The function of DNA methylation in higher plants was investigated by expression of a complementary DNA encoding a cytosine methyltransferase (MET1) from Arabidopsis thaliana as an antisense RNA in transgenic plants. This expression resulted in a 34 to 71 percent reduction in total genomic cytosine methylation. Loss of methylation was observed in both repetitive DNA and single-copy gene sequences. Developmental effects included altered heterochrony, changes in meristem identity and organ number, and female sterility. Cytosine demethylation prolonged both vegetative and reproductive phases of development. These findings implicate DNA methylation in establishing or maintaining epigenetic developmental states in the meristem.

Antisera were raised against peptides corresponding to the N-termini of capsid proteins VP1 and VP2 from the parvovirus minute virus of mice. Epitopes in the 142-amino-acid VP1-specific region were not accessible in the great majority of newly released viral particles, and sera directed against them failed to neutralize virus directly or deplete stocks of infectious virions. However, brief exposure to temperatures of 45 degreesC or more induced a conformational transition in a population of full virions, but not in empty viral particles, in which VP1-specific sequences became externally accessible. In contrast, the VP2 N-terminus was antibody-accessible in all full, but not empty, particles without prior treatment. An electrophoretic mobility shift assay, in which particles were heat-treated and/or preincubated with antibodies prior to electrophoresis, confirmed this pattern of epitope accessibility, showing that the heat-induced conformational transition produces a retarded form of virion that can be supershifted by incubation with VP1-specific sera. The proportion of virions undergoing transition increased with temperature, but at all temperatures up to 70 degreesC viral particles retained structure-specific antigenic determinants and remained essentially intact, without shedding individual polypeptide species or subunits. However, despite the apparent integrity of its protective coat, the genome became accessible to externally applied enzymes in an increasing proportion of virions through this temperature range, suggesting that the conformational transitions that expose VP1 likely also allow access to the genome. Heating particles to 80 degreesC or above finally induced disassembly to polypeptide monomers.

Colorectal cancer is the second most leading cause of cancer death among adult Americans. Two autosomal dominant hereditary forms of the disease, familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer, together account for perhaps 5% of all cases. However, in Ϸ20% of additional colon cancer cases, the affected individuals report a family history of colon cancer in a first-degree relative. Similar familial clusters of colon cancer and early-onset colon adenomas have also been reported. To determine whether such familial aggregations arise by chance or reflect a hereditary colon cancer susceptibility, we conducted a whole genome scan to test for genetic linkage in 53 kindreds in which two or more siblings were affected by age 65 or younger with colon cancer or with advanced colon adenomas that were >1 cm in size or that showed high-grade dysplasia. In this cohort we found genetic linkage of disease (P ‫؍‬ 0.00045) to chromosomal region 9q22.2-31.2 in a pattern consistent with autosomal dominant disease alleles. These data suggest that a single locus can contribute to disease susceptibility in a subset of patients with nonsyndromic forms of familial colorectal neoplasia.colorectal cancer ͉ colon adenomas ͉ genetic linkage ͉ familial cancers C olorectal cancer is the second most leading cause of cancer death among adult Americans (1). Strongly penetrant autosomal dominant hereditary forms of the disease, familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), together account for perhaps 5% of all cases (2-4). However, in Ϸ20% of additional colon cancer cases the affected individuals report a family history of colon cancer in a first-degree relative, and a genetic basis for these familial disease clusters has also been hypothesized (5).Colorectal cancers themselves develop from precursor colon adenomas (3). Thus, the inherited FAP syndrome, which is caused by inactivating mutations in the adenomatous polyposis coli (APC) gene, is marked by development of a profusion of hundreds of colon adenomas that then confer a near 100% risk of colon cancer development by an average age of 40 (2, 3). A subtler adenoma and cancer phenotype is associated with an APC I1307K polymorphism present in the Ashkenazi Jewish population (2). The APC I1307K variant DNA sequence demonstrates enhanced vulnerability to development of inactivating somatic APC mutations, resulting in a slightly less than 2-fold increased risk of colon adenoma and cancer development (2). The existence of additional disease genes associated with increased risk of developing colon adenomas and cancers is suggested by observations of an Ϸ3-fold increased risk of colon cancer among first-degree relatives of individuals who develop colon adenomas before age 60 (6), and by one study that suggested that 19% of persons in the general population might carry autosomal dominant disease alleles conferring susceptibility for colon adenoma or cancer development (7). However, more recent endoscopic-based studies have sug...

