Essential role of the Vp2 and Vp3 DNA-binding domain in simian virus 40 morphogenesis.

Both a DNA-binding domain and a Vp1 interactive determinant have been mapped to the carboxy-terminal 40 residues of the simian virus 40 (SV40) minor capsid proteins, Vp2 and Vp3 (Vp2/3), with the last 13 residues being necessary for these activities. The role of this DNA-binding domain in SV40 morphogenesis and the ability to separate these two signals were investigated by mutagenesis and assessment of the activity and viability of the mutants. The carboxy-terminal 40 residues of Vp2/3 were expressed as a polyhistidine fusion protein, and five basic residues at the extreme carboxy terminus (Vp3 residues K226, R227, R228, R230, and R233) were mutagenized. The wild-type fusion protein bound DNA with a Kd of 3 x 10(-8) identical to that of the full-length Vp3. Mutant proteins containing either one to three or four amino acid substitutions bound DNA 4- to 7-fold or 20- to 30-fold less well, respectively, than the wild-type protein did. The most severe point mutants showed residual DNA binding similar to that of a truncated protein which lacks the entire 13 carboxy-terminal residues. All of the point mutants were able to interact with Vp1, indicating that the two signals within this region are mediated by different residues. When the mutations were placed into the context of the viral DNA and introduced into cells, all the structural proteins were expressed and localized correctly. Not all, however, were viable: mutant genomes whose Vp2/3 bound DNA with intermediate affinities formed plaques just as well as wild-type SV40 DNA did, but three mutants showing greatly reduced DNA binding failed to form plaques at all. These results are consistent with the hypothesis that Vp2/3 plays an essential role in SV40 virion assembly in the nucleus.     

SEXUAL DIFFERENTIATION OF GRIFFITHSIA (CERAMIALES, RHODOPHYTA): NUCLEAR PLOIDY LEVEL OF MIXED-PHASE PLANTS IN G. JAPONICA1

Mixed-phase plants of Griffithsia japonica Okamura spontaneously occurred in a laboratory culture. Four female plants produced tetrasporangia and spermatangia in addition to their normal female reproductive structures (bisexual/mixed-phase plants), and four male plants produced tetrasporangia as well as spermatangia (male/mixed-phase plants). To determine the nuclear ploidy level of these mixed-phase plants, relative nuclear sizes of male, female, tetrasporangial, and mixed-phase plants were measured using a microscopic image analysis system. Haploid gametophytes could be distinguished from diploid tetrasporophytes by relative nuclear sizes, with the later having nuclei twice the size of the former. Relative nuclear sizes of the mixed-phase plants were similar to those of the haploid plants. Thus, the mixed-phase plants were determined to be haploid. Haploid mixed-phase plants of G. japonica have a potential to produce male, female and tetrasporangial reproductive structures. Sex determination models are discussed to explain "haploid" mixed-phase phenomena in red algae .     

OBSERVATIONS ON A CHAMAESIPHONACEOIS ALGA (CYANOPHYCEAE) WITH SPECIAL REFERENCE TO EXOCYTE FORMATION1,2

A unicellular cyanophycean culture contaminant had features of both Geitleribactron Kom. and Cyanophanon Geitl. The cells were elongated, sheathless, mostly similar in diameter throughout their length, and attached polarly in rosettes or groups and produced only a single elongated exocyte (=exospore). Young cells were moderately elongated and resembled Geitleribactron. As cells aged, they greatly elongated and then resembled Cyanophanon. Some cells formed Y-shaped bifurcations, features of C. mirabile Geitl. and C. minus Geitl., but they lacked the basal sheath (pseudovagina) of C. mirabile. During exocyte formation, a thick and localized L-II wall layer protuberance extended the exocyte away from the parent cell. This terminal wall thickening then appeared to move to one side from subsequent and unequal cell wall growth. Cells sovnetimes bent abruptly, occasionally opposite a thickening in the L-II wall layer. Further studies in culture of putative Geitleribactron and Cyanophanon isolates are necessary to ascertain the breadth of their structural diversity and the identity of the present taxon.     

