We wanted to identify genes that are controlled by the circadian clock in the prokaryotic cyanobacterium Synechococcus sp. strain PCC 7942. To use luciferase as a reporter to monitor gene expression, bacterial luciferase genes (luxAB) were inserted randomly into the Synechococcus genome by conjugation with Escherichia coli and subsequent homologous recombination. The resulting transformed clones were then screened for bioluminescence using a newly developed cooled-CCD camera system. We screened -30,000transformed Synechococcus colonies and recovered -800 clones whose bioluminescence was bright enough to be easily monitored by the screening apparatus. Unexpectedly, the bioluminescence expression patterns of almost all of these 800 colonies clearly manifested circadian rhythmicity. These rhythms exhibited a range of waveforms and amplitudes, and they also showed a variety of phase relationships. We also found bioluminescence rhythms expressed by cyanobacterial colonies in which the luciferase gene set was coupled to the promoters of several known genes. Together, these results indicate that control of gene expression by circadian clocks may be more widespread than expected thus far. Moreover, our results show that screening organisms in which promoterless luciferase genes have been inserted randomly throughout the genome by homologous recombination provides an extremely sensitive method to explore differential gene expression.

A diverse set of circadian clock mutants was isolated in a cyanobacterial strain that carries a bacterial luciferase reporter gene attached to a clock-controlled promoter. Among 150,000 clones of chemically mutagenized bioluminescent cells, 12 mutants were isolated that exhibit a broad spectrum of periods (between 16 and 60 hours), and 5 mutants were found that show a variety of unusual patterns, including arrhythmia. These mutations appear to be clock-specific. Moreover, it was demonstrated that in this cyanobacterium it is possible to clone mutant genes by complementation, which provides a means to genetically dissect the circadian mechanism.

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microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

We isolated mutants affected in the circadian expression of the psbAI gene in Synechococcus sp. strain PCC 7942 using a strategy that tags the genomic locus responsible for the mutant phenotype. The search identified one short period (22 h) mutant (M2) and two low amplitude mutants, one of which showed apparent arhythmia (M11) and one that was still clearly rhythmic (M16). We characterized the disrupted locus of the low amplitude but still rhythmic mutant (M16) as the rpoD2 gene, a member of a gene family that encodes sigma70‐like transcription factors in Synechococcus. We also inactivated rpoD2 in a number of reporter strains and showed that the circadian expression of some genes is not modified by the loss of this sigma factor. Therefore, we conclude that rpoD2 is a component of an output pathway of the biological clock that affects the circadian expression of a subset of genes in Synechococcus. This work demonstrates a direct link between a transcription factor and the manifestation of circadian gene expression.

A glnB gene is identified in the cyanobacterium Synechococcus sp. PCC 7942, and its gene product is found to be covalently modified as a result of imbalance in electron transfer in photosynthesis, where photosystem II is favored over photosystem I. The gene was cloned and sequenced and found to encode a polypeptide of 112 amino acid residues, whose sequence shows a high degree of similarity to the Escherichia coli regulatory protein, PI,. In E. coli, PI, is involved in signal transduction in transcriptional and posttranslational regulation of nitrogen assimilation. Increase in ammonium ion concentration is shown to decrease covalent modification of the Synechococcus PI, protein, as in enteric bacteria. We therefore propose that the photosynthetic electron transport chain may regulate the pathway of nitrogen assimilation in cyanobacteria by means of posttranslational, covalent modification of the glnB gene product. The existence of the glnB gene in different strains of cyanobacteria is demonstrated and its implications are discussed.

Purpose:This report describes a fast online tool to accelerate and improve clinical interpretation of single nucleotide polymorphism array results for diagnostic purposes, when consanguinity or inbreeding is identified.Methods:We developed a web-based program that permits entry of regions of homozygosity and, using OMIM, UCSC, and NCBI databases, retrieves genes within these regions as well as their associated autosomal recessive disorders. Relevant OMIM Clinical Synopses can be searched, using key clinical terms permitting further filtering for candidate genes and disorders.Results:The tool aids the clinician by arriving at a short list of relevant candidate disorders, guiding the continued diagnostic work-up. Its efficacy is illustrated by presenting seven patients who were diagnosed using this tool.Conclusion:The online single nucleotide polymorphism array evaluation tool rapidly and systematically identifies relevant genes and associated conditions mapping to identified regions of homozygosity. The built-in OMIM clinical feature search allows the user to further filter to reach a short list of candidate conditions relevant for the diagnosis, making it possible to strategize more focused diagnostic testing. The tabulated results can be downloaded and saved to the desktop in an Excel format. Its efficacy is illustrated by providing a few clinical examples.

Parkinson's disease (PD) has had six genome-wide association studies (GWAS) conducted as well as several gene expression studies. However, only variants in MAPT and SNCA have been consistently replicated. To improve the utility of these approaches, we applied pathway analyses integrating both GWAS and gene expression. The top 5000 SNPs (p<0.01) from a joint analysis of three existing PD GWAS were identified and each assigned to a gene. For gene expression, rather than the traditional comparison of one anatomical region between sets of patients and controls, we identified differentially expressed genes between adjacent Braak regions in each individual and adjusted using average control expression profiles. Over-represented pathways were calculated using a hyper-geometric statistical comparison. An integrated, systems meta-analysis of the over-represented pathways combined the expression and GWAS results using a Fisher's combined probability test. Four of the top seven pathways from each approach were identical. The top three pathways in the meta-analysis, with their corrected p-values, were axonal guidance (p = 2.8E-07), focal adhesion (p = 7.7E-06) and calcium signaling (p = 2.9E-05). These results support that a systems biology (pathway) approach will provide additional insight into the genetic etiology of PD and that these pathways have both biological and statistical support to be important in PD.