RESTRICTION FRAGMENT ANALYSIS OF PINE PHYLOGENY

We used restriction fragment analysis of chloroplast, nuclear, and mitochondrial DNA to study phylogeny in the genus Pinus. Total genomic DNA of 18 to 19 pine species that spanned 14 of the 15 subsections in the genus was cut with 8 restriction enzymes, blotted, and then probed with up to 17 cloned DNA fragments—which were mostly from the chloroplast genome of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). A total of 116 shared characters, the majority representing single point mutations, were subjected to Wagner and Dollo parsimony analyses, coupled with bootstrapping and construction of consensus trees. The hard (subgenus Pinus) and soft pines (subgenus Strobus) were distinct. The soft pines in section Parrya, represented by P. longaeva, edulis, monophylla, and gerardiana, were the group closest to the hypothesized root of the genus. They were also more diverse and more closely related to the hard pines than were their descendents in section Strobus, represented by P. koraiensis, albicaulis, griffithii, and lambertiana, all of which were remarkably similar. Except for a strong clade involving P. canariensis and pinea (section Ternatae), the hard pines were weakly differentiated. The high similarity within the most speciose groups of pines (sections Strobus and Pinus) suggests that the bulk of the genus radiated relatively recently. In contrast to a recent classification, P. leiophylla was not associated with section Ternatae; instead, it appears to belong in section Pinus, and showed a high similarity to P. taeda of subsection Australes. Subsection Oocarpae, represented by P. oocarpa and radiata, appears to be a natural group, and is related to subsection Contortae, represented by P. contorta. More extensive restriction fragment studies will yield many new insights into evolution in the genus. Other methods, however, such as DNA sequencing or fine structure analysis of restriction site mutations, are likely to be necessary for rooting pine phylogenies with respect to other coniferous genera, and for estimating divergence times.     

The Computerized Laboratory Notebook concept for genetic toxicology experimentation and testing

We describe a microcomputer system utilizing the Computerized Laboratory Notebook (CLN) concept developed in our laboratory for the purpose of automating the Battery of Leukocyte Tests (BLT). The BLT was designed to evaluate blood specimens for toxic, immunotoxic, and genotoxic effects after in vivo exposure to putative mutagens. A system was developed with the advantages of low cost, limited spatial requirements, ease of use for personnel inexperienced with computers, and applicability to specific testing yet flexibility for experimentation. This system eliminates cumbersome record keeping and repetitive analysis inherent in genetic toxicology bioassays. Statistical analysis of the vast quantity of data produced by the BLT would not be feasible without a central database. Our central database is maintained by an integrated package which we have adapted to develop the CLN. The clonal assay of lymphocyte mutagenesis (CALM) section of the CLN is demonstrated. PC-Slaves expand the microcomputer to multiple workstations so that our computerized notebook can be used next to a hood while other work is done in an office and instrument room simultaneously. Communication with peripheral instruments is an indispensable part of many laboratory operations, and we present a representative program, written to acquire and analyze CALM data, for communicating with both a liquid scintillation counter and an ELISA plate reader. In conclusion we discuss how our computer system could easily be adapted to the needs of other laboratories.     

A dynamic global vegetation model for use with climate models: concepts and description of simulated vegetation dynamics

Changes in vegetation structure and biogeography due to climate change feedback to alter climate by changing fluxes of energy, moisture, and momentum between land and atmosphere. While the current class of land process models used with climate models parameterizes these fluxes in detail, these models prescribe surface vegetation and leaf area from data sets. In this paper, we describe an approach in which ecological concepts from a global vegetation dynamics model are added to the land component of a climate model to grow plants interactively. The vegetation dynamics model is the Lund–Potsdam–Jena (LPJ) dynamic global vegetation model. The land model is the National Center for Atmospheric Research (NCAR) Land Surface Model (LSM). Vegetation is defined in terms of plant functional types. Each plant functional type is represented by an individual plant with the average biomass, crown area, height, and stem diameter (trees only) of its population, by the number of individuals in the population, and by the fractional cover in the grid cell. Three time‐scales (minutes, days, and years) govern the processes. Energy fluxes, the hydrologic cycle, and carbon assimilation, core processes in LSM, occur at a 20 min time step. Instantaneous net assimilated carbon is accumulated annually to update vegetation once a year. This is carried out with the addition of establishment, resource competition, growth, mortality, and fire parameterizations from LPJ. The leaf area index is updated daily based on prevailing environmental conditions, but the maximum value depends on the annual vegetation dynamics. The coupling approach is successful. The model simulates global biogeography, net primary production, and dynamics of tundra, boreal forest, northern hardwood forest, tropical rainforest, and savanna ecosystems, which are consistent with observations. This suggests that the model can be used with a climate model to study biogeophysical feedbacks in the climate system related to vegetation dynamics.     