SPONTANEOUS AND STIMULATED BIOLUMINESCENCE OF THE DINOFLAGELLATE CERATOCORYS HORRZDA (PERIDINIALES)1

This is the first report of spontaneous bioluminescence in the autotrophic dinoflagellate Ceratocorys horrida von Stein. Bioluminescence was measured, using an automated data acquisition system, in a strain of cultured cells isolated from the Sargasso Sea. Ceratocorys horrida is only the second dinoflagellate species to exhibit rhythmicity in the rate of spontaneous flashing, flash quantum flux (intensity), and level of spontaneous glowing. The rate of spontaneous flashing was maximal during hours 2–4 of the dark phase [i.e. circadian time (CT)16–18 for a 14:10 h LD cycle (LD14:10)], with approximately 2% of the population flashing-min^?1, a rate approximately one order of magnitude greater than that of the dinoflagellate Gonyaulax polyedra. Flash quantum flux was also maximal during this period. Spontaneous flashes were 134 ms in duration with a maximum flux (intensity) of 3.1×10⁹ quanta-s^?1. Light emission presumably originated from blue fluorescent microsources distributed in the cell periphery and not from the spines. Values of both spontaneous flash rate and maximum flux were independent of cell concentration. Isolated cells also produced spontaneous flashes. Spontaneous glowing was dim except for a peak of 6.4× 10⁴quanta-s^?1 cell^?1, which occurred at CT22.9 for LD14:10 and at CT22.8 for LD12:12. The total integrated emission of spontaneous flashing and glowing during the dark phase was 4×10⁹ quantacell^?1, equivalent to the total stimulable luminescence. The rhythms for C. horrida flash and glow behavior were similar to those of Gonyaulax polyedra, although flash rate and quantum flux were greater. Spontaneous bioluminescence in C. horrida may be a circadian rhythm because it persisted for at least three cycles in constant dark conditions. This is also the first detailed study of the stimulated bioluminescence of C. horrida, which also displayed a diurnal rhythm. Cultures exhibited >200 times more mechanically stimulated bioluminescence during the dark phase than during the light phase. Mechanical stimulation during the dark phase resulted in 6.7 flashes. cell^?1; flashes were brighter and longer in duration than spontaneous flashes. Cruise-collected cells exhibited variability in quantum flux with few differences in flash kinetics. The role of dinoflagellate spontaneous bioluminescence in the dynamics of near-surface oceanic communities is unknown, but it may be an important source of natural in situ bioluminescence.     

Extreme fitness differences in mammalian and insect hosts after continuous replication of vesicular stomatitis virus in sandfly cells.

Continuous, persistent replication of a wild-type strain of vesicular stomatitis virus in cultured sandfly cells for 10 months profoundly decreased virus replicative fitness in mammalian cells and greatly increased fitness in sandfly cells. After persistent infection of sandfly cells, fitness was over 2,000,000-fold greater than that in mammalian cells, indicating extreme selective differences in the environmental conditions provided by insect and mammalian cells. The sandfly-adapted virus also showed extremely low fitness in mouse brain cells (comparable to that in mammalian cell cultures). It also showed an attenuated phenotype, requiring a nearly millionfold higher intracranial dose than that of its parent clone to kill mice. A single passage of this adapted virus in BHK-21 cells at 37 degrees C restored fitness to near neutrality and also restored mouse neurovirulence. These results clearly illustrate the enormous capacity of RNA viruses to adapt to changing selective environments.     

