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Carla B Green

Publications and source records attributed to Carla B Green.

16 recordsLinked to original sources
PubMed2026

Caloric restriction modulates genome-wide somatic mutation in mice.

Somatic mutations accumulate throughout life in every cell, and this process constitutes one of the hallmarks of aging-genomic instability. Caloric restriction (CR) has been shown to extend lifespan across diverse species. Using high-fidelity duplex DNA sequencing of bulk liver, bulk kidney, hepatocytes, and cerebellar neurons, we found that CR in mice reduces genome-wide somatic mutation burdens across multiple tissues and cell types. CR reduced both substitution and insertion/deletion burdens, with the magnitude of these effects varying across sample types. CR also decreased the activity of the enigmatic single-base substitution (SBS) mutational process SBS5 that gives rise to most mutations in mammals. Surprisingly, the mutation burden reduction from CR was greatest in transcriptionally inactive regions. This work illuminates links between diet, aging, and genomic integrity and establishes genomic integrity as a modifiable axis of aging.

DNA↗
PubMed2007

Structure/function analysis of Xenopus cryptochromes 1 and 2 reveals differential nuclear localization mechanisms and functional domains important for interaction with and repression of CLOCK-BMAL1.

Circadian rhythms control the temporal arrangement of molecular, physiological, and behavioral processes within an organism and also synchronize these processes with the external environment. A cell autonomous molecular oscillator, consisting of interlocking transcriptional/translational feedback loops, drives the approximately 24-hour duration of these rhythms. The cryptochrome protein (CRY) plays a central part in the negative feedback loop of the molecular clock by translocating to the nucleus and repressing CLOCK and BMAL1, two transcription factors that comprise the positive elements in this cycle. In order to gain insight into the inner workings of this feedback loop, we investigated the structure/function relationships of Xenopus laevis CRY1 (xCRY1) and xCRY2 in cultured cells. The C-terminal tails of both xCRY1 and xCRY2 are sufficient for their nuclear localization but achieve it by different mechanisms. Through the generation and characterization of xCRY/photolyase chimeras, we found that the second half of the photolyase homology region (PHR) of CRY is important for repression through facilitating interaction with BMAL1. Characterization of these functional domains in CRYs will help us to better understand the mechanism of the known roles of CRYs and to elucidate new intricacies of the molecular clock.

ARNTL Transcription Factors↗
PubMed2006

Xenobiotic metabolism in the fourth dimension: PARtners in time.

A significant portion of the transcriptome in mammals, including the PAR bZIP transcription factors DBP, HLF, and TEF, is under circadian clock control. In this issue of Cell Metabolism, Gachon and colleagues (Gachon et al., 2006) show that disruption of these three genes in mice alters gene expression patterns of many proteins involved in drug metabolism and in liver and kidney responses to xenobiotic agents. Triple mutant mice have severe physiological deficits, including increased hypersensitivity to xenobiotic agents and premature aging, highlighting the profound effect the circadian clock has on this important response system.

Animals↗
PubMed2006

Functional analysis of nocturnin: a circadian clock-regulated gene identified by differential display.

Within the retina there is a circadian clock that controls the 24-h timing of processes such as hormone release, cell movement, and gene transcription. In an effort to better understand the molecular nature of this retinal clock, a differential display (DD) screen was performed to isolate a gene with high amplitude circadian rhythmicity in the Xenopus retina. A novel gene expressed in the early evening in photoreceptor cells was isolated and named nocturnin for night factor. This article outlines the steps we took to study a protein of unknown function, particularly highlighting the analyses one can perform when little more than the primary sequence of a gene is known. In addition, we describe the results of sequence analysis that assisted in predicting the function of nocturnin. We have shown that nocturnin acts as a deadenylase in vitro, removing the poly(A) tail from a mature messenger RNA in a process that either leads to degradation or translational silencing of a message. Although the role of nocturnin in the retina is unknown, future studies to identify target mRNAs that are deadenylated by nocturnin will assist in elucidating its physiological role in this tissue.

Animals↗
PubMed2005

Cryptochromes.

Explore the source record for details and available documents.

Animals↗
PubMed2005

The circadian clock-containing photoreceptor cells in Xenopus laevis express several isoforms of casein kinase I.

The frog (Xenopus laevis) retina has been an important model for the analysis of retinal circadian rhythms. In this paper, several isoforms of X. laevis casein kinase I (CKI) were analyzed to address whether they are involved in the phosphorylation and degradation of period protein (PER), as they are in the circadian oscillators of other species. cDNAs encoding two splice variants of CKI(delta) (a full-length form and deletion isoform, which is missing an exon that encodes a putative nuclear localization signal and two evolutionarily conserved protein kinase domains) were isolated and analyzed, together with a previously isolated CKI(epsilon) isoform. Both CKI(delta) and CKI(epsilon) were shown to be constitutively expressed in the photoreceptors of the retina, where a circadian clock has been localized. Both the full-length CKI(delta) and CKI(epsilon) were shown to have kinase activity in vitro, and the full-length CKI(delta) phosphorylated and degraded Drosophila PER when expressed in Drosophila S2 cells. The expression and biochemical characteristics of these CKIs are consistent with an evolutionarily conserved role for CKI in the Xenopus retinal clock. The CKI(delta) deletion isoform did not exhibit kinase activity and did not trigger degradation of PER. Subcellular localization of both CKI(delta) isoforms was cytoplasmic in several cell culture lines, but the full-length CKI(delta) , and not the deletion CKI(delta) isoform, was localized to both the nucleus and the cytoplasm in Drosophila S2 cells. These results indicate that the sequences missing in the deletion CKI(delta) isoform are important for the nuclear localization and kinase activity of the full-length isoform and that one or both of these features are necessary for degradation of Drosophila PER.

