Biomedical subjects

Thomas L Casavant

Publications and source records attributed to Thomas L Casavant.

At least 19 recordsLinked to original sources

PubMed2006

Eukaryotic operon-like transcription of functionally related genes in Drosophila.

Complex biological processes require coordinated function of many genes. One evolutionary solution to the problem of coordinately expressing functionally related genes in bacteria and nematodes is organization of genes in operons. Surprisingly, eukaryotic operons are considered rare outside the nematode lineage. In Drosophila melanogaster, we found lounge lizard (llz), which encodes a degenerin/ENaC cation channel, cotranscribed with CheB42a, a nonhomologous gene of unknown function residing <100 bp upstream. These two genes were transcribed from a single promoter as one primary transcript and were processed posttranscriptionally to generate individual mRNAs. The mechanism did not involve alternative splicing, and it differed from the trans splicing used in nematode operons. Both genes were expressed in the same tissues, and previous work suggested that both may be involved in courtship behavior. A bioinformatic approach identified numerous additional loci as potential Drosophila operons. These data reveal eukaryotic operon-like transcription of functionally related genes in Drosophila. The results also suggest that operon-based transcription may be more common in eukaryotes than previously appreciated.

Animals↗

PubMed2006

Regulation of gene expression in the mammalian eye and its relevance to eye disease.

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

Analysis of Variance↗

PubMed2006

Analysis of ESTs from Lutzomyia longipalpis sand flies and their contribution toward understanding the insect-parasite relationship.

An expressed sequence tag library has been generated from a sand fly vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed from whole adults and 16,608 clones were sequenced from both ends and assembled into 10,203 contigs and singlets. Of these 58% showed significant similarity to known genes from other organisms, <4% were identical to described sand fly genes, and 42% had no match to any database sequence. Our analyses revealed putative proteins involved in the barrier function of the gut (peritrophins, microvillar proteins, glutamine synthase), digestive physiology (secreted and membrane-anchored hydrolytic enzymes), and the immune response (gram-negative binding proteins, thioester proteins, scavenger receptors, galectins, signaling pathway factors, caspases, serpins, and peroxidases). Sequence analysis of this transcriptome dataset has provided new insights into genes that might be associated with the response of the vector to the development of Leishmania.

Animals↗

PubMed2006

An annotated cDNA library of juvenile Euprymna scolopes with and without colonization by the symbiont Vibrio fischeri.

BACKGROUND: Biologists are becoming increasingly aware that the interaction of animals, including humans, with their coevolved bacterial partners is essential for health. This growing awareness has been a driving force for the development of models for the study of beneficial animal-bacterial interactions. In the squid-vibrio model, symbiotic Vibrio fischeri induce dramatic developmental changes in the light organ of host Euprymna scolopes over the first hours to days of their partnership. We report here the creation of a juvenile light-organ specific EST database. RESULTS: We generated eleven cDNA libraries from the light organ of E. scolopes at developmentally significant time points with and without colonization by V. fischeri. Single pass 3' sequencing efforts generated 42,564 expressed sequence tags (ESTs) of which 35,421 passed our quality criteria and were then clustered via the UIcluster program into 13,962 nonredundant sequences. The cDNA clones representing these nonredundant sequences were sequenced from the 5' end of the vector and 58% of these resulting sequences overlapped significantly with the associated 3' sequence to generate 8,067 contigs with an average sequence length of 1,065 bp. All sequences were annotated with BLASTX (E-value < -03) and Gene Ontology (GO). CONCLUSION: Both the number of ESTs generated from each library and GO categorizations are reflective of the activity state of the light organ during these early stages of symbiosis. Future analyses of the sequences identified in these libraries promise to provide valuable information not only about pathways involved in colonization and early development of the squid light organ, but also about pathways conserved in response to bacterial colonization across the animal kingdom.

Aliivibrio fischeri↗

PubMed2006

Protein expression in a transformed trabecular meshwork cell line: proteome analysis.

