Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Phylogenetic evidence for the herbaceous origin of angiosperms

The ancestral angiosperm is commonly interpreted as an arborescent to shrubby magnolialean with large, multiparted, complex flowers. We examined this hypothesis using a phylogenetic analysis of new and reevaluated characters polarizabled with outgroup comparison. Our cladistic analysis of basal angiosperms placed the nonmagnolialeanChloranthaceae andPiperaceae at the bottom of the tree. We further inferred the probable ancestral states of characters not polarizable with outgroup comparison by examining their distribution among taxa at the base of our cladogram. The sum of ancestral character states suggests that the protoangiosperm was a diminutive, rhizomatous to scrambling perennial herb, with small, simple flowers.     

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.     

Modification of delayed-incubation procedure for detection of fecal coliforms in water.

Three holding media, including the vitamin-free Casitone holding medium (m-VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-ST holding medium (containing ethanol, sulfanilamide, and Tris [pH 8.6] was significantly greater than recovery with m-VFC (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-VFC, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-ST by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-ST be used in place of m-VFC for the delayed-incubation membrane filter procedure.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Perturbation of syntrophic isobutyrate and butyrate degradation with formate and hydrogen

The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and isobutyrate was investigated using a defined methanogenic culture, consisting of syntrophic isobutyrate-butyrate degrader strain IB, Methanobacterium formicicum strain T1N, and Methanosarcina mazeii strain T18. Formate and hydrogen were used to perturb syntrophic butyrate and isobutyrate degradation by the culture. The reversible isomerization between isobutyrate and butyrate was inhibited by the addition of either formate or hydrogen, indicating that the isomerization was coupled with syntrophic butyrate degradation for the culture studied. Energetic analysis indicates that the direction of isomerization between isobutyrate and butyrate is controlled by the ratio between the two acids, and the most thermodynamically favorable condition for the degradation of butyrate or isobutyrate in conjunction with the isomerization is at almost equal concentrations of isobutyrate and butyrate. The degradation of isobutyrate and butyrate was completely inhibited in the presence of a high hydrogen partial pressure (>2000 Pa) or a measurable level of formate (10 muM or higher). Significant formate (more than 1 mM) was detected during the perturbation with hydrogen (17 to 40 kPa). Resumption of butyrate and isobutyrate degradation was related to the removal of formate. Energetic analysis supported that formate was another electron carrier, besides hydrogen, during syntrophic isobutyrate-butyrate degradation by this culture. (c) 1996 John Wiley & Sons, Inc.     

Enhanced mineralization of polychlorinated biphenyls in soil inoculated with chlorobenzoate-degrading bacteria.

An Altamont soil containing no measurable population of chlorobenzoate utilizers was examined for the potential to enhance polychlorinated biphenyl (PCB) mineralization by inoculation with chlorobenzoate utilizers, a biphenyl utilizer, combinations of the two physiological types, and chlorobiphenyl-mineralizing transconjugants. Biphenyl was added to all soils, and biodegradation of 14C-Aroclor 1242 was assessed by disappearance of that substance and by production of 14CO2. Mineralization of PCBs was consistently greatest (up to 25.5%) in soils inoculated with chlorobenzoate degraders alone. Mineralization was significantly lower in soils receiving all other treatments: PCB cometabolizer (10.7%); chlorobiphenyl mineralizers (8.7 and 14.9%); and mixed inocula of PCB cometabolizers and chlorobenzoate utilizers (11.4 and 18.0%). However, all inoculated soils had higher mineralization than did the uninoculated control (3.1%). PCB disappearance followed trends similar to that observed with the mineralization data, with the greatest degradation occurring in soils inoculated with the chlorobenzoate-degrading strains Pseudomonas aeruginosa JB2 and Pseudomonas putida P111 alone. While the mechanism by which the introduction of chlorobenzoate degraders alone enhanced biodegradation of PCBs could not be elucidated, the possibility that chlorobenzoate inoculants acquired the ability to metabolize biphenyl and possibly PCBs was explored. When strain JB2, which does not utilize biphenyl, was inoculated into soil containing biphenyl and Aroclor 1242, the frequency of isolates able to utilize biphenyl and 2,5-dichlorobenzoate increased progressively with time from 3.3 to 44.4% between 15 and 48 days, respectively. Since this soil contained no measurable level of chlorobenzoate utilizers yet did contain a population of biphenyl utilizers, the possibility of genetic transfer between the latter group and strain JB2 cannot be excluded.