Measures of nematode community structure and sources of variability among and within agricultural fields

Whole nematode communities, extracted from soil samples taken from agricultural fields, were enumerated by taxonomic family and trophic group (i.e., bacterivores, fungivores, omnivores, plant-parasites, and predators) to evaluate nematode community structure as an indicator for monitoring ecological condition of soil. No differences were found in mixing treatments or methods of packing or shipping samples. However, extraction using Cobb's sifting and gravity method, followed by sucrose centrifugation, gave greater recovery of free-living nematodes than elutriation followed by sucrose centrifugation. Population means and variance of the sampled area were similar when sampled using different strategies for collecting soil samples within fieds, including several patterns, directions and repetitions of transects. Components of variation associated with ratios among the five trophic groups of nematodes and selected indices of community structure were quantified as variation among regions, among counties, among agricultural fields (2-ha area), among transects within agricultural fields, and within composite soil samples. The variance component for'within composite soil samples' was relatively large compared to the other components of variance. Variation within composite soil samples was less for maturity indices (based on life-history strategy characteristics), ratio of bacterivores to plant-parasites, sum of bacterivores and fungivores, populations of plant-parasites, and populations of bacterivores than for trophic diversity indices, populations of fungivores, populations of omnivores, populations of predators, or the ratio of fungivores to bacterivores. With a single composite sample per field, the ability to differentiate ecological condition of soils among fields within a region improved if the variance among and within fields exceeded the variance within composite samples. Given the variance components, power curves indicated that detection of a 10% change (with 0.8 power) in the ecological condition of soils within a region between two time periods would require sampling a minimum of 25 and 50 fields with one composite soil sample analyzed per field for the maturity and trophic diversity index, respectively. More than 100 fieldsper region would be required to detect temporal change in populations of individual trophic groups. Biplots of maturity indices, but not of trophic diversity or populations of individual trophic groups, identified clear differences among fields. Thus, maturity indices, which differentiated among sampling sites better and more efficiently than trophic diversity indices or measures based on populations of individual trophic groups, may be appropriate for use in a regional and/or national monitoring program.     

Nest building by lowland gorillas in the Lopé Reserve,Gabon: Environmental influences and implications for censusing

We analyzed data from 373 fresh nest-sites (containing 2435 nests) of lowland gorillas (Gorilla g. gorilla)during a 4-year period in the Lopé Reserve, Gabon, to determine whether the observed variability in nest building was due to environmental influences. We recognized and defined seven types of nest in terms of the degree of construction and the raw materials used. Overall, nests built on the ground from herbaceous plants are the most common type (40%), followed by tree nests (35%). Frequencies of the different nest-types vary significantly between eight habitat-types. In habitat-types with high densities of understory herbs, ground nests predominated, but when herbs were rare, the majority of nests were in trees. A general preference for sleeping in herbaceous ground nests is indicated since trees are abundant in all habitat-types, except savanna. The frequency of nesting in trees shows a significant positive correlation with rainfall, but effects of climate are confounded by seasonal variation in use of different habitat-types. When elephants were attracted to the same localized food sources as gorillas, many tree nests were built even when herbs were available. We conclude that different nest-types reflect a variety of solutions to maximize comfort, depending on available raw materials and the probability of rainfall or disturbance by elephants or both factors. Nests are a powerful tool for population censuses and demographic studies of great apes, but problems exist in interpreting data on lowland gorilla nests. Results from this analysis show that only a third of nest-sites accurately reflects group size (of weaned individuals) and that 26% of all gorilla nest-sites could be mistaken for those of chimpanzees, as all nests, or all those visible from a transect, were in trees. Gorilla nests at Lopé were nonrandomly distributed with respect to habitat-types, and nest construction varied seasonally, thereby introducing sources of bias to transect nest counts. We discuss these problems and ones related to assessing the decay rate of nest-sites and make recommendations relevant to census work.     

Cloning,characterization and overproduction of nuclease S1 gene (nucS) from Aspergillus oryzae

The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced. It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in lenght. The nucS gene had an open-reading frame of 963 base pairs and coded for a protein of 287 amino acid residues, comprising the signal peptide of 20 amino acids and a mature protein of 267 amino acids. The deduced amino acid sequence agreed well with the published amino acid sequence except for one substitution. Southern hybridization analysis showed that the nucS gene existed as a single copy in the A. oryzae chromosome. When the structural gene of nucS was fused with the promoter of the glaA gene and introduced into A. oryzae, the yield od secreted nuclease S1 increased about 100-fold compared with the recipient strain.