PURPOSE: Characterization of the human trabecular meshwork (TM) proteome is hindered by the small mass of intact tissue and the slow growth of cultured cell strains. We have previously characterized a transformed TM cell strain (GTM3) that demonstrates many of the same protein expression and cell signaling systems of nontransformed cell strains. The aim of this study was to initiate a proteomic survey of GTM3 cells as the initial step toward characterization of the complete human TM proteome. METHODS: GTM3 cells were cultured to confluence, harvested and solubilized in urea/Nonidet. The protein extract (600 mug) was focused in immobilized isoelectric focusing (IEF) strips, separated by 10% SDS PAGE, and visualized with colloidal Coomassie Blue. Spots of interest were excised, destained, and the contained proteins subjected to in-gel reduction, derivatization, and tryptic digestion. Tryptic peptides were extracted and analyzed by electrospray LC/MS/MS. Protein identification was made using the TurboSequest search algorithm and a recent version of the nonredundant human protein database downloaded from the National Center for Biotechnology Information (NCBI). RESULTS: Eighty-seven (87) primary proteins and 93 variants of these proteins were identified. A website was created (TM proteome) that combines data such as graphic spot location within the gel, peptide sequence, apparent and calculated pI, apparent and calculated mass, percentage of coverage, and protein informatic website links. CONCLUSIONS: Proteomic analysis of a transformed human TM cell line has been initiated combining preparative two-dimensional PAGE separation, LC/MS/MS analysis of major proteins, and bioinformatic cataloging of the data. Further investigation of data from the transformed cell strain will be used in a comparative fashion for spot identification of analytical proteomic gels of human TM tissue and cultured normal cells. These initial data will form the base from which the characterization of protein expression in the normal and glaucomatous TM can be accomplished.

Cell Line, Transformed↗

PubMed2006

Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet-Biedl syndrome gene (BBS11).

The identification of mutations in genes that cause human diseases has largely been accomplished through the use of positional cloning, which relies on linkage mapping. In studies of rare diseases, the resolution of linkage mapping is limited by the number of available meioses and informative marker density. One recent advance is the development of high-density SNP microarrays for genotyping. The SNP arrays overcome low marker informativity by using a large number of markers to achieve greater coverage at finer resolution. We used SNP microarray genotyping for homozygosity mapping in a small consanguineous Israeli Bedouin family with autosomal recessive Bardet-Biedl syndrome (BBS; obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal and cardiac abnormalities, and cognitive impairment) in which previous linkage studies using short tandem repeat polymorphisms failed to identify a disease locus. SNP genotyping revealed a homozygous candidate region. Mutation analysis in the region of homozygosity identified a conserved homozygous missense mutation in the TRIM32 gene, a gene coding for an E3 ubiquitin ligase. Functional analysis of this gene in zebrafish and expression correlation analyses among other BBS genes in an expression quantitative trait loci data set demonstrate that TRIM32 is a BBS gene. This study shows the value of high-density SNP genotyping for homozygosity mapping and the use of expression correlation data for evaluation of candidate genes and identifies the proteasome degradation pathway as a pathway involved in BBS.

Animals↗

PubMed2006

Prioritizing regions of candidate genes for efficient mutation screening.

The availability of the complete sequence of the human genome has dramatically facilitated the search for disease-causing sequence variations. In fact, the rate-limiting step has shifted from the discovery and characterization of candidate genes to the actual screening of human populations and the subsequent interpretation of observed variations. In this study we tested the hypothesis that some segments of candidate genes are more likely than others to contain disease-causing variations and that these segments can be predicted bioinformatically. A bioinformatic technique, prioritization of annotated regions (PAR), was developed to predict the likelihood that a specific coding region of a gene will harbor a disease-causing mutation based on conserved protein functional domains and protein secondary structures. This method was evaluated by using it to analyze 710 genes that collectively harbor 4,498 previously identified mutations. Nearly 50% of the genes were recognized as disease-associated after screening only 9% of the complete coding sequence. The PAR technique identified 90% of the genes as containing at least one mutation, with less than 40% of the screening resources that traditional approaches would require. These results suggest that prioritization strategies such as PAR can accelerate disease-gene identification through more efficient use of screening resources.

Computational Biology↗

PubMed2006

Genome-wide identification of pseudogenes capable of disease-causing gene conversion.

