The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural compo... more 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...
Two proteins that bind penicillin were observed in Escherichia coli infected with lambda phages 1... more 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 disap...
Many oviparous reptiles exhibit temperature-dependent sex determination (TSD); i.e., the temperat... more 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.
The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural compo... more 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...
Two proteins that bind penicillin were observed in Escherichia coli infected with lambda phages 1... more 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 disap...
Many oviparous reptiles exhibit temperature-dependent sex determination (TSD); i.e., the temperat... more 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.
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