Jack R. Collins

The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists

The DAVID Gene Functional Classification Tool http://david.abcc.ncifcrf.gov uses a novel agglomeration algorithm to condense a list of genes or associated biological terms into organized classes of related genes or biology, called biological modules. This organization is accomplished by mining the complex biological co-occurrences found in multiple sources of functional annotation. It is a powerful method to group functionally related genes and terms into a manageable number of biological modules for efficient interpretation of gene lists in a network context.

Flap opening in HIV-1 protease simulated by ‘activated’ molecular dynamics

We have used an ‘activated’ molecular dynamics approach to simulate flap opening in HIV-1 protease. An initial impulse for flap opening was provided by applying harmonic constraints to non-flap residues. After an initial ‘melting’ phase, the two β-hairpin structures that constitute the flaps opened to a 25 Å gap within 200 ps of simulation. Analysis of backbone torsion angles suggests that flap opening is related to conformational changes at Lys 45, Met 46, Gly 52 and Phe 53. In contrast, similar molecular dynamics simulations on the M461 mutant, which is associated with drug resistance, indicates that this mutation stabilizes the flaps in a closed conformation.

Structural conservation of interferon gamma among vertebrates

Interferon gamma (IFN-gamma), being the hallmark of the T-cell T(H)1 response, has been extensively studied with respect to its expression and regulation of immune function. This gene has been extensively characterized in many mammalian species, making it one of the most widely cloned immunoregulatory genes. Recently, the gene has been identified in avian and piscine species and we have identified the gene in the frog genome. Based on these identified DNA sequences, we have constructed an evolutionary history of IFN-gamma that shows this molecule can be traced back more than 450 million years ago. Our analysis shows that type II interferon (IFN-gamma) function evolved before the tetrapod-fish split, a finding that contrasts earlier studies showing its origins in tetrapods. The IFN-gamma gene has undergone a further duplication event in teleosts after the tetrapod-fish split suggesting a specific-evolutionary adaptation in fish. The analyses of IFN-gamma, IL-22 and IL-26 genomic region in mammals, chicken, frog and fish reveal an evolutionary conservation of the loci and several regulatory elements controlling IFN-gamma gene transcription. Furthermore, across the vertebrata, the first intron of IFN-gamma gene contains a polymorphic microsatellite that has been closely correlated with disease susceptibility. Comparative-modeling of IFN-gamma structure revealed differences among the representative species but with an overall conservation of the fold, dimer interface and some interactions with the receptor. The structural and functional conservation of IFN-gamma suggests the presence of an innate, natural killer (NK) like response or even an adaptive T(H)1 immune response in lower vertebrates.

Abundance and length of simple repeats in vertebrate genomes are determined by their structural properties

Microsatellites are abundant in vertebrate genomes, but their sequence representation and length distributions vary greatly within each family of repeats (e.g., tetranucleotides). Biophysical studies of 82 synthetic single-stranded oligonucleotides comprising all tetra- and trinucleotide repeats revealed an inverse correlation between the stability of folded-back hairpin and quadruplex structures and the sequence representation for repeats > or =30 bp in length in nine vertebrate genomes. Alternatively, the predicted energies of base-stacking interactions correlated directly with the longest length distributions in vertebrate genomes. Genome-wide analyses indicated that unstable sequences, such as CAG:CTG and CCG:CGG, were over-represented in coding regions and that micro/minisatellites were recruited in genes involved in transcription and signaling pathways, particularly in the nervous system. Microsatellite instability (MSI) is a hallmark of cancer, and length polymorphism within genes can confer susceptibility to inherited disease. Sequences that manifest the highest MSI values also displayed the strongest base-stacking interactions; analyses of 62 tri- and tetranucleotide repeat-containing genes associated with human genetic disease revealed enrichments similar to those noted for micro/minisatellite-containing genes. We conclude that DNA structure and base-stacking determined the number and length distributions of microsatellite repeats in vertebrate genomes over evolutionary time and that micro/minisatellites have been recruited to participate in both gene and protein function.

Cbl-b interacts with ubiquitinated proteins ; differential functions of the UBA domains of c-Cbl and Cbl-b

Cbl proteins are ubiquitin protein ligases, which ubiquitinate activated tyrosine kinases and target them for degradation. Both c-Cbl and Cbl-b have an ubiquitin associated (UBA) domain at their C-terminal end. We observed that high molecular weight ubiquitinated proteins constitutively coimmunoprecipitated with transfected and endogenous Cbl-b, but not c-Cbl. The binding site for these ubiquitinated proteins was mapped to the UBA domain of Cbl-b (UBAb). GST-fusion proteins containing the UBAb interacted with ubiquitinated proteins and polyubiquitin chains in vitro, whereas those containing the UBA domain of c-Cbl (UBAc) did not. The UBAb had a much greater affinity for polyubiquitin chains than for monoubiquitin. Analysis of the UBAb and UBAc demonstrate that the affinity for ubiquitin is determined by multiple amino-acid differences between the two domains. Overexpression of the UBAb, but not overexpression of the UBAc, inhibited a variety of ubiquitin-mediated processes such as degradation of ubiquitinated proteins (i.e. EGFR, Mdm-2, and Siah-1). This in vivo result is consistent with the differences in ubiquitin binding observed in vitro between the UBAb and UBAc. This difference in ubiquitin-binding may reflect distinct regulatory functions of c-Cbl and Cbl-b.

