RESUMEN
In reptiles with temperature-dependent sex determination (TSD), the temperature at which the eggs are incubated determines the sex of the offspring. The molecular switch responsible for determining sex in these species has not yet been elucidated. We have examined the dynamics of yolk steroid hormones during embryonic development in the snapping turtle, Chelydra serpentina, and the alligator, Alligator mississippiensis, and have found that yolk estradiol (E(2)) responds differentially to incubation temperature in both of these reptiles. Based upon recently reported roles for E(2) in modulation of steroidogenic factor 1, a transcription factor known to be significant in the sex differentiation process, we hypothesize that yolk E(2) is a link between temperature and the gene expression pathway responsible for sex determination and differentiation in at least some of these species. Here we review the evidence that supports our hypothesis.
Asunto(s)
Estradiol/metabolismo , Procesos de Determinación del Sexo , Diferenciación Sexual/fisiología , Temperatura , Tortugas/embriología , Animales , Proteínas de Unión al ADN/metabolismo , Yema de Huevo/metabolismo , Femenino , Factores de Transcripción Fushi Tarazu , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Masculino , Receptores Citoplasmáticos y Nucleares , Factor Esteroidogénico 1 , Factores de Transcripción/metabolismoRESUMEN
Although yolk steroids appear to play important roles in the development, growth, and behavior of some birds, their effects in oviparous reptiles are largely unknown. These investigations were initiated to determine initial levels of steroid hormones in the yolks of eggs from two turtle species. Clutches of snapping turtle (Chelydra serpentina) and of painted turtle (Chrysemys picta) eggs were collected and individual egg yolks were analyzed for estradiol-17beta (E(2)) and testosterone (T) using specific RIAs. E(2) and T levels differed significantly between species, the mean E(2) value in snapping turtles was 2.78+/-0.095 (ng/g) compared to 0.89+/-0.064 (ng/g) for painted turtles, and the mean value for T in snapping turtle yolks was 2.56+/-0.098 (ng/g) compared to 0.68+/-0.045 (ng/g) for painted turtles. In addition, E(2) levels were greater than T levels in both species. Within each species, there were significant differences among clutches from different females. E(2) levels in the snapping turtle yolks varied from a clutch mean of 1.38 to 4.55 ng/g and in painted turtles, the clutch means for E(2) varied from 0.34 to 1.34 ng/g. T levels demonstrated similar phenomena within species, with levels in snapping turtles varying from a clutch mean of 0.68 to 4.71 ng/g. Painted turtle levels of T varied from a clutch mean of 0.22 to 0.72 ng/g. There were also significant differences in the E(2)/T ratio, however, E(2)/T ratios did not differ between species. Painted turtle follicles of different sizes showed significant differences in levels of both E(2) and T, and these differences may reflect differing deposition patterns of these steroids in the egg yolk of this turtle during vitellogenesis. The differences in E(2) and T concentration reported here could have important implications for development, growth, and behavior in oviparous reptilian species.
Asunto(s)
Yema de Huevo/química , Estradiol/análisis , Testosterona/análisis , Tortugas , Animales , Femenino , Especificidad de la EspecieRESUMEN
Many oviparous reptiles exhibit temperature-dependent sex determination (TSD); i.e., the temperature at which the egg is incubated determines the sex of the offspring. In TSD reptiles, yolk steroids not only may influence sex determination, but also may mediate hormonal effects on subsequent growth and behavior, as in some avian species. We investigated changes in the levels of estradiol (E(2)) and testosterone (T) during development in yolks of snapping turtle eggs, examined how incubation temperature affects hormone levels, and determined how hormones in turtle eggs are influenced by individual females (=clutch effects). Results indicate significant decreases in both hormones (>50% decline) by the end of the sex-determining period, when two-thirds of the development is complete. The declines in both E(2) and T were significantly affected by incubation temperature, but in different ways. Eggs incubated at female-producing temperatures maintained high levels, those incubated at male-producing temperatures had low E(2) values, and eggs incubated at pivotal temperatures had intermediate levels of E(2). At all three temperatures, T values underwent significant but approximately equal declines, except during the developmental stages just after the sex-determining period, when T levels decreased more at the male-producing temperature than at either of the other two temperatures. Initially, there were significant clutch effects in both hormones, but such differences, attributable to individual females, were maintained only for E(2) later in development. Here we report for the first time that incubation temperature significantly affects the hormonal environment of the developing embryo of a turtle with temperature-dependent sex determination. Based on this and related findings, we propose that yolk sex steroids influence sexual differentiation in these TSD species and play a role in sex determination at pivotal temperatures.
Asunto(s)
Yema de Huevo/química , Estradiol/análisis , Procesos de Determinación del Sexo , Temperatura , Testosterona/análisis , Tortugas/embriología , Animales , Femenino , Masculino , Factores de TiempoRESUMEN
The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/fisiología , Dipeptidasas , Proteínas de Escherichia coli , Escherichia coli/fisiología , Hexosiltransferasas/fisiología , Complejos Multienzimáticos/fisiología , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutación , Peptidoglicano/biosíntesis , Peptidil Transferasas/fisiología , beta-Lactamasas/fisiología , Bacteriólisis/genética , Endopeptidasas , Evolución Molecular , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Modelos Biológicos , Presión Osmótica , Proteínas de Unión a las PenicilinasRESUMEN
Two proteins that bind penicillin were observed in Escherichia coli infected with lambda phages 141, 142, 650, and 651 from the Kohara genomic library. These phages carry chromosomal DNA fragments that do not contain any known penicillin binding protein (PBP) genes, indicating that unrecognized gene products were exhibiting penicillin binding activity. The genes encoding these proteins were subcloned, sequenced, and identified. One gene was ampC, which encodes a chromosomal class C beta-lactamase. The second gene was located at about 8.5 min on the E. coli genomic map and is a previously uncharacterized open reading frame, here named ampH, that encodes a protein closely related to the class C beta-lactamases. The predicted AmpH protein is similar in length to AmpC, but there are extensive alterations in the amino acid sequence between the SXXK and YXN motifs of the two proteins. AmpH bound strongly to penicillin G, cefoxitin, and cephalosporin C; was temperature sensitive; and disappeared from cells after overnight incubation in stationary phase. Although closely related to AmpC and other class C beta-lactamases, AmpH showed no beta-lactamase activity toward the substrate nitrocefin. Mutation of the ampC and/or ampH genes in E. coli lacking PBPs 1a and 5 produced morphologically aberrant cells, particularly in cell filaments induced by aztreonam. Thus, these two members of the beta-lactamase family exhibit characteristics similar to those of the classical PBPs, and their absence affects cell morphology. These traits suggest that AmpC and AmpH may play roles in the normal course of peptidoglycan synthesis, remodeling, or recycling.