Computational complexity of inferring phylogenies from dissimilarity matrices

Molecular biologists strive to infer evolutionary relationships from quantitative macromolecular comparisons obtained by immunological, DNA hybridization, electrophoretic or amino acid sequencing techniques. The problem is to find unrooted phylogenies that best approximate a given dissimilarity matrix according to a goodness-of-fit measure, for example the least-squares-fit criterion or Farris'sf statistic. Computational costs of known algorithms guaranteeing optimal solutions to these problems increase exponentially with problem size; practical computational considerations limit the algorithms to analyzing small problems. It is established here that problems of phylogenetic inference based on the least-squares-fit criterion and thef statistic are NP-complete and thus are so difficult computationally that efficient optimal algorithms are unlikely to exist for them. The Natural Sciences and Engineering Research Council of Canada partially supported this research through an individual operating grant (A4142) to W.H.E. Day.     

Exogenous O6-methylguanine inhibits adduct removal and sensitizes human cells to killing by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine

We partially depleted the O6-methylguanine-DNA methyltransferase activity in four O6-methylguanine (O6-mGua) repair-proficient (Mer+) human cell strains with exogenous O6-mGua (2 mM for 3 h, a non-toxic regimen) and then challenged them with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT-partially depleted HT29 cells removed O6-mGua from DNA at about half the rate of control cells, while removal of 3-methyladenine was unaffected. In spite of partial depletion of MT, however, cell killing by MNNG in a colony-forming assay with HT29, A549, A498 or KD cells was not greatly affected. (This is in contrast to the dramatic potentiation of CNU cytotoxicity observed previously.) In an attempt to sensitize Mer+ strains to killing by MNNG, we treated cells with O6-mGua following MNNG exposure (0.4 mM for 4 days), in addition to the pre-MNNG treatment of 2 mM O6-mGua for 3 h. This sensitized KD and HT29 cells 2-fold to killing by MNNG, based on the dose at 10% survival, but did not sensitive Mer- A1336. However, post-treatment alone was as effective as combined pre- and post-treatment in sensitizing KD cells to killing. Thus, when the O6-mGua post-treatment was begun, greater than 50% of O6-mGua was already removed from cell DNA. Our findings may be accounted for by at least two schemes, one in which nonlethal O6-mGua are removed from DNA rapidly, while potentially lethal O6-mGua are repaired later. The other scheme proposes that exogenous O6-mGua increases the lethality of a non-O6-mGua lesion by reducing its repair both in Mer+ and Mer- cells. Both schemes are consistent with the hypothesis that O6-mGua may be a lethal DNA lesion in human cells.     

Acceleration of surface-dependent autocatalytic activation of blood coagulation factor XII by divalent metal ions

The effect of divalent metal ions on the rate of dextran sulfate dependent autocatalytic activation of human blood coagulation factor XII was studied at pH 7.4 and 25 degrees C. Zn2+ and Cu2+, but not Co2+, increased the rate of factor XII activation induced by dextran sulfate with optimum effects at approximately 5 and 1 microM, respectively, while Ca2+ acceleration required much higher concentrations (millimolar). Further investigation of the effect of Zn2+ on factor XII activation demonstrated a complete dependence on the presence of dextran sulfate, lack of inhibition by soybean trypsin inhibitor, the appearance of alpha-XIIa as the primary reaction product, and reaction kinetics characteristic of an autocatalytic process. These results were consistent with Zn2+ affecting only the rate of surface-mediated factor XII autoactivation. The initial turnover velocity of dextran sulfate induced factor XII autoactivation increased linearly with factor XII concentration in the absence of Zn2+ up to 0.9 microM factor XII but showed saturation behavior over this same concentration range in the presence of 5 microM Zn2+, indicating that Zn2+ increased the reaction rate primarily by lowering the apparent Km. Comparison of the kinetics of autoactivation at mu = 0.15 and 0.24 revealed that the enhancement in the apparent kcat/Km brought about by Zn2+ increased from 19-fold to 520-fold, respectively, due to a differential dependence of the Zn2+-stimulated and unstimulated reactions on ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical specificity of antisera raised against the synthetic encephalitogenic peptide SH624, residues 59–74 of the myelin basic protein

