Michael E. Vayda

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.

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.

Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates

Grape berries contain compounds that aggregate with and precipitate RNA in the presence of chaotropic agents or phenol. The procedure described here extracts RNA from finely ground tissues using mild denaturants, and selectively precipitates the aggregate-forming material with 30% ethanol. The resulting RNA is suitable for northern blot analysis and translationin vitro.

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.

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

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°C. Maximal induction of HSP70 mRNA was observed when the alga was incubated at 10°C for 1 h. Incubations at higher temperatures or for longer periods reduced the amount of HSP70 mRNA detected. Incubations at 20°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°C above usual growth conditions.

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.

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

SummaryThe 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.

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

Translation elongation factor EF-1α 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-1α 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-1α, and expression of an EF-1α/GUS transgene was observed. These results indicate that translational arrest results from to the failure of EF-1α to dissociate from ribosomes during the elongation cycle, and that restoration of protein synthesis is coordinated with expression of EF-1α.

Field performance of transgenic Russet Burbank and Lemhi Russet potatoes

Transformed Russet Burbank and Lemhi Russet clones which contained three different transgene constructs were evaluated for performance under field conditions in Idaho. The transgenic lines were characterized over two growing seasons, using plants grown from both greenhouse produced minitubers (first year) and field grown seed (second year). Individual clones were evaluated for a variety of agronomic and quality properties. Many of the transformed clones showed reduced yield and increases in percent malformed and undersized tubers. Other characteristics, such as specific gravity and fry color, showed less variability. The variation observed in non-transgenic clones regenerated from tissue culture was less than that of the transformed lines. Out of an original population of 57 transgenic lines in tissue culture, maintenance of key agronomic and quality properties of the parental material was observed in only four clones. These results suggest that experiments designed to generate transgenic lines for the marketplace should be initiated with a large number of transgenic clones.CompendioClones transformados de Russet Burbank y Lemhi Russet, que contenían tres diferentes prototipos de transgenes fueron evaluados para comportamiento bajo condiciones de campo en Idaho. Las líneas transgénicas se caracterizaron en dos temporadas de cultivo, utilizando plantas obtenidas tanto de minituberculos producidos en invernadero (primer año) como semilla producida en el campo (segundo año). Se evaluaron clones individuales para diversas propiedades agronomicas y de calidad. Muchos de los clones transformados mostraron rendimientos reducidos y un incremento en el porcentaje de tubérculos deformesy de pequeño tamaño. Otras características, tales como la gravedad específica y el color a la fritura, mostraron una menor variabilidad. La variacíon observada en los clones no transgénicos regenerados de cultivo de tejidos fue menor que aquella de las líneas transformadas. De una población original de 57 líneas transgénicas en cultivo de tejidos, se observó que sólo cuatro de ellas mantenían las principales propiedades agronómicas y de calidad del material progenitor. Estos resultados sugieren que los experimentos diseñados para generar líneas transgenicas para el mercado deberán iniciarse con un gran número de clones transgenicos.