A mass-spectrometric sequence study of the enzyme ribitol dehydrogenase from Klebsiella aerogenes

The first detailed results of the application of a low-resolution mixture analysis approach to the sequence analysis of an enzyme, ribitol dehydrogenase, are given. Examples of the interpretation of the spectra of peptide mixtures derived from this protein are described. Evidence for new fragmentation patterns observed is reported, together with an explanation of the generation of ambiguous sequences by use of a low-specificity enzyme, thermolysin. The overall sequencing strategy evolved is assessed.     

Release of microbial contamination from fractured solids

Estimation of the probability of release of microbial contamination from the interior of solids upon fracture due to impact is essential to the formulation of planetary quarantine and spacecraft sterilization requirements. A model system was designed in which known concentrations of bacterial spores were incorporated in methyl methacrylate plastic. Pieces of plastic were fractured in a uniform manner exposing interior surface areas of consistent and measurable size. Known surface areas were incubated in sets of 20 culture tubes containing liquid growth medium. The subsequent occurrence of visible growth expressed as percent of tubes positive was interpreted as an estimate of the probability of release of at least one viable micro-organism.From these experiments probability of release as a function of microbial concentration in plastic was estimated for exposed interior surface areas of 30.6, 61.2, 91.8 or 122.4 mm². Good agreement of the empirical results with a theoretical mathematical model of the probability of release of contamination from solids was demonstrated. Analysis of the data using the maximum likelihood procedure provided a means of calculating a proportionality constant representing the effective thickness of the exposed area and the characteristics of the recovery procedure.     

Chemical cross-linking reveals a dimeric structure for CTP:phosphocholine cytidylyltransferase

CTP:phosphacholine cytidylyltransferase (EC 2.7.7.15) was purified from rat liver according to the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M. E., and Feldman, D. A. (1986) J. Biol. Chem. 261, 5104-5110). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without beta-mercaptoethanol revealed a single major band of 42,000 daltons. This band corresponds to the 45-kDa catalytic subunit isolated by Feldman and Weinhold (Feldman, D. A., and Weinhold, P. A. (1987) J. Biol. Chem. 262, 9075-9081). A minor component of 84,000 daltons was intensified in nonreducing gels when the sulfhydryl reducing agent, dithiothreitol, was removed from the enzyme preparation by dialysis. Reduction with dithiothreitol and electrophoresis in the second dimension showed that this 84-kDa protein was derived from the 42-kDa protein. This result suggested that the 42 kDa protein can be converted to an 84-kDa protein by disulfide bond formation. Reaction with the thiol-cleavable cross-linking reagents, dithiobis(succimidyl propionate) or dimethyl-3,3'-dithiobispropionimidate, converted the 42-kDa cytidylyltransferase subunit into a diffuse band approximately twice its molecular mass. Disulfide reduction and electrophoresis in the second dimension showed that this band was derived exclusively from the 42-kDa subunit. This cross-linking pattern was observed when cytidylyltransferase was bound to a Triton X-100 micelle or when bound to a membrane vesicle containing phosphatidylcholine, oleic acid, and Triton X-100. Reaction of the fully reduced enzyme with glutaraldehyde also generated a cross-linked dimer. All three cross-linking reagents inactivated the enzyme. Reduction of the disulfide cross-linkers with dithiothreitol partially reactivated the transferase. When Triton was removed from the enzyme preparation by DEAE-Sepharose chromatography, reaction of the detergent-depleted enzyme with glutaraldehyde generated a band corresponding to a hexamer and higher molecular weight aggregates. The dimeric form was regenerated by addition of either Triton X-100 or phosphatidylcholine-oleic acid vesicles. We conclude that the purified, native cytidylyltransferase, when bound to a detergent micelle or membrane vesicle, is a dimer composed of two noncovalently linked 42-kDa subunits. In the absence of a membrane or micelle, the dimers self-aggregate in a reversible manner.     

Sequencing, distribution, and inactivation of the dipeptidase A gene (pepDA) from Lactobacillus helveticus CNRZ32.