Optimal Staining and Sample Storage Time for Direct Microscopic Enumeration of Total and Active Bacteria in Soil with Two Fluorescent Dyes

Direct counting techniques, first developed for aquatic samples, can be used to enumerate bacteria in soil and groundwater sediments. Two fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for actively respiring bacteria and 4(prm1),6-diamidino-2-phenylindole (DAPI) for total bacteria, were tested for their usefulness in epifluorescent direct bacterial enumeration in soil. Both dyes can be used for the same soil sample without affecting enumeration results. Staining for 8 h with CTC and for 40 min with DAPI resulted in maximum numbers of stained cells. The optimal DAPI staining concentration is 10 mg liter(sup-1). After preparation, slides should be stored at 4(deg)C and counted within 2 days for CTC and within 24 h for DAPI. Sodium PP(infi) or sodium chloride solutions were used to desorb bacteria from soil prior to counting. Counts were significantly higher when sodium chloride was used.     

Denaturation of mouse satellite DNA upon melting of chromatin in solution

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.     

Thermodynamics of hydrophobic polyacids

The thermodynamics of deprotonating hydrolyzed 1-1 copolymers of maleic anhydride with pentyl, hexyl and octyl vinyl ether was investigated by calorimetric and potentiometric methods. These polyacids undergo a transition from a compact to a random coil conformation upon ionization in aqueous media. The results were compared with those obtained previously for similar copolymers with smaller alkyl side-chains. The contributions of the enthalpy and entropy to the free energy were analyzed. The major effects appeared to be related to the charging of the compact form of the polyacids, the electrostriction of water by the completely ionized dicarboxylate groups and the reorganization of water around newly exposed alkyl side-chains arising from the conformational transition.     

The genetic fine structure of the complex locus aro3 involved in early aromatic amino acid biosynthesis in Schizosaccharomyces pombe.

Summary The complex locus aro3 of Schizosaccharomyces pombe was subjected to genetical fine structure analysis. By comparing the complementation map and the meiotic recombination map, the aro3 locus could be subdivided into the five adjacent subregions A, B, C, D and E. Out of 115 aro3 alleles, 26 nonsense alleles and 30 missense alleles could be identified by the criteria of nonsense suppressor sensitivity and leakiness, respectively. Most alleles with a pleiotropic complementation pattern are of the nonsense type. We conclude from the polarity of the complementation patterns characterising the nonsense alleles that the translation direction proceeds from subregion A to subregion E. Antipolar effects in complementation are more frequent than in the analogous system of the arom gene cluster of Neurospora crassa.This work formed part of a Ph.D. thesis submitted to the University of Bern     

A toxic amino acid, 2(S)3(R)-2-amino-3-hydroxypent-4-ynoic acid from the fungus Sclerotium rolfsii

An amino acid, lethal to New Hampshire chickens (LD₅₀, 150 mg/kg) was isolated from dried sclerotia of the fungus Sclerotium rolfsii (Sacc.). Purification of the rather unstable compound was effected on a cation exchange column by means of displacement chromatography and the amino acid was crystallised from 80% methanol. A structure was assigned to the compound on the basis of available chemical and physical data, namely 2(S),3(R)-2- amino-3-hydroxypent-4-ynoic acid. Confirmation of this structure was gained by direct and indirect synthetic procedures.