Synthetic peptide SH624 (SHHPARTAHYGSLPQK), residues 59–74 of human myelin basic protein (MBP) was found to be encephalitogenic in the rabbit. Four antisera raised, against the peptide were employed in a liquid-phase equilibrium competitive radioimmunoassay with a series of synthetic peptide analogs of the region to probe the structural requirements of the B-cell determinant subsumed within SH624. The cross-reactivities of the four antisera with intact MBP were also examined. Immunochemical analyses of the four antisera suggested specificities directed against a conformational determinant dependent upon residues from the more phylogenetically conserved carboxyl C-terminal region, residues 65–74 (TAHYGSLPQK) of the synthetic immunogen. Peptide analogs shorter than SH624 from the C-terminal end showed no cross-reactivity with any of the reagent antisera while analogs shorter from the N-terminal end and including the encephalitogenic sequence TTHYGSLPQK, as well as, HYGSLPQK were reactive under equilibrium competitive conditions. SH624-reactive antibodies, cross-reactive with purified heterologous MBPs from 10 different species were also identified in all four reagent antisera. The results of these experiments support previous investigations demonstrating the accessibility of the encephalitogenic 65–74 region in intact MBP. They also underscore the importance of B-cell recognition of organ specific antigenic determinants with respect to MBP immunology and, in particular, the recognition of autoreactive determinants in the neighborhood of encephalitogenic centers.     

Detecting estrus in synchronized heifers-using tailpaint and an aerosol raddle

A synchronization treatment was initiated when each of 1227 heifers (four trials) was tailpainted. The tailpaint was sprayed with an aerosol raddle at the end of the treatment period. The heifers were in herds of 20 to 279 animals. Each herd was observed for estrus at selected post treatment intervals. A heifer was considered to be (or to have been) in estrus when the raddle was rubbed off. In three of the trials, animals which had the raddle removed were inseminated at 48h following the end of the synchronization treatment. The tailpaint of an inseminated animal was scored from 0 (less than 10% of the paint remained) to 5 (more than 90% of the paint remained) and was then reraddled with a second color. The detection-insemination sequence was always repeated at 72 and 96h, and sometimes at 120h. Animals which had been previously inseminated, but then had paint scores reduced by at least 2 units were reinseminated 24h later. Over the four trials, 94.5% of the heifers were detected in estrus through the use of the tailpaint and raddle system. The remaining 67 animals included only 10 (0.8%) which had ovulated without being detected in estrus. The reinsemination rate on consecutive days was 11.3% and was highest among animals that had a tailpaint score of 4 or 5 at 48h. The proportion of animals detected in estrus at selected posttreatment intervals varied with the different synchronization treatments used within one herd, or with the same treatment used in different herds. The combination of tailpaint, raddling, tailpaint scoring and reraddling is a simple sequence which can be effectively used to detect estrus among heifers synchronized in research or commercial herds.     

Sequence polymorphism of human complement factor H

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

Glutamate Oxidation by Soybean Cotyledon and Leaf Mitochondria

Mitochondria purified from cotyledons of soybean seedlings fiveto ten days old have the capacity to rapidly oxidize glutamate(measured as glutamate dependent oxygen consumption). This capacitywas greatest at ten days after planting but was very low priorto emergence of cotyledons from the vermiculite and during senescence.Solubilized glutamate dehydrogenase activity, on the other hand,was substantial at two days after planting, peaked at sevendays, then declined and rose again during senescence. It issuggested that mitochondrial glutamate oxidation plays a rolein reserve mobilization and amino acid metabolism during seedlinggrowth. Leaf mitochondria and those from senescing cotyledonscould not sustain rapid rates of glutamate oxidation despiteready oxidation of other substrates and high solubilized glutamatedehydrogenase activity, suggesting an alternative role for theenzyme in these tissues. Possible controlling factors are discussed. ² Present address, Garvan Institute, Darlinghurst, N. S. W.,Australia. ³ Permanent address, Department de Biologia Vegetal, Facultatde Biologia, Universitat de Barcelona, Barcelona, Spain. (Received May 6, 1988; Accepted August 3, 1988)