An adaptable, portable microarray reader for biodetection.

We have developed an inexpensive portable microarray reader that can be applied to standard microscope slide-based arrays and other array formats printed on chemically modified surfaces. Measuring only 19 cm in length, the imaging device is portable and may be applicable to both triage and clinical settings. For multiplexing and adaptability purposes, it can be modified to work with multiple excitation/emission wavelengths.

Applications of functional protein microarrays: identifying protein-protein interactions in an array format.

The use of protein arrays and their importance in proteomic applications continues to be at the forefront of scientific discovery and innovative technology development. To date, array-based approaches have proven to be a powerful tool for protein expression profiling, novel biomarker discovery, and the examination of protein, DNA, and small molecule interactions. Our laboratory has developed several approaches for characterizing protein-protein interactions using protein microarrays for a variety of different biological applications.

Treatment with 1-alpha,25-dihydroxyvitamin D3 (vitamin D3) to inhibit carcinogenesis in the hamster buccal pouch model.

Objective: To investigate whether systemic therapy with 1-alpha,25-dihydroxyvitamin D(3) (vitamin D(3) [hereinafter, VD(3)]) prevents tumor formation in a hamster buccal pouch model of carcinogenesis.

Design: Randomized trial in which a known carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), was applied to the buccal pouch of 40 hamsters. Animals were randomized to receive systemic VD(3) or no treatment and killed at 2, 6, and 14 weeks after the initiation of DMBA exposure.

The ATPase motif in RAD51D is required for resistance to DNA interstrand crosslinking agents and interaction with RAD51C.

Homologous recombination (HR) is a mechanism for repairing DNA interstrand crosslinks and double-strand breaks. In mammals, HR requires the activities of the RAD51 family (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1), each of which contains conserved ATP binding sequences (Walker Motifs A and B). RAD51D is a DNA-stimulated ATPase that interacts directly with RAD51C and XRCC2.

Towards a proteome-scale map of the human protein-protein interaction network.

Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions.

Nuclear localization of Rad51B is independent of Rad51C and BRCA2.

Rad51B is one of the five paralogs of human Rad51 and is found in a multiprotein complex with three other Rad51 paralogs, Rad51C, Rad51D and Xrcc2. Participation of Rad51B in this complex depends on its direct interaction with Rad51C. Examination of EGFP-Rad51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines reveal the nuclear localization of Rad51B.

Human ORFeome version 1.1: a platform for reverse proteomics.

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products.

Comparison of multiplexed techniques for detection of bacterial and viral proteins.

Immobilized antibody microarrays were compared to the Luminex flow cytometry system that utilizes suspensions of polystyrene microbeads covalently coupled with capture antibodies. The two immunoassays were performed for comparison of reproducibility, limits of detection and dynamic range. The Luminex system showed lower limits of detection and increased dynamic range among samples whereas the protein microarrays could be more amenable to miniaturization.

Protein expression arrays for proteomics.

As biology approaches the 50th year of deciphering the DNA code, the next frontier toward understanding cell function has protein biochemistry in the form of structural and functional proteomics. To accomplish the needs of proteomics, novel strategies must be devised to examine the gene products or proteins, emerged as en masse. The authors have developed a high-throughput system for the expression and purification of eukaryotic proteins to provide the resources for structural studies and protein functional analysis.

Domain mapping of the Rad51 paralog protein complexes.

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and Rad51C-Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs.

Identification of chromatin-related protein interactions using protein microarrays.

Dynamic structural changes in chromatin are mediated by protein interactions that modulate multiple cellular processes including replication, transcription, recombination and DNA repair. Complexes that recognize chromatin are defined by several distinct groups of proteins that either directly modify histones or interact with histone-DNA complexes. A protein microarray format was used to analyze the interaction of various DNA repair proteins with chromatin components.

Array-based proteomics: the latest chip challenge.

Array-based protein technologies are emerging for basic biological research, molecular diagnostics and therapeutic development with the potential of providing parallel functional analysis of hundreds or perhaps hundreds of thousands of proteins simultaneously. Array-based methods are becoming prevalent within proteomics research due to the desire to analyze proteins in an analogous format to that of the DNA microarray. Novel protein biochips are under development in academic laboratories and emerging biotechnology companies to advance the pace and scope of scientific discovery.

Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells.

Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and Rad51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or Rad51C.

Accelerating code to function: sizing up the protein production line.

High-throughput biology has been pioneered by genomics through the application of robotics to expedite DNA-sequencing projects. Advances in high-throughput protein methods are needed to drive the protein production line for high-throughput structural and functional analysis of newly discovered genes. This will require the development and application of a variety of recombinant-protein expression systems to produce the diversity of proteins from both humans and model organisms.

Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange.

Five Rad51-like proteins, referred to as Rad51 paralogs, have been described in vertebrates. We show that two of them, Rad51B and Rad51C, are associated in a stable complex. Rad51B-Rad51C complex has ssDNA binding and ssDNA-stimulated ATPase activities.

RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51.

RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins.

From genes to proteins: high-throughput expression and purification of the human proteome.

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection.

The Rad51 paralog Rad51B promotes homologous recombinational repair.

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out.

Array-based proteomics: high-throughput expression and purification of IMAGE consortium cDNA clones.

As the sequencing efforts of the Human Genome Project approach closure, the next frontier in the quest to understand how specific genes function will rely upon technical advances in protein analysis. An understanding of protein function will be essential to this process. The development of high-throughput genomic strategies has led to the design of new schemes for genome-scale protein science.

Isolation and characterization of RAD51C, a new human member of the RAD51 family of related genes.

The yeast and human RAD51 genes encode strand-transfer proteins that are thought to be involved in both recombinational repair of DNA damage and meiotic recombination. In yeast, the Rad51 family of related proteins also includes Rad55, Rad57 and Dmc1. In mammalian cells, five genes in this family have been identified (HsRAD51, XRCC2, XRCC3, RAD51B/hREC2 and HsDMC1), and here we report the isolation of the sixth member, RAD51C.

Identification of a novel human RAD51 homolog, RAD51B.

The highly conserved Saccharomyces cerevisiae RAD51 protein functions in both mitotic and meiotic homologous recombination and in double-strand break repair. Screening of the public cDNA sequence database for RAD51-like genes led to the identification of a partial sequence from a breast tissue library present in the I.M.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells.

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and gamma-radiation.

Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms.

We have determined that the gene for human microtubule-associated protein 2 (MAP-2) spans 19 exons, including 6 exons identified in this study, 1-4, 8, and 13; all six of these exons are transcribed. The alternative splicing of coding exons generates a greater diversity of MAP-2 transcripts and isoforms. The first three exons encode alternate 5' untranslated regions that can be spliced to additional untranslated sequences contained in exons 4 and 5.