The UL20 gene of herpes simplex virus 1 encodes a function necessary for viral egress.

A recombinant virus from which the start codon and 53% of the UL20 open reading frame had been deleted was constructed and characterized. We report the following: (i) The UL20- mutant formed small plaques in 143 tk- cells but failed to form plaques in Vero cells. Virus yields were approximately 10- to 100-fold lower than those of wild-type virus in all cell lines tested. (ii) Electron microscopic examination of Vero cells infected with the UL20- mutant revealed that enveloped and unenveloped capsids accumulated in the cytoplasm, possibly in the space between the inner and outer lamellae of the nuclear membrane, and that virtually no virus was present in the extracellular space. (iii) Glycoproteins B, C, D, E, H, and I recovered from lysates of cells infected with the UL20- mutant could not be differentiated from those present in lysates of cells infected with the wild-type parent virus with respect to the electrophoretic mobility of mature and precursor forms. (iv) Repair of the deleted sequences restored the wild-type phenotype. (v) The gene product of the UL20 gene was shown to be associated with cellular membranes and to possess characteristics of integral membrane proteins. We conclude that the UL20 gene encodes an integral membrane protein with a hitherto unrecognized function in that it enables the transit of virions to the extracellular space. The function of the UL20 gene product is complemented by some cell lines but not by Vero cells. The vesicles which serve to transport virions may have an origin different from those associated with transport of normal cellular proteins.     

Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2

Summary The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequencing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 protein sequence was found to be related to a family of proteins including hevein and Win-1, which have an amino-terminal lectin domain and a carboxy-terminal domain of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses characterized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dramatically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins.     

Gene expression in nematode-infected plant roots

Summary A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.     

Size-dependent drift responses of mayflies to experimental hydrologic variation: active predator avoidance or passive hydrodynamic displacement?

Summary Larger nymphs within aquatic insect taxa have been frequently observed to be transported down-stream in the stream drift only at night. Others have hypothesized this pattern results primarily from large nymphs' behavioural avoidance of entering drift during daylight, when size-selective, visually-feeding fish predators are most active. This hypothesis assumes that animals can actively control their entry into the drift, which may not be the case under all flow conditions. We experimentally induced streamflow increases and decreases in adjacent riffles in a hydrologically-stable stream during the daytime to examine whether changes in diel patterns of drift abundance and size-distribution of mayflies were consistent with the hypothesis of active avoidance of diurnal drift. We assessed the likelihood of active vs. passive mechanisms of diurnal drift entry and transport for four taxa that differ with respect to body size, morpho-behavioural attributes, microhabitat use, and general propensity to drift. In each of three seasons, diurnal and nocturnal drift samples were collected in three riffles over two diel cycles. Background drift patterns were established on the first day (no flow manipulation). Six h before sunset on the second day, flow was experimentally increased in one riffle, decreased in the second, and not altered in the third (control). Between-day differences in diurnal and nocturnal drift rate and size composition were then compared among the treatment and reference riffles. Responses of two taxa were consistent with active control over drift entry, transport, or both. For Baetis spp., drift-prone mayflies typically preyed upon by fish, diurnal drift rates immediately increased following both flow reduction and flow elevation in all seasons, but only small individuals comprised the drift. Drift by large individuals was delayed until nighttime. Epeorus longimanus also exhibited significant increases in drift rates following flow reduction and elevation, but responses of this large-bodied species were restricted to nighttime. Drift responses for these two taxa were largely independent of direction of hydrologic change, thus indicating a strong behavioural control over drift. By contrast, numbers and sizes of drifting Paraleptophlebia heteronea and Ephemerella infrequens depended strongly on direction of flow change. Drift rates for both species generally declined after flow reduction and increased after flow elevation. Moreover, after flow elevation, larger individuals often drifted diurnally, a finding consistent with expectations under a passive hydrodynamic model. These experiments indicate that size-dependent mayfly drift reflects not only presumed risk from visual fish predators, but also functional attributes of species such as morphology, behaviour, and microhabitat affiliation, which influence aspects of drift entry and transport under variable hydrologic conditions.     

Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions

Detailed studies were carried out on the effects of nitrogen source, phosphate, sodium chloride, growth factors, precursors, CO2, temperature, initial pH, and inoculum size on biomass and eicosapentaenoic acid (EPA) production by Phaeodactylum tricornutum. The EPA content of total fatty acids increased with increasing concentrations of nitrate and urea. Sodium chloride was not required for growth or EPA production. While vitamins B1 and B12 did not affect growth significantly, EPA yield was increased by 65% by B12 supplementation. Maximum EPA production occurred when the air gassing supply was supplemented with 1% CO2. Optimum culture temperature and initial pH for EPA production were 21.5 to 23 degrees C and 7.6, respectively. EPA yields of up to 133 mg/liter of culture were observed. EPA constituted up to 30 to 40% of total fatty acids.     

Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. Princess Diana (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.