A radiation hybrid map of sheep chromosome 23 based on ovine BAC-end sequences

More than 375,000 BAC-end sequences (BES) of the CHORI-243 ovine BAC library have been deposited in public databases. blastn searches with these BES against HSA18 revealed 1806 unique and significant hits. We used blastn-anchored BES for an in silico prediction of gene content and chromosome assignment of comparatively mapped ovine BAC clones. Ovine BES were selected at approximately 1.3-Mb intervals of HSA18 and incorporated into a human-sheep comparative map. An ovine 5000-rad whole-genome radiation hybrid panel (USUoRH5000) was typed with 70 markers, all of which mapped to OAR23. The resulting OAR23 RH map included 43 markers derived from BES with high and unique BLAST hits to the sequence of the orthologous HSA18, nine EST-derived markers, 16 microsatellite markers taken from the ovine linkage map and two bovine microsatellite markers. Six new microsatellite markers derived from the 43 mapped BES and the two bovine microsatellite markers were linkage-mapped using the International Mapping Flock (IMF). Thirteen additional microsatellite markers were derived from other ovine BES with high and unique BLAST hits to the sequence of the orthologous HSA18 and also positioned on the ovine linkage map but not incorporated into the OAR23 RH map. This resulted in 24 markers in common and in the same order between the RH and linkage maps. Eight of the BES-derived markers were mapped using fluorescent in situ hybridization (FISH), to thereby align the RH and cytogenetic maps. Comparison of the ovine chromosome 23 RH map with the HSA18 map identified and localized three major breakpoints between HSA18 and OAR23. The positions of these breakpoints were equivalent to those previously shown for syntenic BTA24 and HSA18. This study presents evidence for the usefulness of ovine BES when constructing a high-resolution comprehensive map for a single sheep chromosome. The comparative analysis confirms and refines knowledge about chromosomal conservation and rearrangements between sheep, cattle and human. The constructed RH map demonstrates the resolution and utility of the newly constructed ovine RH panel.

The locus for bovine dilated cardiomyopathy maps to chromosome 18

Bovine dilated cardiomyopathy (BDCMP) is a severe and terminal disease of the heart muscle observed in Holstein-Friesian cattle over the last 30 years. There is strong evidence for an autosomal recessive mode of inheritance for BDCMP. The objective of this study was to genetically map BDCMP, with the ultimate goal of identifying the causative mutation. A whole-genome scan using 199 microsatellite markers and one SNP revealed an assignment of BDCMP to BTA18. Fine-mapping on BTA18 refined the candidate region to the MSBDCMP06-BMS2785 interval. The interval containing the BDCMP locus was confirmed by multipoint linkage analysis using the software loki. The interval is about 6.7 Mb on the bovine genome sequence (Btau 3.1). The corresponding region of HSA19 is very gene-rich and contains roughly 200 genes. Although telomeric of the marker interval, TNNI3 is a possible positional and a functional candidate for BDCMP given its involvement in a human form of dilated cardiomyopathy. Sequence analysis of TNNI3 in cattle revealed no mutation in the coding sequence, but there was a G-to-A transition in intron 6 (AJ842179:c.378+315G>A). The analysis of this SNP using the study's BDCMP pedigree did not conclusively exclude TNNI3 as a candidate gene for BDCMP. Considering the high density of genes on the homologous region of HSA19, further refinement of the interval on BTA18 containing the BDCMP locus is needed.

A region on equine chromosome 13 is linked to recurrent airway obstruction in horses

REASONS FOR STUDY: Equine recurrent airway obstruction (RAO) is probably dependent on a complex interaction of genetic and environmental factors and shares many characteristic features with human asthma. Interleukin 4 receptor a chain (IL4RA) is a candidate gene because of its role in the development of human asthma, confirmation of this association is therefore required. METHODS: The equine BAC clone containing the IL4RA gene was localised to ECA13q13 by the FISH method. Microsatellite markers in this region were investigated for possible association and linkage with RAO in 2 large Warmblood halfsib families. Based on a history of clinical signs (coughing, nasal discharge, abnormal breathing and poor performance), horses were classified in a horse owner assessed respiratory signs index (HOARSI 1-4: from healthy, mild, moderate to severe signs). Four microsatellite markers (AHT133, LEX041, VHL47, ASB037) were analysed in the offspring of Sire 1 (48 unaffected HOARSI 1 vs. 59 affected HOARSI 2-4) and Sire 2 (35 HOARSI 1 vs. 50 HOARSI 2-4), age 07 years. RESULTS: For both sires haplotypes could be established in the order AHT133-LEXO47-VHL47-ASB37. The distances in this order were estimated to be 2.9, 0.9 and 2.3 centiMorgans, respectively. Haplotype association with mild to severe clinical signs of chronic lower airway disease (HOARSI 2-4) was significant in the offspring of Sire 1 (P = 0.026) but not significant for the offspring of Sire 2 (P = 0.32). Linkage analysis showed the ECA13q13 region containing IL4RA to be linked to equine chronic lower airway disease in one family (P<0.01), but not in the second family. CONCLUSIONS: This supports a genetic background for equine RAO and indicates that IL4RA is a candidate gene with possible locus heterogeneity for this disease. POTENTIAL RELEVANCE: Identification of major genes for RAO may provide a basis for breeding and individual prevention for this important disease.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

