Circadian Genes Are Expressed during Early Development in Xenopus laevis

Circadian oscillators are endogenous time-keeping mechanisms that drive twenty four hour rhythmic changes in gene expression, metabolism, hormone levels, and physical activity. We have examined the developmental expression of genes known to regulate circadian rhythms in order to better understand the ontogeny of the circadian clock in a vertebrate.In this study, genes known to function together in part of the core circadian oscillator mechanism (xPeriod1, xPeriod2, and xBmal1) as well as a rhythmic, clock-controlled gene (xNocturnin) were analyzed using in situ hybridization in embryos from neurula to late tailbud stages. Each transcript was present in the developing nervous system in the brain, eye, olfactory pit, otic vesicle and at lower levels in the spinal cord. These genes were also expressed in the developing somites and heart, but at different developmental times in peripheral tissues (pronephros, cement gland, and posterior mesoderm). No difference was observed in transcript levels or localization when similarly staged embryos maintained in cyclic light were compared at two times of day (dawn and dusk) by in situ hybridization. Quantitation of xBmal1 expression in embryonic eyes was also performed using qRT-PCR. Eyes were isolated at dawn, midday, dusk, and midnight (cylic light). No difference in expression level between time-points was found in stage 31 eyes (p = 0.176) but stage 40 eyes showed significantly increased levels of xBmal1 expression at midnight (RQ = 1.98+/-0.094) when compared to dawn (RQ = 1+/-0.133; p = 0.0004).We hypothesize that when circadian genes are not co-expressed in the same tissue during development that it may indicate pleiotropic functions of these genes that are separate from the timing of circadian rhythm. Our results show that all circadian genes analyzed thus far are present during early brain and eye development, but rhythmic gene expression in the eye is not observed until after stage 31 of development

Differential Contribution of Rod and Cone Circadian Clocks in Driving Retinal Melatonin Rhythms in Xenopus

Background: Although an endogenous circadian clock located in the retinal photoreceptor layer governs various physiological events including melatonin rhythms in Xenopus laevis, it remains unknown which of the photoreceptors, rod and/or cone, is responsible for the circadian regulation of melatonin release. Methodology/Principal Findings: We selectively disrupted circadian clock function in either the rod or cone photoreceptor cells by generating transgenic Xenopus tadpoles expressing a dominant-negative CLOCK (XCLDQ) under the control of a rod or cone-specific promoter. Eyecup culture and continuous melatonin measurement revealed that circadian rhythms of melatonin release were abolished in a majority of the rod-specific XCLDQ transgenic tadpoles, although the percentage of arrhythmia was lower than that of transgenic tadpole eyes expressing XCLDQ in both rods and cones. In contrast, whereas a higher percentage of arrhythmia was observed in the eyes of the cone-specific XCLDQ transgenic tadpoles compare to wildtype counterparts, the rate was significantly lower than in rod-specific transgenics. The levels of the transgene expression were comparable between these two different types of transgenics. In addition, the average overall melatonin levels were not changed in the arrhythmic eyes, suggesting that CLOCK does not affect absolute levels of melatonin, only its temporal expression pattern. Conclusions/Significance: These results suggest that although the Xenopus retina is made up of approximately equa

Studies on the Restriction of Murine Leukemia Viruses by Mouse APOBEC3

APOBEC3 proteins function to restrict the replication of retroviruses. One mechanism of this restriction is deamination of cytidines to uridines in (−) strand DNA, resulting in hypermutation of guanosines to adenosines in viral (+) strands. However, Moloney murine leukemia virus (MoMLV) is partially resistant to restriction by mouse APOBEC3 (mA3) and virtually completely resistant to mA3-induced hypermutation. In contrast, the sequences of MLV genomes that are in mouse DNA suggest that they were susceptible to mA3-induced deamination when they infected the mouse germline. We tested the possibility that sensitivity to mA3 restriction and to deamination resides in the viral gag gene. We generated a chimeric MLV in which the gag gene was from an endogenous MLV in the mouse germline, while the remainder of the viral genome was from MoMLV. This chimera was fully infectious but its response to mA3 was indistinguishable from that of MoMLV. Thus, the Gag protein does not seem to control the sensitivity of MLVs to mA3. We also found that MLVs inactivated by mA3 do not synthesize viral DNA upon infection; thus mA3 restriction of MLV occurs before or at reverse transcription. In contrast, HIV-1 restricted by mA3 and MLVs restricted by human APOBEC3G do synthesize DNA; these DNAs exhibit APOBEC3-induced hypermutation

