Jan A. Olsson

Eclipse period without sequestration in Escherichia coli

The classical Meselson–Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy (15NH4+ and 13C6‐glucose) medium were shifted to light (14NH4+ and 12C6‐glucose) medium. The HH‐, HL‐ and LL‐DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene‐specific probes. The eclipse period, estimated from the kinetics of conversion of HH‐DNA to HL‐ and LL‐DNA, turned out to be 0.60 generation times for the wild‐type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.

Selective estrogen receptor α activation disrupts sex organ differentiation and induces expression of vitellogenin II and very low-density apolipoprotein II in Japanese quail embryos

The Japanese quail (Coturnix japonica) is a widely used model species for studying the roles of steroid hormones in avian sex differentiation. The aim of the present study was to elucidate the significance of estrogen receptors alpha and beta (ERalpha and ERbeta) in normal sex differentiation of the reproductive organs in the Japanese quail and in xenoestrogen-induced disruption of reproductive organ differentiation. Real-time PCR indicated that ERalpha (ESR1) mRNA is expressed in both right and left gonads and Müllerian ducts (MDs) in both sexes during early morphological differentiation. ERbeta (ESR2) transcripts were also detected in gonads and MDs, but at very low levels. Both receptor subtypes were expressed in the liver and may therefore mediate the expression of estrogen-regulated egg-yolk proteins. Aromatase mRNA was expressed at much higher levels in female than male gonads as early as embryonic day 5, indicating early sex differences in estrogen synthesis. Treatment with the ERalpha-selective agonist propyl pyrazole triol showed that frequently reported xenoestrogen effects, such as ovotestis formation, abnormal MD development, and hepatic expression of egg-yolk proteins, were induced by selective activation of ERalpha. Taken together, our results suggest that activation of ERalpha is crucial for estrogen-dependent sex differentiation of the reproductive organs and that ERalpha mediates xenoestrogen-induced toxicity during reproductive development in birds.

Activation of estrogen receptor alpha disrupts differentiation of the reproductive organs in chicken embryos

Gonadal estrogen plays an important role in the differentiation of a female phenotype in birds. Exogenous compounds that interfere with estrogen signaling, for instance by binding to the estrogen receptors alpha and beta (ERα and ERβ), are therefore potential disruptors of sexual differentiation in birds. The ERα agonist propyl-pyrazole-triol (PPT), the ERα antagonist methyl piperidino pyrazole (MPP) and the ERβ agonist diarylproprionitrile (DPN) were used in the present study to explore the roles of the ERs in normal and disrupted sex differentiation in the chicken embryo. Activation of ERα by PPT caused disturbed differentiation of the reproductive organs in both sexes. In male embryos, PPT caused left-side ovotestis formation and retention of the Müllerian ducts. In female embryos, PPT caused retention of the right Müllerian duct (which normally regresses) and malformation of both Müllerian ducts. PPT also induced hepatic expression of mRNA for the estrogen-regulated egg yolk protein apoVLDL II. Notably, none of these effects were observed following treatment with DPN. ERα-inactivation by MPP counteracted the action of PPT but had little effect by its own. Our results indicate that ERα plays an important role in sex differentiation of the reproductive tract in female chicken embryos and show that ERα can mediate xenoestrogen-induced disturbances of sex differentiation.

Transcription of genes involved in fat metabolism in chicken embryos exposed to the peroxisome proliferator-activated receptor alpha (PPARα) agonist GW7647 or to perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA)

Perfluoroalkyl acids (PFAAs) such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are developmental toxicants in various animal classes, including birds. Both compounds interact with peroxisome proliferator-activated receptors (PPARs), but it is not known whether activation of PPARs is involved in their embryo toxicity in birds. We exposed chicken embryos via egg injection at a late developmental stage to GW7647, a potent PPARα agonist in mammals, and to PFOS or PFOA. Mortality was induced by PFOS and PFOA but not by GW7647. Transcripts of a number of genes activated by PPARα agonists in mammals were analyzed in liver and kidney of 18-day-old embryos. Several of the genes were induced in both liver and kidney following exposure to GW7647. Treatment with PFOA resulted in induction of acyl-coenzyme A oxidase mRNA in liver, whereas none of the genes were significantly induced by PFOS treatment. No up-regulation of gene transcription was found in kidney following treatment with PFOS or PFOA. Principal component analysis showed that PFOA caused an mRNA expression pattern in liver more similar to the pattern induced by GW7647 than PFOS did. Our findings do not support that the embryo mortality by PFOS and PFOA in chicken embryos involves PPARα activation.

Eclipse period during replication of plasmid R1: contributions from structural events and from the copy- number control system

The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson–Stahl density‐shift experiment. A mini‐plasmid with the wild‐type R1 replicon and a mutant with a thermo‐inducible runaway‐replication phenotype were used in this work. The eclipses of the chromosome and of the wild‐type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30°C to 42°C. The mutant plasmid had a similar eclipse at temperatures up to 38°C. At 42°C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350–400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild‐type plasmid at 42°C). Hence, the copy‐number control system contributed significantly to the eclipse. The eclipse in the absence of copy‐number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.

Cytotoxicity and decreased corticosterone production in adrenocortical Y-1 cells by 3-methylsulfonyl-DDE and structurally related molecules.

The persistent environmental pollutant 3-methylsulfonyl-DDE (3-MeSO2-DDE) undergoes bioactivation by cytochrome P450 11B1 (CYP11B1) in the adrenal cortex of several animal species in vivo and causes decreased glucocorticoid production and cell death in the zona fasciculata. This study presents extended investigations of the cytotoxic and endocrine disrupting effects of 3-MeSO2-DDE and some structurally related molecules in the mouse adrenocortical cell line Y-1. Both 3-MeSO2-DDE and, to a lesser extent, 3,3′(bis)-MeSO2-DDE decreased corticosterone production and produced CYP11B1-dependent cytotoxicity in Y-1 cells. Neither 2-MeSO2-DDE nor p,p′-DDE had any significant effect on either cell viability or corticosterone production, indicating that the presence and position of the methylsulfonyl moiety of 3-MeSO2-DDE is crucial for its biological activity. The adrenocortical toxicant o,p′-DDD decreased corticosterone production but was not cytotoxic in this cell line. None of the compounds altered Cyp11b1 gene expression, indicating that 3-MeSO2-DDE inhibits CYP11B1 activity on the protein level.

Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication

The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.