First Report of Potato mop-top virus on Potato from the United States.

Potato mop-top virus (PMTV) is a tripartite pomovirus vectored by the powdery scab plasmodiophoromycete Spongospora subterranea pv. subterranea (1). PMTV occurs on potato (Solanum tuberosum) in Europe, the Andes, Asia, and Canada. Internal necrotic arc and fleck tuber symptoms ("spraing") may reduce commercial acceptance of some cultivars (3). PMTV symptoms were discovered in 'Shepody' tubers at the Aroostook Research Farm, Presque Isle, ME in May 2002 and subsequently in 'Russet Burbank' tubers in commercial storage from the 2001 Maine crop. Symptomatic tubers exhibited single or multiple concentric necrotic arcs that were partial or complete, but exhibited no distinct external symptoms. The presence of PMTV in eight 'Shepody' tubers was indicated by positive enzyme-linked immunosorbent assay (ELISA; Adgen, Ltd., Auchincruive, Ayr, Scotland) and confirmed by reverse transcription polymerase chain reaction (RT-PCR). 'Russet Burbank' potatoes were visually diagnosed, and the corresponding halves of 128 symptomatic tubers were forwarded to the University of Maine and APHIS (Beltsville, MD). Of these, ELISA readings in Maine were strongly positive (>3 × background) for 88, ambiguous (1.5-3 × background) for 13, and negative for 27. Subsamples from these three categories were positive by PCR in 17 of 17, 9 of 9, and 12 of 14 cases, respectively. A similar rating, positive or ambiguous, in ELISA testing was identical for all but one case at Beltsville. Confirmation of PMTV required PCR testing, resulting in a characteristic PCR product of 401 bp that was generated from the coat protein coding region on RNA 2 (2) using the primer pair PMTV 1 5'-GCAGCCGTCGAGAATAGATA-3' (RNA nucleotides 316-335) and PMTV 4 5'-GCGAGTTGATGTGCC ACATT-3' (complementary to RNA 2 nucleotides 716-697). An immunocapture RT-PCR using this primer set and the coating antibody from the Adgen ELISA kit was also successful in detecting PMTV. In separate reactions, a second product of 646 bp was generated from the triple gene block on RNA 3 (4) using the primer pair PMTV 5 5'-GGTGAACACGAGGACAAGGT-3' (RNA 3 nucleotides 1417-1436) and PMTV 7 5'-AACAGTCCGGTCTTGTGAAC-3' (complementary to RNA 3 nucleotides 2063-2044). The sequence of these products was 98 to 100% identical to PMTV published sequences. The discovery of this virus will result in adjustments to U.S. and Canadian seed potato certification standards and symptom characterization for common North American cultivars. References: (1) R. A. C. Jones and B. D. Harrison. Ann. Appl. Biol 63:1, 1969. (2) S. Kashiwazak et al. Virology 206:701, 1995. (3) M. Sandgren et al. Am. J. Potato Res. 79:205, 2002. (4) K. P. Scott et al. J. Gen. Virol.75:3561, 1994.

A novel vertebrate myoglobin gene containing three A+T-rich introns is conserved among Antarctic teleost species which differ in myoglobin expression.

This report presents the first teleost myoglobin (Mb) genomic DNA sequence, several features of which are distinct from mammalian Mb genes. We have isolated and compared genomic clones of three closely related Antarctic icefish: Chionodraco rastrospinosus, which expresses Mb mRNA and functional polypeptide; Champsocephalus gunnari, which transcribes the Mb gene but does not produce polypeptide due to a 5-base pair (bp) frameshift insertion; and Chaenocephalus aceratus, which lacks both Mb protein and mRNA. The single-copy icefish Mb gene contains three introns: two at positions identical to those found in mammalian Mb and a novel intron located in the 5' untranslated region three nucleotides upstream from the initiator codon. All three introns are shorter than those found in mammalian Mb genes and exhibit a considerably higher A+T content. The entire Mb transcriptional unit is intact in C. aceratus, indicating that the failure to express this gene is not due to aberrations in the coding region, splice junctions, polyadenylation signals, or core promoter elements. The three icefish Mb sequences display an extreme degree of identity in the transcriptional unit and putative promoter region. In contrast, sequences 65 bp downstream from the polyadenylation site bear no homology among the three species, demonstrating that rapid sequence change has occurred in the 1 million years since the divergence of these species.

