotype on Brassica napus test lines can still differ in host range, symptoms, serology, and SSCP; and (iv) there was perfect correlation between the panel of antibodies and SSCP in differentiating among the isolates; ITA1 and ITA3 were indistinguishable by either assay.

Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system.

The yeast LexA interaction trap was used to screen a cDNA library from Arabidopsis thaliana in order to identify proteins that interact with the viral protein genome linked (VPg)-proteinase of turnip mosaic potyvirus. The screen allowed the isolation of four candidate cDNA clones. Clones pHC4, pHC21, and pHC40 were partially sequenced but no homologies to known proteins were found. However, the amino acid sequence deduced from the complete nucleotide sequence of pSW56 revealed that it was the eukaryotic initiation factor (iso) 4E [eIF(iso)4E]. Deletion analysis indicated that the VPg domain was involved in the interaction with the plant protein. Interaction between the viral protein and the cellular protein was confirmed by ELISA-based binding experiments. eIF(iso)4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus.

Subgroup specific protection of mice from respiratory syncytial virus infection with peptides encompassing the amino acid region 174-187 from the G glycoprotein: the role of cysteinyl residues in protection.

We identified subgroup specific protective epitopes represented by the amino acid regions 174-187 and 171-187 of the G glycoproteins from respiratory syncytial virus (RSV), subgroups A and B. Mice immunized with coupled synthetic peptides corresponding to either the region 174-187 containing a Cys186-->Ser substitution or to the native region 171-187 were completely resistant to RSV infection but only to the respective virus. The protective activities of the peptides 174-187 were dependent on the Cys186-->Ser substitution. In addition, a recombinant protein representing the region 125-203 of the A subgroup G glycoprotein expressed in Escherichia coli was capable without further treatment to completely protect animals against RSV subgroup A infection. We show that the combinations of cysteinyl residues (positions 173, 176, 182, and 186) retained within either synthetic peptides or the recombinant protein G125-203 greatly influenced their protective activities. This indicates that the region 171-187 is essential for the protection conferred by the G125-203 protein. Furthermore, our results strongly suggest that the peptides' and recombinant protein's potencies are a function of a loop-like structure which is stabilized by intramolecular disulfide linkages between Cys176-Cys182 and Cys173-Cys186. This is further supported by the observation that chemical blocking of the sulfidryl groups in synthetic peptides completely eliminated their protective activity.

Purification of turnip mosaic potyvirus viral protein genome-linked proteinase expressed in Escherichia coli and development of a quantitative assay for proteolytic activity.

The 49-kDa, nuclear inclusion a-like, viral protein genome-linked proteinase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromatographies. Two forms of VPg-Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N-terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capable of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH2, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gln and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPg-Pro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH-activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.

Evidence for an internal ribosome entry site within the 5' non-translated region of turnip mosaic potyvirus RNA.

The genomic RNA of potyviruses has a characteristic 5' non-translated region (5'NTR) to which a viral protein, VPg, is covalently attached. This suggests that the viral RNA lacks a conventional cap structure and thus its translation may not proceed in the same way as most cellular mRNAs. To investigate the role of the 5'NTR during translation, various derivatives of the turnip mosaic potyvirus (TuMV) leader were fused to the reporter gene beta-glucuronidase (GUS). These constructs were used to monitor the efficiency of translation in vitro in a rabbit reticulocyte lysate and in planta following microprojectile DNA delivery into tobacco cell suspensions. GUS transcripts fused with the TuMV 5'NTR, whether they were capped or not, were efficiently translated, whereas GUS transcripts without the viral leader needed to be capped for expression. When transcripts of the viral leader were supplied in excess over functional transcripts, translation was inhibited in a dose-dependent manner. Similarly, transcripts synthesized from the reverse complement of the 5'NTR inhibited translation to the same extent as the wild-type sequence, indicating that cap independence was not conferred by a specific sequence within the viral leader. A stable hairpin loop was placed in front or after the viral sequence. This hairpin loop normally prevented translation of control GUS transcripts but when the viral leader was positioned after it a significant level of GUS activity was measured, whether the transcripts were capped or not. On the other hand, when the hairpin loop was positioned after the viral leader, no GUS activity was measured. These results suggested that ribosomes bound to an internal site within the TuMV 5'NTR and then presumably scanned the sequence for the initiator AUG.

Nucleic acid-binding properties of the P1 protein of turnip mosaic potyvirus produced in Escherichia coli.

