Thermostabilization and thermosensitization of herpesvirus

Wallis, Craig (Baylor University College of Medicine, Houston, Tex.), and Joseph L. Melnick. Thermostabilization and thermosensitization of herpesvirus. J. Bacteriol. 90:1632-1637. 1965.-Herpesvirus, long considered as one of the most thermolabile of viruses, was stabilized by 1 m Na(2)SO(4) or Na(2)HPO(4) so that it withstood heating at 50 C, but the virus was not protected by 1 m MgCl(2), MgSO(4), or KH(2)PO(4), or 2 m KCl or NaCl; 1 m Na(2)SO(4) also stabilized herpesvirus at 25 and 37 C. In contrast, herpesvirus was made extremely thermosensitive in the presence of isotonic salt concentrations or of isotonic tris(hydroxymethyl)aminomethane buffer, especially at pH 7.2 or above. Partially purified virus was relatively thermostable when suspended in distilled water at pH 7.2, but in Earle's salt solution the virus immediately became thermosensitive. As found in tissue culture harvests, herpesvirus was thermolabile, but the virus was rendered stable at 50 C by simple dilution in distilled water. Protection by proteins or amino acids, generally accepted as virus-stabilizing agents, did not seem to be the result of a direct effect upon herpesvirus. The present data suggest that the added proteins counteract in part thermosensitizing effects of the salts contained in the virus harvest.     

SSCP and segregation analysis of the human type X collagen gene (COL10A1) in heritable forms of chondrodysplasia.

Type X collagen is a homotrimeric, short chain, nonfibrillar collagen that is expressed exclusively by hypertrophic chondrocytes at the sites of endochondral ossification. The distribution and pattern of expression of the type X collagen gene (COL10A1) suggests that mutations altering the structure and synthesis of the protein may be responsible for causing heritable forms of chondrodysplasia. We investigated whether mutations within the human COL10A1 gene were responsible for causing the disorders achondroplasia, hypochondroplasia, pseudoachondroplasia, and thanatophoric dysplasia, by analyzing the coding regions of the gene by using PCR and the single-stranded conformational polymorphism technique. By this approach, seven sequence changes were identified within and flanking the coding regions of the gene of the affected persons. We demonstrated that six of these sequence changes were not responsible for causing these forms of chondrodysplasia but were polymorphic in nature. The sequence changes were used to demonstrate discordant segregation between the COL10A1 locus and achondroplasia and pseudoachondroplasia, in nuclear families. This lack of segregation suggests that mutations within or near the COL10A1 locus are not responsible for these disorders. The seventh sequence change resulted in a valine-to-methionine substitution in the carboxyl-terminal domain of the molecule and was identified in only two hypochondroplasic individuals from a single family. Segregation analysis in this family was inconclusive, and the significance of this substitution remains uncertain.     

Isozyme analysis of Galaxias species (Teleostei: Galaxiidae) from the Taieri River, South Island, New Zealand: a species complex revealed

We examined genetic differentiation among 23 samples of non-migratory river galaxias from 17 streams in the Taieri River system, South Island, New Zealand. Four major genetic types were found, two of which occur in narrow sympatry in one location. These were compared with topotypical material representing Galaxias anomalus from the Clutha system (Otago) and G. vulgaris from the Waimakariri system (Canterbury) in order to establish identity. Morphological examination of these four major genetic types revealed consistent concomitant differences. The results suggest that there are at least three species of river galaxias in the Taieri system: G. anomalus, G. vulgaris and at least one previously undescribed species. We propose that the genetic structuring and subsequent speciation of this group has been promoted by the absence of the marine juvenile phase that is found in five other members of the genus native to New Zealand. This structuring may be exacerbated by population fragmentation over the last century owing to the negative influence of introduced trout. The phylogenetic diversity within the river system mirrors the diverse flora and invertebrate fauna of the region, and has conservation implications that parallel those resulting from our improved knowledge of the New Zealand herpetofauna through the application of genetic analysis.     

Fungal protoplasts as tools for studies on protein secretion

Summary Protoplasts were prepared from mycelium of Aspergillus niger N-402. Sucrose was used to induce the synthesis and secretion of invertase. Protoplasts secreted 2 forms of invertase, different to those secreted by the mycelium. ¹⁴C mannose was shown to be taken up by protoplasts and incorporated into secreted proteins.     

Factors influencing alkali-induced heat resistance in Salmonella enteritidis phage type 4

The culture of cells of Salmonella enteritidis PT4 at either 25, 30 or 37°C in media at pH values between 8.0 and 9.75 resulted in significant increases in heat resistance. At 37°C, induction was rapid and was dependent on protein synthesis, being inhibited by chloramphenicol. Thermotolerance was stable when cells were transferred from pH 9.2 to pH 7.0 and cultures only became heat sensitive again following significant multiplication at the lower pH.     

Quantification of bacterial morphotypes within anaerobic digester granules from transmission electron micrographs using image analysis

Summary Image analysis was applied to sequential transmission electron micrographs of an ultrathin section from the central region of an anaerobic digester granule to quantify the constituent bacterial morphotypes present. Our experience indicates that this procedure is suitable for the determination of populations of small spherical granules only and that it would be a useful technique for monitoring granule development. The cell area data determined in this study should permit rapid future quantification of Methanothrix- and Methanobacterium-like cells from cell counts derived from transmission electron micrographs.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.