Two distinct types of fatty acid-binding protein are expressed in heart ventricle of Antarctic teleost fishes.

This report provides the first evidence for the existence of two distinct types of fatty acid-binding protein (FABP) in cardiac tissue of vertebrates. Four species of Antarctic teleost fish (Chaenocephalus aceratus, Cryodraco antarcticus, Gobionotothen gibberifrons and Notothenia coriiceps) exhibited two FABP mRNAs of 1. 0 kb and 0.8 kb, which we have termed Hh-FABP and Had-FABP (isolated from Heart tissue, with similarity to mammalian heart-type FABP or mammalian adipose-type FABP respectively). These FABP types appear to be products of distinct genes. Both FABP transcripts were abundant in cardiac and aerobic pectoral muscle. However, relative abundance of the two types varied distinctly among other tissues such as kidney, brain, spleen and white muscle. Neither FABP type was expressed in liver or intestine. The coding regions of Hh-FABP and Had-FABP cDNAs from the same species are only approximately 60% identical with one another. However, homologues of each FABP species, which exhibit >98% identity to their respective types, were isolated from three other Antarctic teleosts. Phylogenetic analysis of aligned amino-acid sequences places Hh-FABP with other vertebrate heart-type FABPs, and Had with adipose/cutaneous FABPs. Expression of two distinct FABPs in cardiac tissue of Antarctic teleosts may be related to their ability to both utilize fatty acid as the primary metabolic fuel and to store lipid intracellularly.

Conservation of the myoglobin gene among Antarctic notothenioid fishes.

We determined the myoglobin cDNA sequence for seven Antarctic notothenioid fish species. These data identify mutations in the myoglobin gene for Champsocephalus gunnari and Pagetopsis macropterus, two icefish species that lack detectable quantities of the polypeptide but express myoglobin mRNA. a third species lacking myoglobin polypeptide, Chaenocephalus aceratus, is devoid of myoglobin mRNA and accordingly failed to produce myoglobin products on polymerase chain reaction (PCR) amplification. Myoglobin cDNA sequences were highly conserved among the species the express the protein, particularly in the coding region. Sequence variation among the myoglobin-expressing channichthyid species was 2.0% to 2.9% in the coding region and 2.6% to 3.3% over the entire cDNA. The same extent of variation, 1.6% to 3.2% in the coding sequence and 2.8% to 3.7% overall, was observed between the icefishes and more distantly related, red-blooded nototheniid species. The two species expressing mutant myoglobin mRNA, C. gunnari and P. macropterus, exhibited the highest degree of sequence variation among the fish myoglobins examined. Drift in the myoglobin sequence in these two species, and conservation of myoglobin cDNA among fishes from two distinct families, suggest that a selective pressure operates to maintain myoglobin in the species that express the protein.

Kinetic characterization of myoglobins from vertebrates with vastly different body temperatures.

Fish myoglobins are structurally distinct from the previously characterized mammalian myoglobins. Teleost fishes express generally lower levels of myoglobin than those found in mammals. Although the oxygen binding affinity is essentially the same as mammalian myoglobins, oxygen dissociation rates and carbon monoxide combination rates of the teleost myoglobins studied are significantly faster. Thus, the kinetic parameters of myoglobin from two Antarctic teleost species, measured close to their body temperature of -1 degree C, are comparable to those of mammalian myoglobins with higher body temperatures. These data suggest myoglobins from Antarctic teleosts may function at extreme environmental temperatures.

Variable expression of myoglobin among the hemoglobinless Antarctic icefishes.

The important intracellular oxygen-binding protein, myoglobin (Mb), is thought to be absent from oxidative muscle tissues of the family of hemoglobinless Antarctic icefishes, Channichthyidae. Within this family of fishes, which is endemic to the Southern Ocean surrounding Antarctica, there exist 15 known species and 11 genera. To date, we have examined eight species of icefish (representing seven genera) using immunoblot analyses. Results indicate that Mb is present in heart ventricles from five of these species of icefish. Mb is absent from heart auricle and oxidative skeletal muscle of all species. We have identified a 0.9-kb mRNA in Mb-expressing species that hybridizes with a Mb cDNA probe from the closely related red-blooded Antarctic nototheniid fish, Notothenia coriiceps. In confirmation that the 0.9-kb mRNA encodes Mb, we report the full-length Mb cDNA sequence of the ocellated icefish, Chionodraco rastrospinosus. Of the eight icefish species examined, three lack Mb polypeptide in heart ventricle, although one of these expresses the Mb mRNA. All species of icefish retain the Mb gene in their genomic DNA. Based on phylogeny of the icefishes, loss of Mb expression has occurred independently at least three times and by at least two distinct molecular mechanisms during speciation of the family.