Pseudogenes are remnants of gene duplication (nonprocessed pseudogenes) and retrotransposition (processed pseudogenes) events. This study describes methods for identifying gene conversion candidates from predicted pseudogenes. Pseudogenes may accumulate and harbor sequence variations over time that become disease-causing mutations when transferred to genes by gene conversion. A total of 14,476 pseudogenes were identified, including 3,426 nonprocessed pseudogenes. In addition, 1,945 nonprocessed pseudogenes that are localized near their progenitor gene were evaluated for their possible role in gene conversion and disease. All 11 known, human cases of gene conversion (with deleterious effects) involving pseudogenes were successfully identified by these methods. Among the pseudogenes identified is a retinitis pigmentosa 9 (RP9) pseudogene that carries a c.509A>G mutation which produces a p.Asp170Gly substitution that is associated with the RP9 form of autosomal dominant retinitis pigmentosa (adRP). The c.509A>G mutation in RP9 is a previously unrecognized example of gene conversion between the progenitor gene and its pseudogene. Notably, two processed pseudogenes also contain mutations associated with diseases. An inosine monophosphate dehydrogenase 1 (IMPDH1) pseudogene carries a c.676G>A mutation that produces a p.Asp226Asn substitution that causes the retinitis pigmentosa 10 (RP10) form of adRP; and a phosphoglycerate kinase 1 (PGK1) pseudogene (PGK1P1) carries a c.837T>C mutation that produces a p.Ile252Thr substitution that is associated with a phosphoglycerate kinase deficiency. Ranking of nonprocessed pseudogenes as candidates for gene conversion was also performed based on the sequence characteristics of published cases of pseudogene-mediated gene conversion. All results and tools produced by this study are available for download at: http://genome.uiowa.edu/pseudogenes.

Computational Biology↗

PubMed2006

Integration site choice of a feline immunodeficiency virus vector.

We mapped 226 unique integration sites in human hepatoma cells following gene transfer with a feline immunodeficiency virus (FIV)-based lentivirus vector. FIV integrated across the entire length of the transcriptional units. Microarray data indicated that FIV integration favored actively transcribed genes. Approximately 21% of FIV integrations within transcriptional units occurred in genes regulated by the LEDGF/p75 transcriptional coactivator. DNA in regions of FIV insertion sites exhibited a "bendable" structure and a pattern of duplex destabilization favoring strand separation. FIV integration preferences are more similar to those of primate lentiviruses and distinct from those of Moloney murine leukemia virus, avian sarcoma leukosis virus, and foamy virus.

Animals↗

PubMed2006

First exons and introns--a survey of GC content and gene structure in the human genome.

Most transcriptional regulatory elements are located in non-coding DNA. In particular, some first introns play a vital role in transcriptional control and splicing. The length and GC-content of first exons and introns in complex organisms suggests that these structural units are likely to be important functional elements in large genomes. Hence, in this paper we perform a systematic comparison of exon-intron structure and GC content on all known genes in the human genome. Our in-silico analysis found that the GC content of introns and exons varies significantly depending on their length. On average, the first intron of a gene is significantly longer than other introns in the same gene. Our results also show that first introns and exons are more GC rich than last and internal. This study provides insight into the structure of eukaryotic genes. These results confirm and expand the previously identified regulatory potential of first exons and introns.

Base Composition↗

PubMed2005

Genetic characterization of commonly used glioma cell lines in the rat animal model system.

OBJECT: Animal models have been used extensively to discern the molecular biology of diseases and to gain insight into treatments. Nevertheless, discrepancies in the effects of treatments and procedures have been encountered during the transition from these animal models to application of the information to clinical trials in humans. To assess the genetic similarities between human gliomas and four cell lines used routinely in animal models, the authors used microarray technology to characterize the similarities and differences in gene expression. METHODS: To define the changes in gene expression, normal rat astrocytes were compared with four rat glioma cell lines (C6, 9L, F98, and RG2). The data were analyzed using two different methods: fold-change analysis and statistical analysis with t statistics. The gene products that were highlighted after intersecting the lists generated by the two methods of analysis were scrutinized against changes in gene expression reported in the literature. Tumorigenesis involves three major steps: the accumulation of genetic alterations, uncontrolled growth, and selected survival of transformed cells. The discussion of the results focuses attention on genes whose primary function is in pathways involved in glioma proliferation, infiltration, and neovascularization. A comparative microarray analysis of differentially expressed genes for four of the commonly used rat tumor cell lines is presented here. CONCLUSIONS: Due to the variances between the cell lines and results from analyses in humans, caution must be observed in interpreting as well as in the translation of information learned from animal models to its application in human trials.