The N-terminal P1 protein of turnip mosaic potyvirus (TuMV) polyprotein was overexpressed in Escherichia coli, purified by metal chelation chromatography under denaturing conditions and renatured. U.v. cross-linking experiments indicated that the recombinant protein interacted with RNA, and gel retardation electrophoresis demonstrated that more than one molecule of P1 bound one molecule of RNA. Formation of the protein-RNA complexes was dependent on the conformational state of P1 and was stable at relatively high concentrations of NaCl. P1 had the ability to bind ssRNA and ssDNA, with similar affinity, but was not able to bind to dsDNA. The TuMV protein had the additional characteristic of binding dsRNA with affinity similar to that observed with single-stranded nucleic acids.

Genetic mapping of turnip mosaic virus resistance in Lactuca sativa.

Presence of the dominant Tu gene in Lactuca sativa is sufficient to confer resistance to infection by turnip mosaic virus (TuMV). In order to obtain an immunological assay for the presence of TuMV in inoculated plants, the TuMV coat protein (CP) gene was cloned by amplification of a cDNA corresponding to the viral genome using degenerate primers designed from conserved potyvirus CP sequences. The TuMV CP was overexpressed in Escherichia coli, and polyclonal antibodies were produced. To locate Tu on the L. sativa genetic map, F3 families from a cross between cvs "Cobbham Green" (resistant to TuMV) and "Calmar" (susceptible) were genotyped for Tu. Families known to be recombinant in the region containing Tu were infected with TuMV and tested by the indirect enzyme-linked immunosorbent assay (ELISA) using the anti-CP serum. This assay placed Tu between two random amplified polymorphic DNA (RAPD) markers and 3.2 cM from Dm5/8 (which confers resistance to Bremia lactucae). Also, bulked segregant analysis was used to screen for additional RAPD markers tightly linked to the Tu locus. Five new markers linked to Tu were identified in this region, and their location on the genetic map was determined using informative recombinants in the region. Six markers were identified as being linked within 2.5 cM of Tu.

The complete nucleotide sequence of turnip mosaic potyvirus RNA.

The complete RNA genome of turnip mosaic potyvirus (TuMV) was amplified by seven consecutive reverse transcriptase-polymerase chain reactions and cloned into pUC9. The viral RNA is 9830 nucleotides long and contains a single open reading frame (ORF) of 9489 bases encoding a large polyprotein of 3863 amino acids with a calculated M(r) of 358,000. The non-coding region (NCR) preceding the ORF is 129 nucleotides long and has a high AU content (70%). Its predicted secondary structure is characterized by a hairpin loop with a free energy loss of -69.9 kJ/mol. The termination codon is followed by an AU-rich NCR of 209 bases, excluding the poly(A) tail. Seven potential nuclear inclusion a proteinase (NIa-Pro) recognition heptapeptides are found in the polyprotein. Their sequences agree with consensus potyviral NIa-Pro cleavage sequences except for that at the 6K-VPg site, which is characterized by a glutamic acid residue preceding the hydrolysed peptide bond. The TuMV proteins are similar to their corresponding potyviral proteins.

Release of a 22-kDa protein derived from the amino-terminal domain of the 49-kDa NIa of turnip mosaic potyvirus in Escherichia coli.

The coding region for the precursor 6K-small nuclear inclusion a (NIa) protein and for the NIa protein of turnip mosaic potyvirus (TuMV) were introduced into the plasmid pET-11d for high-level expression in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses of E. coli proteins showed that the NIa protein underwent endoproteolysis and released a 22-kDa polypeptide. NH2-terminal amino acid sequencing of the recombinant 22-kDa protein was performed and was identical to the predicted amino end of the NIa protein. Site-directed mutagenesis confirmed that the hydrolysis was associated with the NIa proteolytic activity and that the proteinase recognized a Glu residue within an amino acid sequence found in the NIa protein which fitted the TuMV consensus cleavage site sequence. Fusion of the 6K protein with the NIa protein partially inhibited the hydrolytic reaction. The recombinant 22-kDa protein is likely the VPg of TuMV.

Cloning, characterization, and expression of a cDNA encoding a 50-kilodalton protein specifically induced by cold acclimation in wheat.

