The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose) and repression (by glucose) of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome “remodeler” rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4′s action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription—one that requires the action of SWI/SNF recruited by the activator, and a slower one that does not—clarifies our understanding of the early events of gene activation, and in particular corrects earlier reports that SWI/SNF plays no role in GAL gene induction. Our finding that chromatin structure is irrelevant for repression as studied here—that is, repression sets in as efficiently whether or not promoter nucleosomes are allowed to reform—contradicts the widely held, but little tested, idea that nucleosomes are required for repression. These findings were made possible by our nucleosome occupancy assay. The assay, we believe, will prove useful in studying other outstanding issues in the field.

Juvenile calf oocytes represent an untapped source of germ plasm for reproduction. Reports on the developmental competence of calf oocytes have been controversial. In this research, oocytes were recovered after gonadotropin stimulation from Holstein calves (N = 10) at 2-3 mo of age (2-mo cycle) and again at 4-5 mo of age (4-mo cycle). The in vitro developmental competence was measured, and prestimulation follicle numbers (for 2-mo cycle) and poststimulation follicle numbers (both cycles) were obtained. The number of antral follicles doubled after stimulation (23.4 +/- 6.1 vs. 55.1 +/- 16.1) for the 2-mo cycle and for the 4-mo cycle (47.4 +/- 12.4). The number of follicles observed prior to stimulation in the 2-mo cycle was found to be highly correlated with the poststimulation oocyte recovery for both collection cycles (r = 0.95, 2-mo cycle; r = 0.81, 4-mo cycle). The majority (90-96%) of recovered oocytes were found to be usable for in vitro maturation and fertilization; of these, 41-42% cleaved and 10-11% developed to morulae or blastocysts. Eighty-four in vitro-produced embryos were transferred to synchronized recipients and resulted in 11 pregnancies, leading to 7 live (4 males, 3 females) and 2 dead (one male, one female) calves at full term. No significant differences were observed between the 2-mo and 4-mo collection cycles; however, 73% of the total pregnancies resulted from the 2-mo cycle. All pregnancies resulted from embryos of high-responding donors. The high correlation between the number of follicles prior to stimulation and the poststimulation response suggests the possibility of screening calves prior to stimulation for routine embryo production.

ABSTRACTThe efficacies oftrans-cinnamaldehyde (TC) and eugenol (EG) for reducingSalmonella entericaserovar Enteritidis colonization in broiler chickens were investigated. In three experiments for each compound, 1-day-old chicks (n= 75/experiment) were randomly assigned to five treatment groups (n= 15/treatment group): negative control (-veS. Enteritidis, -ve TC, or EG), compound control (-veS. Enteritidis, +ve 0.75% [vol/wt] TC or 1% [vol/wt] EG), positive control (+veS. Enteritidis, -ve TC, or EG), low-dose treatment (+veS. Enteritidis, +ve 0.5% TC, or 0.75% EG), and high-dose treatment (+veS. Enteritidis, +ve 0.75% TC, or 1% EG). On day 0, birds were tested for the presence of any inherentSalmonella(n= 5/experiment). On day 8, birds were inoculated with ∼8.0 log10CFUS. Enteritidis, and cecal colonization byS. Enteritidis was ascertained (n= 10 chicks/experiment) after 24 h (day 9). Six birds from each treatment group were euthanized on days 7 and 10 after inoculation, and cecalS. Enteritidis numbers were determined. TC at 0.5 or 0.75% and EG at 0.75 or 1% consistently reduced (P< 0.05)S. Enteritidis in the cecum (≥3 log10CFU/g) after 10 days of infection in all experiments. Feed intake and body weight were not different for TC treatments (P> 0.05); however, EG supplementation led to significantly lower (P< 0.05) body weights. Follow-upin vitroexperiments revealed that the subinhibitory concentrations (SICs, the concentrations that did not inhibitSalmonellagrowth) of TC and EG reduced the motility and invasive abilities ofS. Enteritidis and downregulated expression of the motility genesflhCandmotAand invasion geneshilA,hilD, andinvF. The results suggest that supplementation with TC and EG through feed can reduceS. Enteritidis colonization in chickens.

See Scherer (doi:10.1093/awv279) for a scientific commentary on this article.Charcot-Marie-Tooth type 1 neuropathies are inherited disorders of the peripheral nervous system caused by mutations in Schwann cell-related genes. Typically, no causative cure is presently available. Previous preclinical data of our group highlight the low grade, secondary inflammation common to distinct Charcot-Marie-Tooth type 1 neuropathies as a disease amplifier. In the current study, we have tested one of several available clinical agents targeting macrophages through its inhibition of the colony stimulating factor 1 receptor (CSF1R). We here show that in two distinct mouse models of Charcot-Marie-Tooth type 1 neuropathies, the systemic short- and long-term inhibition of CSF1R by oral administration leads to a robust decline in nerve macrophage numbers by ∼70% and substantial reduction of the typical histopathological and functional alterations. Interestingly, in a model for the dominant X-linked form of Charcot-Marie-Tooth type 1 neuropathy, the second most common form of the inherited neuropathies, macrophage ablation favours maintenance of axonal integrity and axonal resprouting, leading to preserved muscle innervation, increased muscle action potential amplitudes and muscle strengths in the range of wild-type mice. In another model mimicking a mild, demyelination-related Charcot-Marie-Tooth type 1 neuropathy caused by reduced P0 (MPZ) gene dosage, macrophage blockade causes an improved preservation of myelin, increased muscle action potential amplitudes, improved nerve conduction velocities and ameliorated muscle strength. These observations suggest that disease-amplifying macrophages can produce multiple adverse effects in the affected nerves which likely funnel down to common clinical features. Surprisingly, treatment of mouse models mimicking Charcot-Marie-Tooth type 1A neuropathy also caused macrophage blockade, but did not result in neuropathic or clinical improvements, most likely due to the late start of treatment of this early onset disease model. In summary, our study shows that targeting peripheral nerve macrophages by an orally administered inhibitor of CSF1R may offer a highly efficacious and safe treatment option for at least two distinct forms of the presently non-treatable Charcot-Marie-Tooth type 1 neuropathies.

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