Sequence-Dependent Gene Conversion: Can Duplicated Genes Diverge Fast Enough to Escape Conversion?

Conversion between duplicated genes limits their independent evolution. Models in which conversion frequencies decrease as genes diverge are examined to determine conditions under which genes can "escape" further conversion and hence escape from a gene family. A review of results from various recombination systems suggests two classes of sequence-dependence models: (1) the "k-hit" model in which conversion is completely inactivated by a few (k) mutational events, such as the insertion of a mobile element, and (2) more general models where conversion frequency gradually declines as genes diverge through the accumulation of point mutants. Exact analysis of the k-hit model is given and an approximate analysis of a more general sequence-dependent model is developed and verified by computer simulation. If mu is the per nucleotide mutation rate, then neutral duplicated genes diverging through point mutants are likely to escape conversion provided 2 mu/lambda much greater than 0.1, where lambda is the conversion rate between identical genes. If 2 mu/lambda much less than 0.1, the expected number of conversions before escape increases exponentially so that, for biological purposes, the genes never escape conversion. For single mutational events sufficient to block further conversions, occurring at rate nu per copy per generation, many conversions are expected if 2 nu/lambda much less than 1, while the genes essentially evolve independently if 2 nu/lambda much greater than 1. Implications of these results for both models of concerted evolution and the evolution of new gene functions via gene duplication are discussed.     

Involvement of the phosphate regulon and the psiD locus in carbon-phosphorus lyase activity of Escherichia coli K-12.

Escherichia coli K-12 can readily mutate to use methylphosphonic acid as the sole phosphorus source by a direct carbon-to-phosphorus (C-P) bond cleavage activity that releases methane and Pi. The in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the phosphate regulon. Since psiD::lacZ(Mu d1) mutants cannot metabolize methylphosphonic acid, psiD may be the structural gene(s) for C-P lyase.     

Bacterial carbon-phosphorus lyase: products, rates, and regulation of phosphonic and phosphinic acid metabolism.

Carbon-phosphorus bond cleavage activity, found in bacteria that utilize alkyl- and phenylphosphonic acids, has not yet been obtained in a cell-free system. Given this constraint, a systematic examination of in vivo C-P lyase activity has been conducted to develop insight into the C-P cleavage reaction. Six bacterial strains were obtained by enrichment culture, identified, and characterized with respect to their phosphonic acid substrate specificity. One isolate, Agrobacterium radiobacter, was shown to cleave the carbon-phosphorus bond of a wide range of substrates, including fosfomycin, glyphosate, and dialkyl phosphinic acids. Furthermore, this organism processed vinyl-, propenyl-, and propynylphosphonic acids, a previously uninvestigated group, to ethylene, propene, and propyne, respectively. A determination of product stoichiometries revealed that both C-P bonds of dimethylphosphinic acid are cleaved quantitatively to methane and, furthermore, that the extent of C-P bond cleavage correlated linearly with the specific growth rate for a range of substrates. The broad substrate specificity of Agrobacterium C-P lyase and the comprehensive characterization of the in vivo activity make this an attractive system for further biochemical and mechanistic experiments. In addition, the failure to observe the activity in a group of gram-positive bacteria holds open the possibility that a periplasmic component may be required for in vivo expression of C-P lyase activity.     

Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.     

New poly(A)+RNAs appear coordinately during the differentiation of Naegleria gruberi amebae into flagellates

We have examined the nature of the requirement for RNA synthesis during the differentiation of Naegleria gruberi amebae into flagellates (Fulton, C., and C. Walsh, 1980, J. Cell Biol., 85:346-360) by looking for poly(A)+RNAs that are specific to differentiating cells. A cDNA library prepared from poly(A)+RNA extracted from cells 40 min after initiation of the differentiation (40-min RNA), the time when formation of flagella becomes insensitive to inhibitors of RNA synthesis, was cloned into pBR322. Recombinant clones were screened for sequences that were complementary to 40-min RNA but not to RNA from amebae (0-min RNA). Ten of these differentiation-specific (DS) plasmids were identified. The DS plasmids were found to represent at least four different poly(A)+RNAs based on cross-hybridization, restriction mapping, and Northern blot analysis. Dot blot analysis was used to quantify changes in DS RNA concentration. The four DS RNAs appeared coordinately during the differentiation. They were first detectable at 10-15 min after initiation, reached a peak at 70 min as flagella formed, and then declined to low levels by 120 min when flagella reached full length. The concentration of the DS RNAs was found to be at least 20-fold higher in cells at 70 min than in amebae. The changes in DS RNA concentration closely parallel changes in tubulin mRNA as measured by in vitro translation (Lai, E.Y., C. Walsh, D. Wardell, and C. Fulton, 1979, Cell, 17:867-878).     

sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence

The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a beta-octylglucoside mixed micellar assay (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and at lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. beta-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. beta-Octylglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.     

Carbon and nitrogen assimilation and partitioning in soybeans exposed to low root temperatures

Low root temperature effects on vegetative growth of soybean (Harosoy 63 × Rhizobium japonicum USDA 16) were examined in 35 day old plants exposed to temperatures of 15°C (shoots at 25°C) for an 11 day period. Duing this period various aspects of C and N assimilation and partitioning were monitored including shoot night and nodulated root respiration, C and N partitioning to six plant parts, C₂H₂ reduction, H₂ evolution, leaf area, transpiration, net photosynthesis, and N₂ fixation. The low temperature treatment resulted in a decrease in the net rate of N₂ fixation but nitrogenase relative efficiency increased. In response, the plant retained N in the tissues of the nodulated root and decreased N partitioning to young shoot tissues, thereby inducing the remobilization of N from older leaves, and reducing leaf area development. The leaf area specific rate of net photosynthesis was not affected over the study period; however, shoot and nodulated root respiration declined. Consequently, C accumulated in mature leaves and stems, partly in the form of increased starch reserves. Three possibilities were considered for increasing low temperature tolerance in nodulated soybeans: (a) decrease in temperature optima for nitrogenase, (b) increased development of nodules and N₂ fixation capacity at low temperature, and (c) alterations in the pattern of C and N partitioning in response to low temperature conditions.     

Methods for quantitative and qualitative evaluation of vaginal microflora during menstruation

The quantitative and qualitative changes in the bacterial flora of the vagina during menstruation have received inadequate study. Similarly, the effect of vaginal tampons on the microbial flora as well as the relationship between the microbial flora of the vagina and that of the tampon has not been adequately evaluated. The purposes of the present study were (i) to develop quantitative methods for studying the vaginal flora and the flora of tampons obtained during menstruation and (ii) to determine whether there were differences between the microflora of the tampon and that of the vaginal vault. Tampon and swab samples were obtained at various times from eight young healthy volunteers for 8 to 10 menstrual cycles. Samples consisted of swabs from women wearing menstrual pads compared with swab and tampon samples taken at various times during the menstrual cycle. Samples were analyzed for total facultative and anaerobic bacterial counts, and the six dominant bacterial species in each culture were identified. Statistical evaluation of the results indicates that total bacterial counts decreased during menstruation and that swab and tampon samples yielded similar total counts per unit weight of sample. The numbers of bacteria in tampons tended to be lower than in swabs taken at the same time. Overall, during menstruation, the concentrations of lactobacilli declined, but otherwise there was little difference among the species found during menstruation compared with those found in intermenstrual samples. Cotton tampons had little discernible effect on the microbial flora.