HLA class I and II diversity contributes to the etiologic heterogeneity of non-Hodgkin lymphoma subtypes

A growing number of loci within the human leukocyte antigen (HLA) region have been implicated in non-Hodgkin lymphoma (NHL) etiology. Here, we test a complementary hypothesis of "heterozygote advantage" regarding the role of HLA and NHL, whereby HLA diversity is beneficial and homozygous HLA loci are associated with increased disease risk. HLA alleles at class I and II loci were imputed from genome-wide association studies (GWAS) using SNP2HLA for: 3,617 diffuse large B-cell lymphomas (DLBCL), 2,686 follicular lymphomas (FL), 2,878 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL), 741 marginal zone lymphomas (MZL), and 8,753 controls of European descent. Both DLBCL and MZL risk were elevated with homozygosity at class I HLA-B and -C loci (OR DLBCL=1.31, 95% CI=1.06-1.60; OR MZL=1.45, 95% CI=1.12-1.89) and class II HLA-DRB1 locus (OR DLBCL=2.10, 95% CI=1.24-3.55; OR MZL= 2.10, 95% CI=0.99-4.45). Increased FL risk was observed with the overall increase in number of homozygous HLA class II loci (p-trend<0.0001, FDR=0.0005). These results support a role for HLA zygosity in NHL etiology and suggests that distinct immune pathways may underly the etiology of the different NHL subtypes

Melatonin release from the XOP-XCLΔQ-GFP and the CAR-XCLΔQ-GFP transgenic eyecups and wild-type controls.

<p>Each pair of eyecups was prepared from individual tadpoles and flow-through culture was performed for 5 days. Media fractions were collected every four hours, and assayed for melatonin by RIA. Each line represents melatonin release from a pair of eyecups. A. Melatonin release rhythms in the individual XOP-XCLΔQ-GFP eyecups (n = 5) and wild-type controls (n = 7). As compared to the wild-type eyes that demonstrate melatonin release in a circadian manner for five days, the majority of the transgenic eyes do not show significant circadian melatonin rhythmicity. B. Melatonin rhythms in the CAR-XCLΔQ-GFP (n = 6) and wild-type eyecups (n = 6). With some exceptions, eyecups release melatonin in a circadian manner.</p

Total melatonin levels of the transgenic eyes and wild-type controls were comparable.

<p>Average of all fractions from the transgenic and wild-type eyes was calculated for the two different genotypes (XOP and CAR). Values on the figure are average melatonin content (picograms per 4hr) +/− SEM. A. The XOP transgenic (n = 25) vs. wild-type eyes (n = 37). B. The CAR transgenic (n = 17) vs. wild-type eyes (n = 73).</p

Arrhythmic melatonin secretion correlates with mRNA levels of XOP-XCLΔQ, but not CAR-XCLΔQ.

<p>qPCR was performed on GFP as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015599#pone-0015599-g004" target="_blank">Figure 4</a>. The GFP mRNA levels from the arrhythmic and rhythmic animal groups in each of the two transgenic animals were averaged. A. Comparison of GFP levels between rhythmic (XOP-R; n = 13) and arrhythmic (XOP-AR; n = 12) groups in the XOP transgenics (<i>P<0.05</i>, Student <i>t-</i>test). B. Rhythmic (CAR-R; n = 8) and arrhythmic (CAR-AR; n = 11) groups in CAR transgenic eyecups expressed comparable levels of GFP. Values are average relative GFP expression levels +/− SEM.</p

Expression levels of the two different transgenes are comparable.

<p>After flow-through culture was complete, each pair of eyes was collected, RNA was extracted and real-time PCR was performed on GFP to compare relative levels of transgene expression. The average GFP levels from the XOP (n = 25) and CAR (n = 26) transgenic eyes were comparable and the difference was not statistically significant. Values are average relative GFP expression levels +/− SEM.</p