Previously, the gene for a general dipeptidase (pepDA) was isolated from a gene bank of Lactobacillus helveticus CNRZ32. The pepDA gene consists of a 1,422-bp open reading frame which could encode a polypeptide of 53.5 kDa. No significant identity was found between the deduced amino acid sequence of the pepDA product and the sequence for other polypeptides reported in GenBank. Southern hybridization studies with a pepDA probe indicated that the nucleotide sequence for pepDA is not well conserved among a variety of lactic acid bacteria. Growth studies indicated that a pepDA deletion had no detectable effect on growth rate or acid production by L. helveticus CNRZ32 in milk. Furthermore, no difference in total cellular dipeptidase activity was detected between the mutant and wild-type strains during logarithmic growth in MRS medium.     

Biogeography of milkweed butterflies (Nymphalidae: Danainae) and mimetic patterns on tropical Paciflc archipelagos

Distributions of danaine butterfly species and associated mimetic patterns were compared among fifteen archipelagos of the tropical Pacific Ocean, and within five major archipelagos (the Bismarcks, Fiji, East and West Solomon Islands, and Vanuatu). Using both simple and stepwise linear regression analysis, variation in the total number of danaine species and number of mimetic patterns was assessed with respect to island size, isolation and elevation. Relative to interarchipelago distributions, the distribution of danaine species and number of mimetic patterns on islands within archipelagos exhibited less dependence upon interisland distance and island area. Geographical features influencing the number of mimetic patterns were similar to those of danaines as a whole. Analysis of residuals from stepwise linear regression suggested that factors influencing danaine distributions were different from those for non-danaine butterflies. This result is consistent with the hypothesis of enhancement of danaine species establishment through Müllerian mimicry, although other factors such as host plant availability and similar habitat use may also be important.     

Linkage disequilibrium in crosses between Illinois maize strains divergently selected for protein percentage

The objectives of this study were two fold: (1) to determine whether divergent selection for kernel protein concentration, which produced the Illinois high protein (IHP), Illinois low protein (ILP), reverse low protein (RLP), and reverse high protein (RHP) maize (Zea mays L.) strains, had generated coupling-phase linkages among genes controlling protein concentration or other traits and (2) to measure the effectiveness of random mating in reducing linkage disequilibrium in segregating generations from crosses between the strains. To achieve these objectives, design III progenies from the F₂ and F₆ (produced by random mating the F₂) from the crosses of IHP × ILP, IHP × RHP, ILP × RLP, and RHP × RLP were evaluated. Estimates of additive variance for percent protein in the crosses of IHP × ILP and ILP × RLP were significantly less in the F₆ than in the F₂ indicating the presence of coupling-phase linkages in the parents and their breakup by random mating. In addition, a significant reduction in dominance variance for grain yield from the F₂ to the F₆ in IHP × ILP suggested the presence of repulsion-phase linkages. No other evidence of coupling or repulsion-phase linkages was found for any of the traits measured. These results demonstrate the effectiveness of long-term divergent selection in the development of coupling-phase linkages and of random mating to dissipate linkage disequilibrium.Research supported by the Illinois Agricultural Experiment Station     

Fine root growth phenology,production, and turnover in a northern hardwood forest ecosystem

A large part of the nutrient flux in deciduous forests is through fine root turnover, yet this process is seldom measured. As part of a nutrient cycling study, fine root dynamics were studied for two years at Huntington Forest in the Adirondack Mountain region of New York, USA. Root growth phenology was characterized using field rhizotrons, three methods were used to estimate fine root production, two methods were used to estimate fine root mortality, and decomposition was estimated using the buried bag technique. During both 1986 and 1987, fine root elongation began in early April, peaked during July and August, and nearly ceased by mid-October. Mean fine root ( 3 mm diameter) biomass in the surface 28-cm was 2.5 t ha^–1 and necromass was 2.9 t ha^–1. Annual decomposition rates ranged from 17 to 30% beneath the litter and 27 to 52% at a depth of 10 cm. Depending on the method used for estimation, fine root production ranged from 2.0 to 2.9 t ha^–1, mortality ranged from 1.8 to 3.7 t ha^–1 yr^–1, and decomposition was 0.9 t ha^–1 yr^–1. Thus, turnover ranged from 0.8 to 1.2 yr^–1. The nutrients that cycled through fine roots annually were 4.5–6.1 kg Ca, 1.1–1.4 kg Mg, 0.3–0.4 kg K, 1.2–1.7 kg P, 20.3–27.3 kg N, and 1.8–2.4 kg S ha^–1. Fine root turnover was less important than leaf litterfall in the cycling of Ca and Mg and was similar to leaf litterfall in the amount of N, P, K and S cycled.