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Supplementary Data from Correcting miR-15a/16 genetic defect in New Zealand Black mouse model of CLL enhances drug sensitivity

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G1 in non–New Zealand Black B cell and New Zealand Black–derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G1 accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G1/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3′ untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease. [Mol Cancer Ther 2009;8(9):2684–92]

The purpose of the current study was to describe the perceptions and experiences of New Horizons Band assistants in terms of learning, skill transfer and pedagogical preparation. The participant population for this study was 35 past student teacher-assistants from three New Horizons bands, one in Texas, one in Florida and one in Virginia. There were ten open-ended items on the questionnaire that asked about student assistant perceptions and experiences. Participants noted the growth they experienced from serving as a band teaching assistant in a New Horizons programme, describing their appreciation for lifelong learning, exposure to the complexity of how to teach to a variety of learners, and experience with the challenges of pacing with adults and blunt feedback from programme participants. The article concludes with a discussion of possible collegiate preparatory experiences that could broaden the teacher training experience for college programmes.

Objective: Celiac disease is a genetic disease affecting people of all ages, resulting in small intestine enteropathy. It is considered to be a clinical chameleon. Average prevalence of cel...

Purpose/Objective(s): The additional personnel and imaging procedures required for Adaptive Radiation Therapy (ART) pose a challenge for a broad implementation. We hypothesize that a change in transit fluence during the treatment course is correlated with the change of quality of life and thus can be used as a replanning trigger. Materials/Methods: Twenty-one head and neck cancer (HNC) patients filled out an MD Anderson Dysphagia Inventory (MDADI) questionnaire, before-and-after the radiotherapy treatment course. The transit fluence was measured by the Watchdog (WD) in-vivo portal dosimetry system. The patients were monitored with daily WD and weekly CBCTs. The region of interest (ROI) of each patient was defined as the outer contour of the patient between approximate spine levels C1 to C4, essentially the neck and mandible inside the beam’s eye view. The nth day integrated transit fluence change, Δϕn, and the volume change, ΔVROI, of the ROI of each patient was calculated from the corresponding WD and CBCT measurements. The correlation between MDADI scores and age, gender, planning mean dose to salivary glands sg>, weight change ΔW, ΔVROI, and Δϕn, were analyzed using the ranked-Pearson correlation. Results: No statistically significant correlation was found for age, gender and ΔW. sg> was found to have clinically important correlation with functional MDADI (ρ = −0.39, P = 0.081). ΔVROI was found to have statistically significant correlation of 0.44, 0.47 and 0.44 with global, physical and functional MDADI ( P-value < 0.05). Δϕn was found to have statistically significant ranked-correlation (−0.46, −0.46 and −0.45) with physical, functional and total MDADI ( P-value < 0.05). Conclusion: A transit fluence based decision support metric (DSM) is statistically correlated with the dysphagia risk. It can not only be used as an early signal in assisting clinicians in the ART patient selection for replanning, but also lowers the resource barrier of ART implementation.

Background Prioritization of breast cancer patients based on the risk of resistance to tamoxifen plays a significant role in personalized therapeutic planning and improving disease course and outcomes. Methods In this work, we demonstrate that a genome-wide pathway-centric computational framework elucidates molecular pathways as markers of tamoxifen resistance in ER+ breast cancer patients. In particular, we associated activity levels of molecular pathways with a wide spectrum of response to tamoxifen, which defined markers of tamoxifen resistance in patients with ER+ breast cancer. Findings We identified five biological pathways as markers of tamoxifen failure and demonstrated their ability to predict the risk of tamoxifen resistance in two independent patient cohorts (Test cohort1: log-rank p-value = 0.02, adjusted HR = 3.11; Test cohort2: log-rank p-value = 0.01, adjusted HR = 4.24). We have shown that these pathways are not markers of aggressiveness and outperform known markers of tamoxifen response. Furthermore, for adoption into clinic, we derived a list of pathway read-out genes and their associated scoring system, which assigns a risk of tamoxifen resistance for new incoming patients. Interpretation We propose that the identified pathways and their read-out genes can be utilized to prioritize patients who would benefit from tamoxifen treatment and patients at risk of tamoxifen resistance that should be offered alternative regimens. Funding This work was supported by the Rutgers SHP Dean's research grant, Rutgers start-up funds, Libyan Ministry of Higher Education and Scientific Research, and Katrina Kehlet Graduate Award from The NJ Chapter of the Healthcare Information Management Systems Society.