A high-throughput system for segmenting nuclei using multiscale techniques.

Automatic segmentation of cell nuclei is critical in several high‐throughput cytometry applications whereas manual segmentation is laborious and irreproducible. One such emerging application is measuring the spatial organization (radial and relative distances) of fluorescence in situ hybridization (FISH) DNA sequences, where recent investigations strongly suggest a correlation between nonrandom arrangement of genes to carcinogenesis. Current automatic segmentation methods have varying performance in the presence of nonuniform illumination and clustering, and boundary accuracy is seldom assessed, which makes them suboptimal for this application. The authors propose a modular and model‐based algorithm for extracting individual nuclei. It uses multiscale edge reconstruction for contrast stretching and edge enhancement as well as a multiscale entropy‐based thresholding for handling nonuniform intensity variations. Nuclei are initially oversegmented and then merged based on area followed by automatic multistage classification into single nuclei and clustered nuclei. Estimation of input parameters and training of the classifiers is automatic. The algorithm was tested on 4,181 lymphoblast nuclei with varying degree of background nonuniformity and clustering. It extracted 3,515 individual nuclei and identified single nuclei and individual nuclei in clusters with 99.8 ± 0.3% and 95.5 ± 5.1% accuracy, respectively. Segmented boundaries of the individual nuclei were accurate when compared with manual segmentation with an average RMS deviation of 0.26 μm (∼2 pixels). The proposed segmentation method is efficient, robust, and accurate for segmenting individual nuclei from fluorescence images containing clustered and isolated nuclei. The algorithm allows complete automation and facilitates reproducible and unbiased spatial analysis of DNA sequences. Published 2008 Wiley‐Liss, Inc.

Are there neutral helium compounds which are stable in their ground state?: A theoretical investigation of HeBCH and HeBeO

At the MP4(SDTQ)/6-311G★★//MP2/6-31G★★+ ZPE level of theory, HeBCH is predicted to be unstable towards helium dissociation by −6.0 kcalmol, but HeBeO is calculated to be a stable molecule by + 3.2 kcalmol. Convened to the reaction enthalpy, the latter value increases to + 3.9 kcalmol.

Long homopurine•homopyrimidine sequences are characteristic of genes expressed in brain and the pseudoautosomal region

Homo(purine•pyrimidine) sequences (R•Y tracts) with mirror repeat symmetries form stable triplexes that block replication and transcription and promote genetic rearrangements. A systematic search was conducted to map the location of the longest R•Y tracts in the human genome in order to assess their potential function(s). The 814 R•Y tracts with ≥250 uninterrupted base pairs were preferentially clustered in the pseudoautosomal region of the sex chromosomes and located in the introns of 228 annotated genes whose protein products were associated with functions at the cell membrane. These genes were highly expressed in the brain and particularly in genes associated with susceptibility to mental disorders, such as schizophrenia. The set of 1957 genes harboring the 2886 R•Y tracts with ≥100 uninterrupted base pairs was additionally enriched in proteins associated with phosphorylation, signal transduction, development and morphogenesis. Comparisons of the ≥250 bp R•Y tracts in the mouse and chimpanzee genomes indicated that these sequences have mutated faster than the surrounding regions and are longer in humans than in chimpanzees. These results support a role for long R•Y tracts in promoting recombination and genome diversity during evolution through destabilization of chromosomal DNA, thereby inducing repair and mutation.

An exhaustive DNA micro-satellite map of the human genome using high performance computing.

The current pace of the generation of sequence data requires the development of software tools that can rapidly provide full annotation of the data. We have developed a new method for rapid sequence comparison using the exact match algorithm without repeat masking. As a demonstration, we have identified all perfect simple tandem repeats (STR) within the draft sequence of the human genome. The STR elements (chromosome, position, length and repeat subunit) have been placed into a relational database. Repeat flanking sequence is also publicly accessible at http://grid.abcc.ncifcrf.gov. To illustrate the utility of this complete set of STR elements, we documented the increased density of potentially polymorphic markers throughout the genome. The new STR markers may be useful in disease association studies because so many STR elements manifest multiallelic polymorphism. Also, because triplet repeat expansions are important for human disease etiology, we identified trinucleotide repeats that exist within exons of known genes. This resulted in a list that includes all 14 genes known to undergo polynucleotide expansion, and 48 additional candidates. Several of these are non-polyglutamine triplet repeats. Other examinations of the STR database demonstrated repeats spanning splice junctions and identified SNPs within repeat elements.

Design of sensitive fluorogenic substrates for human cathepsin D

Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimers disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E‐NH2, where X=cysteine, methylcysteine, ethylcysteine, tert‐butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k cat/K m ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure‐based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.