Chromosomal deletions ("deficiencies') are powerful tools in the genetic analysis of complex genomes. They have been exploited extensively in Drosophila melanogaster, an organism in which deficiencies can be efficiently induced and selected. Spontaneous deletions in humans have facilitated the dissection of phenotypes in contiguous gene syndromes and led to the positional cloning of critical genes. In mice, deletion complexes created by whole animal irradiation experiments have enabled a systematic characterization of functional units along defined chromosomal regions. However, classical mutagenesis in mice is logistically impractical for generating deletion sets on a genome-wide scale. Here, we report a high-throughput method for generating radiation-induced deletion complexes at defined regions in the genome using ES cells. Dozens of deletions of up to several centiMorgans, encompassing a specific locus, can be created in a single experiment and transmitted through the germline. The ability to rapidly create deletion complexes along chromosomes will facilitate systematic functional analyses of the mammalian genome.

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PURPOSEThe paradox of primary care is the observation that primary care is associated with apparently low levels of evidence-based care for individual diseases, but systems based on primary care have healthier populations, use fewer resources, and have less health inequality. The purpose of this article is to explore, from a complex systems perspective, mechanisms that might account for the effects of primary care beyond disease-specific care. METHODSIn an 8-session, participatory group model-building process, patient, caregiver, and primary care clinician community stakeholders worked with academic investigators to develop and refine an agent-based computer simulation model to test hypotheses about mechanisms by which features of primary care could affect health and health equity.RESULTS In the resulting model, patients are at risk for acute illness, acute lifechanging illness, chronic illness, and mental illness. Patients have changeable health behaviors and care-seeking tendencies that relate to their living in advantaged or disadvantaged neighborhoods. There are 2 types of care available to patients: primary and specialty. Primary care in the model is less effective than specialty care in treating single diseases, but it has the ability to treat multiple diseases at once. Primary care also can provide disease prevention visits, help patients improve their health behaviors, refer to specialty care, and develop relationships with patients that cause them to lower their threshold for seeking care. In a model run with primary care features turned off, primary care patients have poorer health. In a model run with all primary care features turned on, their conjoint effect leads to better population health for patients who seek primary care, with the primary care effect being particularly pronounced for patients who are disadvantaged and patients with multiple chronic conditions. Primary care leads to more total health care visits that are due to more disease prevention visits, but there are reduced illness visits among people in disadvantaged neighborhoods. Supplemental appendices provide a working version of the model and worksheets that allow readers to run their own experiments that vary model parameters. CONCLUSION This simulation model provides insights into possible mechanisms for the paradox of primary care and shows how participatory group model building can be used to evaluate hypotheses about the behavior of such complex systems as primary health care and population health. INTRODUCTIONM ultiple studies have found that primary care is associated with poorer quality care for individual diseases than is care provided by clinicians focused primarily on those diseases. [1][2][3][4][5][6] Yet, other evidence shows that systems based on primary care have better quality of care, better population health, greater equity, and lower cost. [7][8][9][10][11] This discrepancy between apparently poor disease-specific care and advantageous outcomes at the level of the whole person and system has been called t...

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The Cleveland Clinic Lerner College of Medicine (CCLCM) was established as one of three tracks in the Case Western Reserve University (CWRU) School of Medicine Program to prepare physician investigators through rigorous, comprehensive, longitudinal research training in a five-year undergraduate medical education setting. The curriculum includes specific research activities and dedicated research time, which culminates in a masterslevel thesis. The track has an administrative structure to facilitate student access to research-related opportunities. Basic scientists and clinicians participate in all curricular activities thereby fostering communication across the clinical medicine/research divide. The track features a centralized, portfolio-based assessment system. In this track, students submit portfolios to demonstrate their progress toward mastering nine competencies including research. A comprehensive program evaluation is in place to monitor the curricular impact on students. Results show that the CCLCM track has attracted a subset of students interested in research careers. Two cohorts of students (n = 56) have graduated. The Promotions Committee determined that all graduates successfully met the performance standards for the research competency. These graduates scored above mean USMLE Step 1 subscores in biostatistics and epidemiology. They successfully completed a research thesis where approximately 60% of the topics addressed basic science and translational research. Over 40% of graduates also obtained a masters degree concurrently with their medical degrees. Furthermore, 23% of CCLCM graduates received competitive awards to support their medical school research activities. Recommendations are included to expose students to research in multiple venues to create and sustain a passion for rigorous problemsolving. Conclusions offer lessons learned from our experience.