Depression is a widespread psychological issue. Research suggests that attachment theory provides a useful lens through which to understand the occurrence of depression, as attachment anxiety and attachment avoidance is associated with greater reports of depression. However, less is known about how attachment-motivated perceptions and cognitions relate to depression. In the current study, we examined self-compassion, and perceptions of belonging and burdensomeness as potential mediators of the association between depression and attachment anxiety and avoidance, using a sample (N = 370) of students from a large southern metropolitan university. Results suggested that self-compassion, and perceived belonging and burdensomeness, mediated the association between attachment anxiety and depression. Those with greater attachment anxiety reported less self-compassion, feeling less belonging, and more burdensomeness. These factors were associated with increased depressive symptoms. With respect to attachment avoid...

Personalized normative feedback (PNF) has been used extensively to reduce alcohol consumption, particularly among heavy drinkers. However, the majority of PNF studies have used only descriptive norms (real or perceived pervasiveness of a given behavior). The purpose of the current study was to explore the efficacy of PNF both with and without an injunctive message indicating approval or disapproval based on the participants' standing relative to other students' drinking levels. This randomized trial evaluated two brief web-based alcohol intervention conditions (descriptive-norms-feedback-only condition versus a descriptive-plus-injunctive-message condition relative to an assessment-only control condition).Participants included 176 students who had reported at least one heavy drinking episode in the past month. Participants completed baseline and follow-up assessments of perceived norms and drinking. Follow-up assessments were completed at 2 weeks post-intervention by 165 (94%) participants.Analyses were conducted using zero-inflated negative binomial regression models. As expected, the descriptive-norms-only condition was effective in reducing drinking among heavier baseline drinkers at follow-up relative to the control condition. However, contrary to expectations, the descriptive-plus-injunctive-message condition did not predict less drinking at follow-up.This study was unique in using an injunctive message as an adjunct to descriptive-norms feedback within the context of drinking. Findings highlight the need for additional research into the role of defensiveness, which may serve as an impediment to using injunctive norms/messages in interventions for problematic substance use and other potentially stigmatizing behaviors.

Striking levels of spatial organization exist among and within interphase cell chromosomes, raising the possibility that other nuclear molecular components may also be organized in ways that facilitate nuclear function. To further examine molecular distributions and organization within cell nuclei, we utilized Raman spectroscopy to map distributions of molecular components, with a focus on cellular lipids. Although the vast majority of cellular lipids are associated with membranes, mapping the 2870/2850 cm−1 lipid peak ratios revealed that the most highly ordered lipids within interphase cells are found within cell nuclei. This finding was seen in cells from multiple tissue types, noncancerous cells, and in cancer cell lines of different metastatic potential. These highly ordered lipids colocalize with nuclear chromatin, are present throughout the nuclear volume, and remain colocalized with chromatin through mitosis, when the nuclear envelope has dissociated. Phosphatidylinositol is a major component of the highly ordered lipids. The presence of phosphatidylinositol and other lipids in the nuclear interior is well established, but their highly ordered packing has not been reported and represents a unique finding. The molecular interactions involved in the formation and maintenance of these highly ordered lipids, and their potential effects on nuclear activities, remain to be discovered.

This chapter examines three approaches to teaching and learning that resonate with community music principles and that can help inform the theoretical bases for community music practice, because there are similarities between the facilitating behaviours of community musicians and the teaching behaviours of educators. Specifically, this chapter portrays a continuum of viewpoints about guiding others—pedagogy, andragogy, and heutagogy—and illustrates how aspects of each approach can be applied to community music practice. These approaches range from authoritarian ideas that are teacher-centred and learner-dependent to more autonomous ideas that embrace learner-centred and self-directed learning. The New Horizons Band of Iowa City, Iowa, in the United States, is presented as an illustration.

The physiological form of vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), has a role in normal hematopoiesis, enhancing monocyte–macrophage differentiation. It also has antiproliferative and prodifferentiation effects against various myeloid leukemia cell lines and acute myeloid leukemia (AML) blasts ex vivo. Nevertheless, hematopoiesis in mice with vitamin D receptor (VDR) deletion is essentially normal, indicating that in mammals the vitamin D pathway appears to have a nonessential but perhaps a contributory role in blood formation. This chapter reviews the general mechanisms by which vitamin D derivatives (VDDs) can drive cell differentiation, the associated cell cycle traverse arrest, and alter cell survival. However, in vivo these activities of 1,25(OH) 2 D 3 require supraphysiological levels of 1,25(OH) 2 D 3 . The important distinctions between different mechanisms of 1,25(OH) 2 D 3 -induced differentiation that are cell type and cell context–specific and the different pathways that participate in 1,25(OH) 2 D 3 -induced signal propagation are described here. Clinical trials with 1,25(OH) 2 D 3 have been performed for the treatment of preleukemia/myelodysplastic syndrome and AML, but the doses effective in vitro caused hypercalcemia in vivo. However, numerous vitamin D analogs have been synthesized, which, in model systems, have reduced calcemic activity but have increased ability to induce cell differentiation and to inhibit proliferation of leukemic cells. Perhaps the available or new analogs combined with other differentiating or antiproliferative agents, each working through different pathways, will demonstrate synergistic activity and offer improved therapy for AML.