Translational arrest in hypoxic potato tubers is correlated with the aberrant association of elongation factor EF-1 alpha with polysomes.

Translation elongation factor EF-1 alpha became stably associated with potato tuber polysomes at the onset of hypoxia, coincident with a sharp rise in lactate and decrease in tissue pH. This aberrant association of EF-1 alpha with polysomes also occurred when aerobic tuber extracts were acidified in vitro. Upon resumption of protein synthesis, an increase in the steady-state levels of EF-1 alpha, and expression of an EF-1 alpha/GUS transgene was observed. These results indicate that translational arrest results from to the failure of EF-1 alpha to dissociate from ribosomes during the elongation cycle, and that restoration of protein synthesis is coordinated with expression of EF-1 alpha.

Biphasic Stimulation of Translational Activity Correlates with Induction of Translation Elongation Factor 1 Subunit [alpha] upon Wounding in Potato Tubers.

Potato (Solanum tuberosum) tubers exhibit an increase in translational activity in response to mechanical wounding. The response is biphasic, with an initial stimulation apparent within the first 2 h after wounding and a second increase occurring 12 to 24 h after wounding. Increased activity is apparent by measurement of protein synthesis both in vivo and in vitro using a cell-free extract. Accumulation of the translational elongation factor 1 subunit [alpha] (EF-1[alpha]) parallels translational activity. Changes in the steady-state level of EF-1[alpha] mRNA, and expression of a chimeric EF-1[alpha] promoter/[beta]-glucuronidase construct in transgenic potato tubers, indicate that the gene encoding EF-1[alpha] is transcribed during both periods of translational stimulation. These results indicate that stimulation of translational activity is coordinated with increased expression and accumulation of translation factors.

The heat shock response of an antarctic alga is evident at 5 degrees C.

A subtidal seaweed collected in antarctic waters, Plocamium cartilagineum (L. Dix.), displayed induction of mRNAs encoding the 70 kDa heat shock protein (HSP70) and the ubiquitin polyprotein (UBI) when incubated at 5 degrees C. Maximal induction of HSP70 mRNA was observed when the alga was incubated at 10 degrees C for 1 h. Incubations at higher temperatures or for longer periods reduced the amount of HSP70 mRNA detected. Incubations at 20 degrees C or greater resulted in cell death. These data indicate that dispite the unusually low temperature of induction, this macrophyte exhibits a heat shock response similar to that of other organisms at temperatures 5 to 10 degrees C above usual growth conditions.

Stress-Induced Translational Control in Potato Tubers May Be Mediated by Polysome-Associated Proteins.

Potato tubers exhibit distinct responses to wounding and hypoxia that include selective translation of stress-induced mRNAs. Newly synthesized wound-response mRNAs are bound to polysomes, whereas preexisting mRNAs are displaced and degraded. mRNAs that are induced and translated during hypoxic conditions are bound to ribosomes as expected. However, preexisting wound-response mRNAs whose translation is inhibited during hypoxia remain bound to polysomes, indicating that there are at least two distinct mechanisms by which translation is regulated in response to stress conditions. A 32-kD phosphoprotein is associated with polyribosomes from wounded tubers. This protein remains polysome bound as long as wound-response mRNAs are present, even during hypoxia when these mRNAs are no longer translated. However, association of the 32-kD protein with polysomes is not elicited by hypoxic stress alone. The kinase that phosphorylates this protein is active only for the first 24 hr after wounding and is not active during periods of hypoxia. This protein may mediate recognition of the wound-response mRNAs by ribosomes.

Hypoxic stress inhibits multiple aspects of the potato tuber wound response.