We have isolated, sequenced, and expressed a cold-specific cDNA clone, Wcs120, that specifically hybridizes to a major mRNA species of approximately 1650 nucleotides from cold-acclimated wheat (Triticum aestivum L.). The accumulation of this mRNA was induced in less than 24 hours of cold treatment, and remained at a high steady-state level during the entire period of cold acclimation in the two freezing-tolerant genotypes of wheat tested. The expression of Wcs120 was transient in a less-tolerant genotype even though the genomic organization of the Wcs120 and the relative copy number were the same in the three genotypes. The mRNA level decreased rapidly during deacclimation and was not induced by heat shock, drought, or abscisic acid. The Wcs120 cDNA contains a long open reading frame encoding a protein of 390 amino acids. The encoded protein is boiling stable, highly hydrophilic, and has a compositional bias for glycine (26.7%), threonine (16.7%), and histidine (10.8%), although cysteine, phenylalanine, and tryptophan were absent. The WCS120 protein contains two repeated domains. Domain A has the consensus amino acid sequence GEKKGVMENIKEKLPGGHGDHQQ, which is repeated 6 times, whereas domain B has the sequence TGGTYGQQGHTGTT, which is repeated 11 times. The two domains were also found in barley dehydrins and rice abscisic acid-induced protein families. The expression of this cDNA in Escherichia coli, using the T(7) RNA polymerase promoter, produced a protein of 50 kilodaltons with an isoelectric point of 7.3, and this product comigrated with a major protein synthesized in vivo and in vitro during cold acclimation.

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants.

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.

The use of PCR for cloning of large cDNA fragments of turnip mosaic potyvirus.

A method is described whereby turnip mosaic virus RNA (TuMV RNA) was reverse transcribed and the resulting cDNA amplified enzymatically using the Taq DNA polymerase and degenerate oligonucleotide primers. Two degenerate oligonucleotide primers based on regions of homology in the amino acid sequence of the cytoplasmic inclusion protein and the nuclear inclusion b protein from five potyviruses were synthesized. Polymerase chain reactions utilizing these degenerate primers in association with specific primers amplified a 1.2 kb and a 3.3 kb fragment. These amplified fragments were dC-tailed and cloned into pUC9. Their partial sequence, when compared to potyvirus sequences, showed that they were derived from TuMV RNA and approximately 4.4 kb of viral genome was cloned.

Sequence of the 3'-terminal region of turnip mosaic virus RNA and the capsid protein gene.

A sequence of 1801 nucleotides originating from the 3' end region of turnip mosaic virus (TuMV) RNA was cloned using the polymerase chain reaction and found to contain one long open reading frame (ORF). The amino acid sequence of three different regions of the isolated TuMV capsid protein (including the NH2 terminus) was determined and these partial sequences were found in the translation product predicted to be encoded by the large ORF. The data suggested that the TuMV capsid protein was a product arising from the maturation of a larger polyprotein, as observed for other potyviruses. Furthermore, the putative cleavage site corresponded to a glutamine-alanine dipeptide, a site commonly used in plant virus polyprotein processing. The capsid protein cistron was composed of 864 nucleotides and corresponded to a region encoding 288 amino acids with a calculated Mr of 33,186; the adjacent 3' non-coding region was 667 nucleotides long. The deduced amino acid sequence of the TuMV capsid protein is closely related to other potyvirus capsid proteins, with most of the variation being found within the NH2-terminal region.

Ruthenium ammine complexes as electron acceptors for growth stimulation by plasma membrane electron transport.

Ammineruthenium(III) complexes have been found to act as electron acceptors for the transplasmalemma electron transport system of animal cells. The active complexes hexaammineruthenium(III), pyridine pentammineruthenium(III), and chloropentaammineruthenium(III) range in redox potential (E'0) from 305 to -42 mV. These compounds also act as electron acceptors for the NADH dehydrogenase of isolated plasma membranes. Stimulation of HeLa cell growth, in the absence of calf serum, by these compounds provides evidence that growth stimulation by the transplasma membrane electron transport system is not entirely based on reduction and uptake of iron.

Detection in BHK cells of a precursor form for lamin A.

Lamins are structural proteins found in the fibrous lamina underlining the nuclear envelope. In vitro translation of polyadenylated RNA or polysomes followed by immunoprecipitation with a serum raised against BHK nuclear matrix proteins showed that lamin A (72 kD) is synthesized as a high molecular weight precursor (74 kD) (Laliberté et al., J Cell Biol 98 (1984) 980) [23]. We have thus investigated the presence in BHK cells of this putative precursor by in vivo labelling with [35S]methionine and immunoprecipitation of lamin proteins. Short labelling times, ranging from 5 to 60 min reveal the presence of the 74 kD protein. Pulse-chase experiments indicate that the half-life of the precursor is about 60 min. On two-dimensional gel, the 74 kD protein is resolved in a cluster of isovariants between pH 7.4 and 6.6, which are generally slightly more alkaline than their counterparts in lamin A. These results indicate that lamin A is synthesized as a precursor of 74 kD; the long half-life further suggests that pre-lamin A might accumulate in some sort of cellular pool before undergoing post-transcriptional modification(s) to give the mature form of lamin A.

Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A.

The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.