Synaptotagmin (Syt) family proteins contain tandem C2 domains, C2A and C2B, which insert into anionic membranes in response to increased cytosolic Ca2+ concentration and facilitate exocytosis in neuronal and endocrine cells. The C2A domain from Syt7 binds lipid membranes much more tightly than the corresponding domain from Syt1, but the implications of this difference for protein function are not yet clear. In particular, the ability of the isolated Syt7 C2A domain to initiate membrane apposition and/or aggregation has been previously unexplored. Here, we demonstrate that Syt7 C2A induces apposition and aggregation of liposomes using Forster resonance energy transfer (FRET) assays, dynamic light scattering, and spectroscopic techniques involving lipid-coated gold nanoparticles (LCAuNPs). Protein–membrane binding, membrane apposition, and macroscopic aggregation are three separate phenomena with distinct Ca2+ requirements: the threshold Ca2+ concentration for membrane binding is lowest, followed by apposit...

Against the background of a dearth of studies examining the properties of the scale scores of the Personality Assessment Inventory-Adolescent (PAI-A; Morey, 2007), this study was conducted to evaluate evidence of construct validity for the Anxiety (ANX) and Depression (DEP) scales of the PAI-A. Convergent and discriminant validity of the ANX and DEP scale scores were investigated using a sample of adolescents admitted to the adolescent program of a private tertiary care inpatient treatment facility. Multiple methods assessing anxious and depressive symptomology and diagnoses were included. Construct validity of the ANX and DEP scales was mostly supported. Advantages of using the PAI-A for the assessment of anxiety and depression were discussed.

Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein–dependent and β-arrestin–dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein–dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance–based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical “two-site model” for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor-mediated signal transduction.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that was initially identified by its ability to inhibit the movement of macrophages. Cell migration is a highly complex process involving changes to the cytoskeleton and cell adhesion molecules, and is regulated by the Rho GTPases. A simple model using human monocytic U-937 cells to elicit the classic MIF response was implemented to examine the mechanism of MIF-induced migration inhibition. Our results demonstrate that MIF inhibits migration of these U-937 cells through a non-canonical receptor, CXCR4, in the absence of the putative primary MIF receptor CD74. Migration inhibition is dependent upon a series of temporal perturbations of the activities of the Rho GTPases: initial activation followed by subsequent inactivation of RhoA, inactivation of Rac1, and cyclic activation of Cdc42. MIF-mediated changes in the activities of the Rho GTPases jointly contributed to migration inhibition in these cells. Collectively, these data suggest that the MIF-mediated migration inhibition is mediated by the outcome of G-protein signaling, and in less adherent cells such as those of the monocyte/macrophage lineage, RhoA directly affects net translocation through its ability to induce cell body contraction. These findings demonstrate that CXCR4 can mediate MIF signaling in the absence of CD74 in addition to serving as a MIF co-receptor along with CD74. These results correlate MIF activity to specific and sequential Rho GTPase activity perturbations, and given that CXCR4 functions in numerous processes, suggests potential roles for the modulation of cell movement in those events including development, cell survival and viral infection.

