Illegitimate recombination in Xenopus: characterization of end-joined junctions.

When linear DNAs are injected into Xenopus laevis eggs, they are converted into several different kinds of recombination products. Some molecules undergo homologous recombination by a resection-annealing mechanism; some ends are precisely ligated; and some ends are joined by illegitimate means. The homologous and illegitimate products are also generated in nuclear extracts from stage VI Xenopus oocytes. In order to gain insight into the mechanism(s) of illegitimate end joining, we amplified, cloned and sequenced a number of junctions from eggs and from oocyte extracts. The egg junctions fell into three categories: some with no homology at the join point that may have been produced by blunt-end ligation; some based on small, but significant homologies (5-10 bp); and some with matches of only 1 or 2 nucleotides at the joint. Junctions made in oocyte extracts were largely of the latter type. In the extracts, formation of illegitimate joints required the addition of all four deoxyribonucleoside triphosphates and was inhibited by aphidicolin. This indicates that this process involves DNA synthesis, and mechanisms incorporating this feature are considered. The spectrum of recombination products formed in Xenopus eggs is very reminiscent of those produced from DNA introduced into mammalian cells.     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.     

Genomic structure,expression and evolution of the alfalfa aspartate aminotransferase genes

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.     

Interactions between size and temperature influence fecundity and longevity of a tortricid moth,Zeiraphera canadensis

Females of Zeiraphera canadensis Mut. & Free., the spruce bud moth, were reared in the laboratory at constant and alternating temperatures, and in an outdoor insectary, to (1) determine the effects of temperature, age and size on several reproductive parameters and, (2) to test the hypothesis that body size-temperature interactions influence longevity and realized fecundity. Egg maturation was linearly related to age and large moths developed eggs at a higher rate than small ones. Mcan lifetime oviposition rate reached a maximum and remained stable at temperatures 20° C while the mean lifetime rate of egg maturation increased linearly with temperature, indicating that higher temperatures adversely affect oviposition. The production of nonviable eggs increased with age but also with temperature, suggesting high temperature (25° C) reduces egg quality and/or hinders fertilization. The realized fecundity and longevity of females reared under an alternating temperature regime (mean 20° C) was significantly less than that of females reared at constant 20° C. Similar realized fecundity, longevity and mean lifetime oviposition rates for females reared at temperatures alternating between 10 and 25° C (mean 20° C) and those at constant 25° C reflected the inability of females to recover from elevated diurnal temperatures. Longevity was positively related to female body size at constant 15 and 20° C but the relationships were negative for moths exposed to diurnal temperatures equal to or exceeding 25° C. Due to the reduced longevity of large moths at high temperatures, linear regressions between size and realized fecundity were only significant at constant temperatures 20° C. At higher temperatures, the size-fecundity relationship became curvilinear as a result of the diminished reproductive output of large individuals. Reduced fecundity and longevity of large females at high temperatures may have been due to elevated internal temperatures of large-bodied moths. Large females in a controlled-environment chamber maintained at 25° C developed an internal temperature excess (i.e. temperature above ambient) of nearly 2° C while small-bodied females exceeded ambient by only 0.3° C. However, when held at 20° C, the temperature excess of large-bodied moths was much less than 1° C and small-bodied females did not differ from ambient. Such interactions between temperature and body size suggest that there should be stabilizing selection toward moderate-sized individuals and may explain the absence of size-related effects on fecundity and longevity previously reported for several other lepidopterans.     

PROGENY SEX RATIOS IN DIOECIOUS SILENE LATIFOLIA (CARYOPHYLLACEAE)

Most sex ratios reported for Silene latifolia are female biased. As a result of experiments performed by Correns in the early 1900s, pollen tube competition has generally been accepted as the primary cause of these skewed ratios. We did four sets of hand pollinations in which we varied the size of pollen loads and placement of pollen along the filamentous stigma. The effect of pollen load size on progeny sex ratios was not statistically significant. Of 32 maternal families, 17 contained more females than males (one ratio deviated statistically from 1:1), and 13 contained more males than females. Paternal families exhibited a greater range of sex ratios, including three with a significant female bias and one with a significant male bias. Within experiments, neither the maternal parent nor where pollen was placed had a statistically significant effect on progeny sex ratios; the paternal effect was significant in one experiment. We suggest that sex ratios in Silene latifolia are not necessarily affected by the level of pollen competition. Other factors, including variation among males and sex-linked mortality, may help explain the skewed sex ratios that characterize populations of this species. Further, Correns' observations of excess females may have resulted from his use of interspecific hybrids.     

H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes.

The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.     

Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.