Animals↗
PubMed2005

Genetic manipulation of circadian rhythms in Xenopus.

Xenopus laevis retina is an important experimental model system for the study of circadian oscillator mechanisms, as light input pathways, central oscillator mechanisms, and multiple output pathways are all contained within this tissue. These retinas continue to exhibit robust circadian rhythms even after being maintained in culture for many days. The usefulness of this system has been improved even further by the development of a technique for simple genetic manipulation of these animals, which is complemented by expanded genomics resources (Xenopus genome project, microarray, etc.). By taking advantage of the transgenic technique in Xenopus described in this article, many types of analysis can be done on the primary transgenic animals within a couple of weeks after transgenesis. The availability of many cell-type-specific promoters and well-characterized cell types within the Xenopus retina provides the advantage of cell-specific modification of clock function using this method; in other words, contributions of different cell types within the circadian system can be analyzed independently by "molecular dissociation" of these cells. This article describes both how this transgenic technique is useful and various considerations that should be taken into account when these types of experiments are planned and interpreted. Application of these new techniques to studies of clock function provide an opportunity to rapidly assess gene expression and?or function in the context of the intact retina.

Animals↗
PubMed2004

Cryptochromes: tail-ored for distinct functions.

Cryptochromes are important components of circadian clocks in both plants and animals. Recent work suggests that the carboxy-terminal tails of these conserved proteins are used in drastically different ways in different organisms.

Biological Clocks↗
PubMed2004

Retinal circadian clocks and control of retinal physiology.

Retinas of all classes of vertebrates contain endogenous circadian clocks that control many aspects of retinal physiology, including retinal sensitivity to light, neurohormone synthesis, and cellular events such as rod disk shedding, intracellular signaling pathways, and gene expression. The vertebrate retina is an example of a "peripheral" oscillator that is particularly amenable to study because this tissue is well characterized, the relationships between the various cell types are extensively studied, and many local clock-controlled rhythms are known. Although the existence of a photoreceptor clock is well established in several species, emerging data are consistent with multiple or dual oscillators within the retina that interact to control local physiology. A prominent example is the antiphasic regulation of melaton in and dopamine in photoreceptors and inner retina, respectively. This review focuses on the similarities and differences in the molecular mechanisms of the retinal versus the SCN oscillators, as well as on the expression of core components of the circadian clockwork in retina. Finally, the interactions between the retinal clock(s) and the master clock in the SCN are examined.

Animals↗
PubMed2003

Nuclear localization and transcriptional repression are confined to separable domains in the circadian protein CRYPTOCHROME.

Circadian rhythms are driven by molecular clocks composed of interlocking transcription/translation feedback loops. CRYPTOCHROME (CRY) proteins are critical components of these clocks and repress the activity of the transcription factor heterodimer CLOCK/BMAL1. Unlike the homologous DNA repair enzyme 6-4 PHOTOLYASE, CRYs have extended carboxyl-terminal tails and cannot repair DNA damage (reviewed in ). Unlike mammals, Xenopus laevis contains both CRYs (xCRYs) and 6-4 PHOTOLYASE (xPHOTOLYASE), providing an excellent comparative tool to study CRY repressive function. We can extend findings to CRYs in general because xCRYs share high sequence homology with mammalian CRYs. We show here that deletion of xCRYs' C-terminal domain produces proteins that are, like xPHOTOLYASE, unable to suppress CLOCK/BMAL1 activation. However, these truncations also cause the proteins to be cytoplasmically localized. A heterologous nuclear localization signal (NLS) restores the truncation mutants' nuclear localization and repressive activity. Our results demonstrate that the CRYs' C termini are essential for nuclear localization but not necessary for the suppression of CLOCK/BMAL1 activation; this finding indicates that these two functions reside in separable domains. Furthermore, the functional differences between CRYs and PHOTOLYASE can be attributed to the few amino acid changes in the conserved portions of these proteins.

Amino Acid Sequence↗
PubMed2003

Nocturnin, a deadenylase in Xenopus laevis retina: a mechanism for posttranscriptional control of circadian-related mRNA.