Animals↗

PubMed2005

CFTR DeltaF508 mutation has minimal effect on the gene expression profile of differentiated human airway epithelia.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel regulated by phosphorylation. Most of the disease-associated morbidity is the consequence of chronic lung infection with progressive tissue destruction. As an approach to investigate the cellular effects of CFTR mutations, we used large-scale microarray hybridization to contrast the gene expression profiles of well-differentiated primary cultures of human CF and non-CF airway epithelia grown under resting culture conditions. We surveyed the expression profiles for 10 non-CF and 10 DeltaF508 homozygote samples. Of the 22,283 genes represented on the Affymetrix U133A GeneChip, we found evidence of significant changes in expression in 24 genes by two-sample t-test (P < 0.00001). A second, three-filter method of comparative analysis found no significant differences between the groups. The levels of CFTR mRNA were comparable in both groups. There were no significant differences in the gene expression patterns between male and female CF specimens. There were 18 genes with significant increases and 6 genes with decreases in CF relative to non-CF samples. Although the function of many of the differentially expressed genes is unknown, one transcript that was elevated in CF, the KCl cotransporter (KCC4), is a candidate for further study. Overall, the results indicate that CFTR dysfunction has little direct impact on airway epithelial gene expression in samples grown under these conditions.

Cell Differentiation↗

PubMed2005

Insights into a dinoflagellate genome through expressed sequence tag analysis.

BACKGROUND: Dinoflagellates are important marine primary producers and grazers and cause toxic "red tides". These taxa are characterized by many unique features such as immense genomes, the absence of nucleosomes, and photosynthetic organelles (plastids) that have been gained and lost multiple times. We generated EST sequences from non-normalized and normalized cDNA libraries from a culture of the toxic species Alexandrium tamarense to elucidate dinoflagellate evolution. Previous analyses of these data have clarified plastid origin and here we study the gene content, annotate the ESTs, and analyze the genes that are putatively involved in DNA packaging. RESULTS: Approximately 20% of the 6,723 unique (11,171 total 3'-reads) ESTs data could be annotated using Blast searches against GenBank. Several putative dinoflagellate-specific mRNAs were identified, including one novel plastid protein. Dinoflagellate genes, similar to other eukaryotes, have a high GC-content that is reflected in the amino acid codon usage. Highly represented transcripts include histone-like (HLP) and luciferin binding proteins and several genes occur in families that encode nearly identical proteins. We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X. We speculate this histone may be retained for its role in DNA double-strand break repair. CONCLUSION: This is the most extensive collection to date of ESTs from a toxic dinoflagellate. These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production.

Amino Acid Sequence↗

PubMed2005

Identifying components of the NF-kappaB pathway in the beneficial Euprymna scolopes-Vibrio fischeri light organ symbiosis.

The Toll/NF-kappaB pathway is a common, evolutionarily conserved innate immune pathway that modulates the responses of animal cells to microbe-associated molecular patterns (MAMPs). Because MAMPs have been implicated as critical elements in the signaling of symbiont-induced development, an expressed sequence tag library from the juvenile light organ of Euprymna scolopes was used to identify members of the Toll/NF-kappaB pathway. Full-length transcripts were identified by using 5' and 3' RACE PCR. Seven transcripts critical for MAMP-induced triggering of the Toll/NF-kappaB phosphorylation cascade have been identified, including receptors, signal transducers, and a transcription factor. Further investigations should elucidate the role of the Toll/NF-kappaB pathway in the initiation of the beneficial symbiosis between E. scolopes and Vibrio fischeri.

Aliivibrio fischeri↗

PubMed2004

Isolation and characterization of autotrophic, hydrogen-utilizing, perchlorate-reducing bacteria.

Recent studies have shown that perchlorate (ClO(4) (-)) can be degraded by some pure-culture and mixed-culture bacteria with the addition of hydrogen. This paper describes the isolation of two hydrogen-utilizing perchlorate-degrading bacteria capable of using inorganic carbon for growth. These autotrophic bacteria are within the genus Dechloromonas and are the first Dechloromonas species that are microaerophilic and incapable of growth at atmospheric oxygen concentrations. Dechloromonas sp. JDS5 and Dechloromonas sp. JDS6 are the first perchlorate-degrading autotrophs isolated from a perchlorate-contaminated site. Measured hydrogen thresholds were higher than for other environmentally significant, hydrogen-utilizing, anaerobic bacteria (e.g., halorespirers). The chlorite dismutase activity of these bacteria was greater for autotrophically grown cells than for cells grown heterotrophically on lactate. These bacteria used fumarate as an alternate electron acceptor, which is the first report of growth on an organic electron acceptor by perchlorate-reducing bacteria.