Potato (Solanum tuberosum L.) tubers subjected to wounding under hypoxic stress do not synthesize RNA species that are induced in response to wounding in aerobic conditions. Further, wound-response proteins fail to be synthesized when wounded tubers are transferred to hypoxic conditions although messenger RNAs which encode them persist for many hours after transfer. Hypoxic stress also prevents the incorporation of [(3)H]thymidine by wounded tubers that occurs in aerobic conditions. In contrast, hypoxic tubers accumulate and translate transcripts of genes whose products are involved in anaerobic metabolism whether or not they are wounded. Both the hypoxic response and the aerobic wound response preclude the synthesis of proteins encoded by messenger RNAs which accumulated during the tuberization process and which can be translated in vitro. Finally, wounding elicits the degradation of a subset of these tuberization-associated transcripts. These data indicate a complex and precise regulation of gene expression at several levels of macromolecular synthesis.

Hypoxic stress inhibits the appearance of wound-response proteins in potato tubers.

Potato (Solanum tuberosum L.) tubers respond to environmental stresses by alterations of macromolecular synthesis. In an aerobic environment tubers respond rapidly to wounding by synthesizing a set of proteins, the most prominent of which display apparent molecular weights of 78, 48, 38, and 31 kilodaltons. These proteins become intensely labeled by [(35)S]methionine within 2 hours of wounding. The 78 kilodalton polypeptide has been identified by immunoprecipitation as phenylalanine ammonia-lyase. By contrast, tubers incubated in hypoxic conditions for a period as short as 1.5 hours exhibit significantly reduced incorporation of amino acids such that newly synthesized polypeptides are not detected. However, a second set of proteins is synthesized by wounded tubers after prolonged incubation in a hypoxic environment. One peptide of this set is precipitated by an antibody directed against aldolase; several of these proteins may be enzymes of glycolysis necessary for anaerobic metabolism. The results indicate that there is a complex regulatory mechanism which allows mature potato tubers to respond to changes in the environment.

Isolation and characterization of monoclonal antibodies against the adenovirus core proteins.

Monoclonal antibodies have been prepared that recognize adenovirus core proteins V, VII, and mu in ELISA and Western blot assays. Antibodies produced by all of 87 positive hybridoma colonies obtained from a mouse injected with the precursor to protein VII, pVII, produced antibodies that also reacted with purified protein VII in an ELISA assay and all tested recognized denatured protein VII immobilized on nitrocellulose. Such failure to recover antibodies that specifically recognized only protein pVII suggests that epitopes common to the 174 amino acid protein VII and its 197 amino acid precursor were more effective antigenic determinants than the N-terminal 23 amino acid segment unique to pVII. All antibodies raised against protein mu cross-reacted with protein VII in both assays, but only a small fraction of the anti-protein VII or pVII antibodies recognized protein mu. Such cross-reactivity is discussed in relation to an unusual, arginine-rich sequence present in both protein VII and protein mu.

Isolation and characterization of adenovirus core nucleoprotein subunits.

Digestion of adenovirus type 2 (Ad2) or Ad5 cores with micrococcal nuclease generated four nucleoprotein species that could be resolved by electrophoresis in low-ionic-strength polyacrylamide gels: these nucleoproteins displayed mobilities equivalent to those of DNA fragments of 900 to 1,025, 775 to 850, 650 to 725, and 525 to 600 base pairs (bp) and thus were readily distinguishable from HeLa cell mononucleosomes. The DNA fragments associated with the core nucleoprotein species were more than 250 to 90 bp long. Nucleoproteins containing 150, 120, or 90 bp of DNA were the most stable. Polypeptide VII was associated with each of the nucleoprotein species liberated from Ad2 cores. These data suggest that polypeptide VII and viral DNA of 90 to 150 bp comprise the unit particle of the Ad2 or Ad5 core nucleoproteins.

Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection.

The proteins associated with parental, adenoviral DNA in productively-infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P-labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross-linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co-migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross-linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross-linkable to viral DNA in cells infected by wild-type virus and a non-infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre-early and early viral genes.

Identification of proteins and protein domains that contact DNA within adenovirus nucleoprotein cores by ultraviolet light crosslinking of oligonucleotides 32P-labelled in vivo.