Autoantibodies to diverse antigens escape regulation in systemic lupus erythematosus under the influence of a multitude of predisposing genes. To gain insight into the differential impact of diverse genetic backgrounds on tolerance mechanisms controlling autoantibody production in lupus, we established a single lupus-derived nephritis associated anti-basement membrane Ig transgene on each of four inbred murine lupus strains, including BXSB, (NZBxNZW)F1, NZB, and MRL/lpr, as approved by the Duke University and the Durham Veterans Affairs Medical Centers’ Animal Care and Use Committees. In nonautoimmune C57BL/6 mice, B cells bearing this anti-laminin Ig transgene are stringently regulated by central deletion, editing, and anergy. Here, we show that tolerance is generally intact in unmanipulated Ig transgenic BXSB, (NZBxNZW)F1, and NZB mice, based on absence of serum transgenic anti-laminin autoantibodies and failure to recover spontaneous anti-laminin monoclonal antibodies. Four- to six-fold depletion of splenic B cells in transgenic mice of these strains, as well as in MRL/lpr transgenic mice, and reduced frequency of IgM+ bone marrow B cells suggest that central deletion is grossly intact. Nonetheless the four strains demonstrate distinct transgenic B cell phenotypes, including endotoxin-stimulated production of anti-laminin antibodies by B cells from transgenic NZB mice, and in vitro hyperproliferation of both endotoxin- and BCR-stimulated B cells from transgenic BXSB mice, which are shown to have an enrichment of CD21-high marginal zone cells. Rare anti-laminin transgenic B cells spontaneously escape tolerance in MRL/lpr mice. Further study of the mechanisms underlying these strain-specific B cell fates will provide insight into genetic modification of humoral autoimmunity in lupus.

In adaptive radiation therapy of head and neck cancer, any significant anatomical changes observed are used to adapt the treatment plan to maintain target coverage without elevating the risk of xerostomia. However, the additional resources required for adaptive radiation therapy pose a challenge for broad-based implementation. It is hypothesized that a change in transit fluence is associated with volumetric change in the vicinity of the target and therefore can be used as a decision support metric for adaptive radiation therapy. This was evaluated by comparing the fluence with volumetric changes in 12 patients. Transit fluence was measured by an in vivo portal dosimetry system. Weekly cone beam computed tomography was used to determine volume change in the rectangular region of interest from condyloid process to C6. The integrated transit fluence through the region of interest on the day of the cone beam computed tomography scan was calculated with the first treatment as the baseline. The correlation between fluence change and volume change was determined. A logistic regression model was also used to associate the 5% region of interest volume reduction replanning trigger point and the fluence change. The model was assessed by a chi-square test. The area under the receiver–operating characteristic curve was also determined. A total of 46 pairs of measurements were obtained. The correlation between fluence and volumetric changes was found to be −0.776 ( P value

While generally illegal, corruption is a cost of doing business in many parts of the world. Competing perspectives suggest both positive and negative effects of corruption on entrepreneurial activity. In this study, we do not ask whether corruption has an effect, but rather ask how much of an effect do different forms of corruption communicated in different ways have on pursuing a new opportunity. We use a three-study, replication-based design to explore the relationship between the type and communication of corruption and pursuing a new opportunity. Across samples in the U.S. and Ghana, we find that "favors," or non-pecuniary corruption, explicitly communicated, lowers opportunity pursuit the most, while "monetary," or pecuniary corruption, implicitly communicated has little effect on opportunity pursuit. Our study sheds light on an under-explored area of corruption research; scholars—and policy makers—should look beyond only monetary forms when exploring corruption's impact on entrepreneurship. For transparency, we post all data, code, and study materials online at http://bit.ly/2wipQ8u .

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G1 in non–New Zealand Black B cell and New Zealand Black–derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G1 accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G1/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3′ untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease. [Mol Cancer Ther 2009;8(9):2684–92]

The function of the relatively well-studied DNA replication origins in the yeast Saccharomyces cerevisiae is dependent upon interactions between origin replication complex (ORC) proteins and several defined origin sequence elements, including the 11 bp ARS consensus sequence (ACS). Although the ORC proteins, as well as numerous other protein components required for DNA replication initiation, are largely conserved between yeast and mammals, DNA sequences within mammalian replication origins are highly variable and sequences homologous to the yeast ACS elements are generally not present. We have previously identified several replication initiation sites within the nontranscribed spacer region of the human ribosomal RNA gene, and found that two highly utilized sites each contain a homologue of the yeast ACS embedded within a DNA unwinding element and a matrix attachment region. Here we examine protein binding within these initiation sites, and demonstrate that these ACS homologues specifically bind the alternate splicing factor SF2/ASF as well as GAPDH in vitro, and present evidence that the SF2/ASF interaction also occurs within the nuclei of intact cells. As the moderate upregulation of SF2/ASF has been linked to oncogenesis through the promotion of alternatively spliced forms of several regulatory proteins, our results suggest an additional mechanism by which SF2/ASF may influence the transformed cell phenotype.

