[Show abstract][Hide abstract] ABSTRACT: The Notch signal transduction pathway mediates important cellular functions through direct cell-to-cell contact. Deregulation
of Notch activity can lead to an altered cell proliferation and has been linked to many human cancers. Casein kinase 2 (CK2),
a ubiquitous kinase, regulates several cellular processes by phosphorylating proteins involved in signal transduction, gene
expression, and protein synthesis. In this report we identify NotchICD as a novel target of phosphorylation by CK2. Using mapping and mutational studies, we identified serine 1901, located in
the ankyrin domain of Notch, as the target amino acid. Interestingly, phosphorylation of serine 1901 by CK2 appears to generate
a second phosphorylation site at threonine 1898. Furthermore, threonine 1898 phosphorylation only occurs when Notch forms
a complex with Mastermind and CSL. Phosphorylation of both threonine 1898 and serine 1901 resulted in decreased binding of
the Notch-Mastermind-CSL ternary complex to DNA and consequently lower transcriptional activity. These data indicate that
the phosphorylation of serine 1901 and threonine 1898 negatively regulates Notch function by dissociating the complex from
DNA. This study identifies a new component involved in regulation of NotchICD transcriptional activity, reinforcing the notion that a precise and tight regulation is required for this essential signaling
Preview · Article · Jun 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct
cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (NICD) from the plasma membrane results in the accumulation of deregulated nuclear NICD that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear NICD forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding
protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that
NICD forms a transcriptional activation complex, little is known about how the complex is assembled. In this study, we demonstrate
that NICD multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly,
we demonstrate that the assembly is mediated by NICD multimers interacting with Skip and Mastermind. These interactions form a preactivation complex that is then resolved by
CSL to form the Notch transcriptional activation complex on DNA.
Full-text · Article · Mar 2011 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: ExCyto PCR cells provide a novel and cost effective means to amplify DNA transformed into competent bacterial cells. ExCyto PCR uses host E. coli with a chromosomally integrated gene encoding a thermostable DNA polymerase to accomplish robust, hot-start PCR amplification of cloned sequences without addition of exogenous enzyme.
Because the thermostable DNA polymerase is stably integrated into the bacterial chromosome, ExCyto cells can be transformed with a single plasmid or complex library, and then the expressed thermostable DNA polymerase can be used for PCR amplification. We demonstrate that ExCyto cells can be used to amplify DNA from different templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours. Further, PCR amplification with ExCyto cells is comparable to amplification using commercial DNA polymerases. The ability to transform a bacterial strain and use the endogenously expressed protein for PCR has not previously been demonstrated.
ExCyto PCR reduces pipetting and greatly increases throughput for screening EST, genomic, BAC, cDNA, or SNP libraries. This technique is also more economical than traditional PCR and thus broadly useful to scientists who utilize analysis of cloned DNAs in their research.
[Show abstract][Hide abstract] ABSTRACT: Ichthyoplankton collections provide a valuable means to study fish life histories. However, these collections are greatly underutilized, as larval fishes are frequently not identified to species due to their small size and limited morphological development. Currently, there is an effort underway to make species identification more readily available across a broad range of taxa through the sequencing of a standard gene. This effort requires the development of new methodologies to both rapidly produce and analyse large numbers of sequences. The methodology presented in this paper addresses these issues with a focus on the larvae of large pelagic fish species. All steps of the methodology are targeted towards high-throughput identification using small amounts of tissue. To accomplish this, DNA isolation was automated on a liquid-handling robot using magnetic bead technologies. Polymerase chain reaction and a unidirectional sequencing reaction followed standard protocols with all template cleanup and transferring also automated. Manual pipetting was thus reduced to a minimum. A character-based bioinformatics program was developed to handle the large sequence output. This program incorporates base-call quality scores in two types of sample to voucher sequence comparisons and provides suggested identifications and sequence information in an easily interpreted spreadsheet format. This technique when applied to tuna and billfish larvae collected in the Straits of Florida had an 89% success rate. A single species (Thunnus atlanticus) was found to dominate the catch of tuna larvae, while billfish larvae were more evenly divided between two species (Makaira nigricans and Istiophorus platypterus).
Full-text · Article · Feb 2007 · Molecular Ecology Notes
[Show abstract][Hide abstract] ABSTRACT: While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes.
We describe a bioinformatics pipeline known as FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs) from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P < 0.001) enrichment in genes associated with amine metabolism.
The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for FunnyBase can be accessed at http://genomics.rsmas.miami.edu/funnybase/super_craw4/. Data and source code are available by request at email@example.com.