A new approach to the identification of DNA binding proteins has been developed and used to study the DNA-protein interactions within the nucleoprotein core of subgroup C adenoviruses. Virions labelled in vivo with [32P]orthophosphate were exposed to ultraviolet light and the DNA digested by chemical or enzymatic methods. Labelled phosphoamino acids of the virion phosphoproteins were selectively hydrolysed by alkali, permitting proteins crosslinked to DNA to be identified by virtue of their covalently attached, 32P-labelled nucleotides. In parallel experiments, [3H]arginine-labelled virions were crosslinked by exposure to ultraviolet light and analysed by more conventional methods. The results indicate that proteins VII and V lie in close contact with viral DNA within the core. The compact arrangement of the nucleoprotein core appears to be capable of trapping protein VII molecules that are not covalently attached to DNA after exposure to ultraviolet light, suggesting that viral DNA might be wrapped around clusters of protein VII molecules. The domains of protein VII that lie in contact with DNA were identified by partial proteolytic mapping of the sites of covalent-attachment of the 32P-labelled oligonucleotides. The implications of these data for the nature of the interactions that mediate the packaging of viral DNA within the nucleoprotein core of adenovirions are discussed.

Insertion of the Mu1 transposable element into the first intron of maize Adh1 interferes with transcript elongation but does not disrupt chromatin structure.

The presence of the Mu1 transposable element within the first intervening sequence of the maize Adh1 gene interfered with transcription through that gene. Insertion of the element did not have an apparent effect on transcription initiation or chromatin structure. In nuclei isolated from anaerobically induced roots, in which Adh1 is transcriptionally active, a subset of the Adh1 chromatin is arranged in a unique conformation characterized by a generalized sensitivity to nucleases, specific DNAase I sensitive sites and a nucleosome array distinct from the inactive configuration present in leaf nuclei. The chromatin organization of the Mu1-induced mutant alleles is indistinguishable from that of the progenitor Adh1-S allele and a point mutant allele that is null for ADH1 activity. The initiation of transcription also proved to be unaffected in these mutants. Nuclear runoff experiments indicated that Adh1 sequences upstream from the point of Mu1 insertion were transcribed normally, but sequences downstream to the insertion were drastically reduced relative to a reference gene expressed in anaerobic root nuclei. Thus, it was concluded that the defect in these Mu1-induced mutants does not reside at the level of gene accessibility or transcript initiation. Rather, Mu1 presents an impediment to the progress of the polymerase II complex during transcript elongation.

Interactions among the three adenovirus core proteins.

Interactions among the three adenovirus core polypeptides V, VII, and mu were examined, using the reversible chemical cross-linker dithiobis(succinimidyl propionate) and two-dimensional polyacrylamide gel electrophoresis. Cross-linked species obtained from gradient-purified adenovirus type 2 cores were well represented among the cross-linked products of pentonless virions and crude core preparations. The more efficiently formed cross-linked core species were also identified with the arginine-specific cross-linker, p-azidophenyl glyoxal. In addition to dimers of polypeptides V and VII, efficient cross-linking of V to VII, V to mu, and VII to V to mu was detected in adenovirus cores. Notably absent were cross-linked species corresponding to higher multimers of polypeptide VII. A major core-capsid interaction appeared to be via the association of polypeptide V with a dimer of polypeptide VI.

Transcription of adenovirus cores in vitro.

Transcription in whole HeLa cell extracts of the nucleoprotein core complexes released from adenovirus type 2 or type 5 virions has been examined. The average length of transcripts from deproteinized DNA templates increased steadily during a 90-min reaction in vitro, exhibiting an elongation rate of approximately 70 nucleotides per minute. On the other hand, transcripts made from viral core templates were restricted to a length of less than 2000 nucleotides. Accordingly, efficient transcription of cores (50 nucleotides elongated/min) ceased after 10-20 min of incubation in whole-cell extracts. Deproteinized viral DNA and viral nucleoprotein complexes appeared to support the initiation of a similar number of transcripts per template molecule, but the rate of initiation was faster when cores were provided as templates. Deproteinized viral DNA supported the synthesis of VA-RNA and of transcripts that hybridized to the region of the viral genome containing the 5' portion of the major late transcriptional. Viral cores also directed the synthesis of RNA products which hybridized to fragments of the viral genome containing E1A, E1B, and E4 regions. The results of nuclease protection experiments indicated that the presence of core proteins did not preclude accurate initiation of transcription from the E4 region.