Synaptotagmin 1 (Syt1), a Ca2+ sensor involved in exocytosis in pre-synaptic neurons, contains a characteristic tandem C2 sequence (C2AB) which inserts into anionic membranes in the presence of Ca2+. Previous studies have shown that the C2AB fragment of Syt1 inserts into target membranes more deeply than either individual C2A or C2B domain, suggesting cooperative interaction between C2A and C2B in the membrane-docked state. This behavior stands in apparent contrast to that of another family member, Syt7, whose C2A and C2B domains have been shown to bind membranes independently. To compare the cooperative behaviors of the C2 domains in these two isoforms, the dissociation kinetics (off-rates) of protein-liposome complexes were measured upon addition of the Ca2+ chelator EDTA using stopped-flow fluorescence spectroscopy. Using liposomes composed of a 1:1 mixture of phosphatidylcholine and phosphatidylserine (PC/PS), the Syt1 C2AB tandem domain exhibits a much slower off-rate than either of the single domains. In contrast, dissociation kinetics for Syt7 C2AB were best fit to a two-step model in which each rate constant matches those from the individual domains. This preliminary result supports the presence of interdomain interactions in the membrane-docked state of Syt1 C2AB but not Syt7 C2AB. These experiments were performed with protein domains purified using affinity chromatography with high-salt washes and verified to be >95% free of nucleic acid contaminants; ongoing work aims to assess effects of polyanions on this apparent cooperative behavior, including ion-exchange steps in the protein purifications.

Summary Mouse models are valuable tools in the study of human chronic lymphocytic leukaemia (CLL). The New Zealand Black (NZB) strain is a naturally occurring model of late-onset CLL characterized by B-cell hyperproliferation and autoimmunity early in life, followed by progression to CLL. Other genetically engineered models of CLL that have been developed include (NZB × NZW) F1 mice engineered to express IL5, mice expressing human TCL1A, and mice overexpressing both BCL2 and a tumour necrosis factor receptor-associated factor. The applicability to human CLL varies with each model, suggesting that CLL is a multifactorial disease. Our work with the de novo NZB model has revealed many similarities to the human situation, particularly familial CLL. In NZB, the malignant clones express CD5, zap-70, and have chromosomal instability and germline Ig sequence. We also identified a point mutation in the 3′-flanking sequence of Mirn16-1, which resulted in decreased levels of the microRNA, miR-16 in lymphoid tissue. Exogenous restoration of miR-16 to an NZB malignant B-1 cell line resulted in cell cycle alterations, suggesting that the altered expression of Mirn15a/16-1 is an important molecular lesion in CLL. Future studies utilizing the NZB mouse could ascertain the role of environmental triggers, such as low dose radiation and organic chemicals in the augmentation of a pre-existing propensity to develop CLL.

Numerous studies have demonstrated that DNA replication initiates within the 30 kB non-transcribed spacer (NTS) region of the human ribosomal RNA gene (rDNA). Using a series of closely spaced primer pairs to measure nascent leading strand abundance in mid and late S phase cells isolated by centrifugal elutriation, we find evidence for one highly preferred initiation site and two less utilized sites within a 6 kb region of the NTS. The initiation sites colocalize with significant DNA unwinding elements (DUEs), matrix attachment regions (MARs), and ARS-like sequences. An intrinsic DNA bending site was localized by circular permutation analysis to within several hundred base pairs of one initiation site. While DUE and MAR elements occur elsewhere throughout the 43 kb rDNA sequence, the close association of DUE and MAR elements occurs only near replication initiation sites, a juxtaposition also seen in other well-studied mammalian replication initiation sites. The utilization of rDNA initiation sites close to DUE and MAR elements in mid and late S phase, but not in very early S phase as previously shown, suggests that in rRNA genes, contributions from these sequence-associated properties may be more significant to initiation sites associated with transcriptionally inactive genes, than to initiation sites associated with transcriptionally active genes.

Metazoan replication origins often contain multiple potential initiation sites, and the selection of which of the potential sites are used appears to be dependent upon multiple factors, including the state of differentiation, cell metabolism, and local transcriptional activity. Numerous studies have shown that a replication origin exists within the non-transcribed spacer region of the human ribosomal RNA gene. We here analyze nascent leading strand DNA from S phase human lymphoid cells, and find that while the majority of rDNA replicates in mid- and late S phase and preferentially initiates replication 6 kbp from the transcription start site, in very early S phase the preferred initiation site is much closer to the transcription start site and may involve rDNA promoter sequences. This early site is coincident with a minimum GC skew value, diagnostic for replication origins in bacteria and yeast. These results suggest that replication timing can influence initiation site selection. The timing and nucleolar localization of rDNA further suggest that this site likely participates in the small number of perinucleolar initiation foci observed in very early S phase cells that represent the beginning of cellular DNA replication.