BACKGROUND: Different types of regulation are utilized to produce a robust circadian clock, including regulation at the transcriptional, posttranscriptional, and translational levels. A screen for rhythmic messages that may be involved in such circadian control identified nocturnin, a novel gene that displays high-amplitude circadian expression in the Xenopus laevis retina, with peak mRNA levels in the early night. Expression of nocturnin mRNA is confined to the clock-containing photoreceptor cell layer within the retina. RESULTS: In these studies, we show that nocturnin removes the poly(A) tail from a synthetic RNA substrate in a process known as deadenylation. Nocturnin nuclease activity is magnesium dependent, as the addition of EDTA or mutation of the residue predicted to bind magnesium disrupts deadenylation. Substrate preference studies show that nocturnin is an exonuclease that specifically degrades the 3' poly(A) tail. While nocturnin is rhythmically expressed in the cytoplasm of the retinal photoreceptor cells, the only other described vertebrate deadenylase, PARN, is constitutively present in most retinal cells, including the photoreceptors. CONCLUSIONS: The distinct spatial and temporal expression of nocturnin and PARN suggests that there may be specific mRNA targets of each deadenylase. Since deadenylation regulates mRNA decay and/or translational silencing, we propose that nocturnin deadenylates clock-related transcripts in a novel mechanism for posttranscriptional regulation in the circadian clock or its outputs.

Animals↗
PubMed2002

In vivo disruption of Xenopus CLOCK in the retinal photoreceptor cells abolishes circadian melatonin rhythmicity without affecting its production levels.

Xenopus laevis retinas, like retinas from all vertebrate classes, have endogenous circadian clocks that control many aspects of normal retinal physiology occurring in cells throughout all layers of the retina. The localization of the clock(s) that controls these various rhythms remains unclear. One of the best studied rhythmic events is the nocturnal release of melatonin. Photoreceptor layers can synthesize rhythmic melatonin when these cells are in isolation. However, within the intact retina, melatonin is controlled in a complex way, indicating that signals from many parts of the retina may contribute to the production of melatonin rhythmicity. To test this hypothesis, we generated transgenic tadpoles that express different levels of a dominant negative Xenopus CLOCK specifically in the retinal photoreceptors. Eyes from these tadpoles continued to produce melatonin at normal levels, but with greatly disrupted rhythmicity, the severity of which correlated with the transgene expression level. These results demonstrate that although many things contribute to melatonin production in vivo, the circadian clock localized in the retinal photoreceptors is necessary for its rhythmicity. Furthermore, these data show that the control of the level of melatonin synthesis is separable from the control of its rhythmicity and may be controlled by different molecular machinery. This type of specific "molecular lesion" allows perturbation of the clock in intact tissues and is valuable for dissection of clock control of tissue-level processes in this and other complex systems.

Animals↗
PubMed2002

Circadian regulation of nocturnin transcription by phosphorylated CREB in Xenopus retinal photoreceptor cells.

Although CLOCK/BMAL1 heterodimers have been implicated in transcriptional regulation of several rhythmic genes in vitro through E-box sequence elements, little is known about how the circadian clock regulates rhythmic genes with diverse phases in vivo. The gene nocturnin is rhythmically transcribed in Xenopus retinal photoreceptor cells, which contain endogenous circadian clocks. Transcription of nocturnin peaks in these cells in the middle of the night, while CLOCK/BMAL1 activity peaks during the early morning. We have identified a novel protein-binding motif within the nocturnin promoter, which we designated the nocturnin element (NE). Although the NE sequence closely resembles an E-box, our data show that it functions as a cyclic AMP response element (CRE) by binding CREB. Furthermore, phosphorylated CREB (P-CREB) levels are rhythmic in Xenopus photoreceptors, with a phase similar to that of nocturnin transcription. Our results suggest that P-CREB controls the rhythmic regulation of nocturnin transcription and perhaps that of other night phase genes. The NE may be an evolutionary intermediate between the E-box and CRE sequences, both of which seem to be involved in the circadian control of transcription, but have evolved to drive transcription with different phases in these clock-containing cells.

3T3 Cells↗
PubMed2002

Bulla gouldiana period exhibits unique regulation at the mRNA and protein levels.

The authors cloned the period (per) gene from the marine mollusk Bulla gouldiana, a well-characterized circadian model system. This allowed them to examine the characteristics of the per gene in a new phylum, and to make comparisons with the conserved PER domains previously characterized in insects and vertebrates. Only one copy of the per gene is present in the Bulla genome, and it is most similar to PER in two insects: the cockroach, Periplaneta americana, and silkmoth, Antheraea pernyi. Comparison with Drosophila PER (dPER) and murine PER 1 (mPER1) sequence reveals that there is greater sequence homology between Bulla PER (bPER) and dPER in the regions of dPER shown to be important to heterodimerization between dPER and Drosophila timeless. Although the structure suggests conservation between dPER and bPER, expression patterns differ. In all cells and tissues examined that are peripheral to the clock neurons in Bulla, bPer mRNA and protein are expressed constitutively in light:dark (LD) cycles. In the identified clock neurons, the basal retinal neurons (BRNs), a rhythm in bPer expression could be detected in LD cycles with a peak at zeitgeber time (ZT) 5 and trough expression at ZT 13. This temporal profile of expression more closely resembles that of mPER1 than that of dPER. bPer rhythms in the BRNs were not detected in continuous darkness. These analyses suggest that clock genes may be uniquely regulated in different circadian systems, but lead to similar control of rhythms at the cellular, tissue, and organismal levels.

Animals↗