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1.  Reproducible RNA Preparation from Sugarcane and Citrus for Functional Genomic Applications 
High-throughput functional genomic procedures depend on the quality of the RNA used. Copurifying molecules can negatively impact the functionality of some plant RNA preparations employed in these procedures. We present a simplified, rapid, and scalable SDS/phenol-based method that provides the high-quantity and -quality RNA required by the newly emerging biotechnology applications. The method is applied to isolating RNA from tissues of two biotechnologically important crop plants, sugarcane and citrus, which provide a challenge due to the presence of fiber, polysaccharides, or secondary metabolites. The RNA isolated by this method is suitable for several downstream applications including northern blot hybridization, microarray analysis, and quantitative RT-PCR. This method has been used in a diverse range of projects ranging from screening plant lines overexpressing mammalian genes to analyzing plant responses to viral infection and defense signaling molecules.
PMCID: PMC2817868  PMID: 20148085
2.  Circadian Activation of the Mitogen-Activated Protein Kinase MAK-1 Facilitates Rhythms in Clock-Controlled Genes in Neurospora crassa 
Eukaryotic Cell  2013;12(1):59-69.
The circadian clock regulates the expression of many genes involved in a wide range of biological functions through output pathways such as mitogen-activated protein kinase (MAPK) pathways. We demonstrate here that the clock regulates the phosphorylation, and thus activation, of the MAPKs MAK-1 and MAK-2 in the filamentous fungus Neurospora crassa. In this study, we identified genetic targets of the MAK-1 pathway, which is homologous to the cell wall integrity pathway in Saccharomyces cerevisiae and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mammals. When MAK-1 was deleted from Neurospora cells, vegetative growth was reduced and the transcript levels for over 500 genes were affected, with significant enrichment for genes involved in protein synthesis, biogenesis of cellular components, metabolism, energy production, and transcription. Additionally, of the ∼500 genes affected by the disruption of MAK-1, more than 25% were previously identified as putative clock-controlled genes. We show that MAK-1 is necessary for robust rhythms of two morning-specific genes, i.e., ccg-1 and the mitochondrial phosphate carrier protein gene NCU07465. Additionally, we show clock regulation of a predicted chitin synthase gene, NCU04352, whose rhythmic accumulation is also dependent upon MAK-1. Together, these data establish a role for the MAK-1 pathway as an output pathway of the circadian clock and suggest a link between rhythmic MAK-1 activity and circadian control of cellular growth.
PMCID: PMC3535850  PMID: 23125351
3.  Transcription Factors in Light and Circadian Clock Signaling Networks Revealed by Genomewide Mapping of Direct Targets for Neurospora White Collar Complex ▿† 
Eukaryotic Cell  2010;9(10):1549-1556.
Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.
PMCID: PMC2950426  PMID: 20675579
Nature reviews. Genetics  2005;6(7):544-556.
The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.
PMCID: PMC2735866  PMID: 15951747
5.  Circadian genomics of the chick pineal gland in vitro 
BMC Genomics  2008;9:206.
Chick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis. These properties make the in vitro pineal a particularly useful model for exploring circadian control of gene transcription in a pacemaker tissue, as well as regulation of the transcriptome by primary inputs to the clock (both photic and noradrenergic).
We used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD. We report that a reduced subset of genes was rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar. We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night. As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially enhanced or suppressed specific sets of genes in a fashion that was dependent upon time of day.
Our combined approach of utilizing a temporal, photic and pharmacological microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal. We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in circadian physiology. Further, we hypothesize that the pineal circadian transcriptome is reduced but functionally conserved in vitro, and supports an endogenous role for the pineal in regulating local rhythms in metabolism, immune function, and other conserved pathways.
PMCID: PMC2405806  PMID: 18454867
6.  Gene expression in the developing mouse retina by EST sequencing and microarray analysis 
Nucleic Acids Research  2001;29(24):4983-4993.
Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (–/–) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.
PMCID: PMC97568  PMID: 11812828

Results 1-6 (6)