Bacteria↗

PubMed2004

Missense variations in the fibulin 5 gene and age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the developed world. The study of a rare mendelian form of macular degeneration implicated fibulin genes in the pathogenesis of more common forms of this disease. We evaluated five fibulin genes in a large series of patients with AMD. METHODS: We studied 402 patients with AMD and 429 control subjects from the same clinic population. Patients were examined by means of indirect ophthalmoscopy, slit-lamp microscopy, and fundus photography to establish the presence and phenotypic pattern of AMD. DNA samples were screened for sequence variations in five members of the fibulin gene family. RESULTS: Amino acid-altering sequence variations were found in all five fibulin genes, many of which were observed only in patients with AMD. Several of the altered residues have been conserved during evolution. Seven of the 402 patients with AMD had amino acid-altering sequence variations in the fibulin 5 gene, whereas none were observed among 429 control subjects (P<0.01). In addition, these seven patients all had small, circular drusen, which are commonly referred to as basal laminar or cuticular drusen. CONCLUSIONS: Missense mutations in the fibulin 5 gene were found in 1.7 percent of patients with AMD. Many variations in other fibulin genes were also found in these patients, and the evolutionary conservation of the affected residues suggests that several of these variations may also be involved in AMD.

Aged↗

PubMed2004

Gene expression profiling of potential PPARgamma target genes in mouse aorta.

Diminished activity of peroxisome proliferator-activated receptor-gamma (PPARgamma) may play a role in the pathogenesis of hypertension and vascular dysfunction. To better understand what genes are regulated by PPARgamma, an experimental data set was generated by microarray analysis, in duplicate, of pooled aortic mRNA isolated from mice treated for 21 days with a PPARgamma agonist (rosiglitazone) or vehicle. Of the 12,488 probe sets present on the array (Affymetrix MG-U74Av2), 181 were differentially expressed between groups according to a statistical metric generated using Affymetrix software. A significant correlation was observed between the microarray results and real-time RT-PCR analysis of 39 of these genes. Cluster analysis revealed 3 expression patterns, 29 transcripts of moderate abundance that were decreased (-93%) to very low levels, 106 transcripts that were downregulated (-42%), and 46 transcripts that were upregulated (+70%). Functional groups that were decreased included inflammatory response (-93%, n = 6), immune response (-86%, n = 7), and cytokines (-82%, n = 7). There was an overall upregulation in the oxidoreductase activity group (+47%, n = 9). Individually, six transcripts in this group were increased (+72%), and three were decreased (-34%). Fourteen of the genes map to regions in the rat genome that have been linked to increased blood pressure, and of 142 upstream regions analyzed, sequences resembling the DNA binding site for PPARgamma were identified in 101 of the differentially expressed genes.

Animals↗

PubMed2004

A comprehensive nonredundant expressed sequence tag collection for the developing Rattus norvegicus heart.

Congenital heart defects affect approximately 1,000,000 people in the United States, with 40,000 new births contributing to that number every year. A large percentage of these defects can be attributed to septal defects. We assembled a nonredundant collection of over 12,000 expressed sequence tags (ESTs) from a total of 30,000 ESTs, with the ultimate goal of identifying spatially and/or temporally regulated genes during heart septation. These ESTs were compiled from nonnormalized, normalized, and serially subtracted cDNA libraries derived from two sets of tissue samples. The first includes microdissected rat hearts from embryonic (E) days E13, E15, and E16.5-E18.5 and adult heart. The second includes hearts from embryonic days E17, E19, and E21 and postnatal (P) days P1, P12, P74, and P200. Over 6,000 novel ESTs were identified in the libraries derived from these two sets of tissues, all of which have been contributed to the NCBI rat UniGene collection. It is anticipated that such EST and cDNA clone resources will prove invaluable to gene expression studies aimed at the understanding of the molecular mechanisms underlying heart septation defects.

Animals↗