Leslie C. Lane

A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis

Abstract Sulfhydryl-containing proteins can oxidize during SDS-polyacrylamide gel electrophoresis to produce broad bands with irreproducible mobilities. Oxidation affects molecular weight estimates and decreases resolution. Alkylation of reduced proteins prior to electrophoresis eliminates the problems caused by reoxidation. A simple protocol involves reducing proteins with low concentrations of dithioerythritol and alkylating with a slight excess of iodoacetamide. These reactions are insensitive to atmospheric oxygen and to reagent concentrations provided that sulfhydryl groups are first completely reduced and then completely alkylated. The reagents need not be removed from the proteins prior to electrophoresis.

DNA Viruses: The Really Big Ones (Giruses)

Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences than on extensive details about individual viruses.

Isolation and characterization of a virus from the intracellular green alga symbiotic with Hydra viridis.

The isolation and characterization of a virus (designated HVCV) from the symbiotic Chlorella-like green alga present in Hydra viridis (Florida strain) are described. The large polyhedral viral particle sedimented at ca. 2600 S in sucrose density gradients and had a buoyant density in CsCl of 1.295 g/ml. The virus contained at least 19 polypeptides (MW range, 10,300 to 82,000) and the major polypeptide (MW, 46,000) was a glycoprotein. The viral genome consisted of a double-stranded DNA with a buoyant density in CsCl of 1.7114 g/ml (52% GC) and a molecular weight of ca. 136 x 10(6).

Detecting structural changes in viral capsids by hydrogen exchange and mass spectrometry.

Amide hydrogen exchange and mass spectrometry have been used to study the pH‐induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D2O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural alteration occurred in the regions around the quasi‐threefold axes, which are located at the center of the asymmetric unit. The increased levels of deuterium indicate loosening of structure in these regions. This observation confirms the previously proposed swelling model for BMV and cowpea chlorotic mottle virus (CCMV) and is consistent with the structure of swollen CCMV recently determined by cryo‐electron microscopy and image reconstruction. Structural changes in the extended N‐ and C‐terminal arms were also detected and compared with the results obtained with other swollen plant viruses. This study demonstrates that protein fragmentation/amide hydrogen exchange is a useful tool for probing structural changes in viral capsids.

Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus

ABSTRACT The 331-kbp chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.

Polyamine content of several RNA plant viruses

Abstract Three polyhedral viruses in the bromovirus group, brome mosaic virus, cowpea chlorotic mottle virus, and broad bean mottle virus, contain no detectable polyamines. Two other polyhedral viruses, turnip yellow mosaic virus and cowpea mosaic virus, contain roughly 1% spermidine by weight. The rod-shaped barley stripe mosaic virus contains no detectable polyamines.

Agarose gel electrophoresis of denatured RNA with silver staining

This paper describes agarose gel electrophoresis and silver staining of denatured RNAs. Glyoxal- or formaldehyde-denatured RNAs are electrophoresed in an agarose gel cast on a plastic support using an inert, low conductivity buffer. Following electrophoresis, the gel is stained with a sensitive silver stain. The method produces sharp, well-resolved bands and yields accurate RNA size estimates. Because of its sensitivity and simplicity, it is suitable for routine laboratory use.

Platte Valley Yellows, a chlorotic condition of soybeans: symptoms and preliminary chemical analyses

SummaryA chlorotic condition of soybeans in the river valleys of eastern Nebraska was studied. The pattern of chlorosis, presence of petiolar necrosis, reductions in rhizobial and mycorrhizal infection, and plant and soil chemical analyses suggest that the condition is more complex than normal iron deficiency chlorosis or other mineral deficiencies or toxicities. Nodulation is almost totally inhibited in affected plants. Mycorrhizal fungi, while able to infect early in the season, do not persist on affected plants. Breakdown of endodermis was observed in the roots of affected plants. The factors contributing to this condition have not yet been determined.