A de novo 1.1-1.6 Mb subtelomeric deletion of chromosome 20q13.33 in a patient with learning difficulties but without obvious dysmorphic features

We report on a de novo submicroscopic deletion of 20q13.33 identified by subtelomeric fluorescence in situ hybridization (FISH) in a 4-year-old girl with learning difficulties, hyperlaxity and strabismus, but without obvious dysmorphic features. Further investigations by array-based comparative genomic hybridization (array-CGH) and FISH analysis allowed us to delineate the smallest reported subterminal deletion of chromosome 20q, spanning a 1.1-1.6 Mb with a breakpoint localized between BAC RP5-887L7 and RP11-261N11. The genes CHRNA4 and KCNQ2 implicated in autosomal dominant epilepsy are included in the deletion interval. Subterminal 20q deletions as found in the present patient have, to our knowledge, only been reported in three patients. We review the clinical and behavioral phenotype of such "pure" subterminal 20q deletions.

Genetic maps of the proximal half of chromosome arm 2L of Drosophila melanogaster

Drosophila mutants have played an important role in elucidating the physiologic function of genes. Large-scale projects have succeeded in producing mutations in a large proportion of Drosophila genes. Many mutant fly lines have also been produced through the efforts of individual laboratories over the past century. In an effort to make some of these mutants more useful to the research community, we systematically mapped a large number of mutations affecting genes in the proximal half of chromosome arm 2L to more precisely defined regions, defined by deficiency intervals, and, when possible, by individual complementation groups. To further analyze regions 36 and 39-40, we produced 11 new deficiencies with gamma irradiation, and we constructed 6 new deficiencies in region 30-33, using the DrosDel system. trans-heterozygous combinations of deficiencies revealed 5 additional functions, essential for viability or fertility.

Comparison of amplification methods for transcriptomic analyses of low abundance prokaryotic RNA sources

Microarrays have established as instrumental for bacterial detection, identification, and genotyping as well as for transcriptomic studies. For gene expression analyses using limited numbers of bacteria (derived from in vivo or ex vivo origin, for example), RNA amplification is often required prior to labeling and hybridization onto microarrays. Evaluation of the fidelity of the amplification methods is crucial for the robustness and reproducibility of microarray results. We report here the first utilization of random primers and the highly processive Phi29 phage polymerase to amplify material for transcription profiling analyses. We compared two commercial amplification methods (GenomiPhi and MessageAmp kits) with direct reverse-transcription as the reference method, focusing on the robustness of mRNA quantification using either microarrays or quantitative RT-PCR. Both amplification methods using either poly-A tailing followed by in vitro transcription, or direct strand displacement polymerase, showed appreciable linearity. Strand displacement technique was particularly affordable compared to in vitro transcription-based (IVT) amplification methods and consisted in a single tube reaction leading to high amplification yields. Real-time measurements using low-, medium-, and highly expressed genes revealed that this simple method provided linear amplification with equivalent results in terms of relative messenger abundance as those obtained by conventional direct reverse-transcription.

Analysis of nonlinear gain-induced effects on short-pulse amplification in doped fibers by use of an extended power equation

Optical pulse amplification in doped fibers is studied using an extended power transport equation for the coupled pulse spectral components. This equation includes the effects of gain saturation, gain dispersion, fiber dispersion, fiber nonlinearity, and amplified spontaneous emission. The new model is employed to study nonlinear gain-induced effects on the spectrotemporal characteristics of amplified subpicosecond pulses, in both the anomalous and the normal dispersion regimes.

Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes

BACKGROUND: Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS: We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS: We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS: We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Losses of chromosome arms 4q, 8p, 13q and gain of 8q are correlated with increasing chromosomal instability in hepatocellular carcinoma

OBJECTIVE: Chromosomal instability is a key feature in hepatocellular carcinoma (HCC). Array comparative genomic hybridization (aCGH) revealed recurring structural aberrations, whereas fluorescence in situ hybridization (FISH) indicated an increasing number of numerical aberrations in dedifferentiating HCC. Therefore, we examined whether there was a correlation between structural and numerical aberrations of chromosomal instability in HCC. METHODS AND RESULTS: 27 HCC (5 well, 10 moderately, 12 lower differentiated) already cytogenetically characterized by aCGH were analyzed. FISH analysis using probes for chromosomes 1, 3, 7, 8 and 17 revealed 1.46-4.24 signals/nucleus, which correlated with the histological grade (well vs. moderately,p < 0.0003; moderately vs. lower, p < 0.004). The number of chromosomes to each other was stable with exceptions only seen for chromosome 8. Loss of 4q and 13q, respectively, were correlated with the number of aberrations detected by aCGH (p < 0.001, p < 0.005; Mann-Whitney test). Loss of 4q and gain of 8q were correlated with an increasing number of numerical aberrations detected by FISH (p < 0.020, p < 0.031). Loss of 8p was correlated with the number of structural imbalances seen in aCGH (p < 0.048), but not with the number of numerical changes seen in FISH. CONCLUSION: We found that losses of 4q, 8p and 13q were closely correlated with an increasing number of aberrations detected by aCGH, whereas a loss of 4q and a gain of 8q were also observed in the context of polyploidization, the cytogenetic correlate of morphological dedifferentiation.

Aurora kinases as anticancer drug targets

The human aurora family of serine-threonine kinases comprises three members, which act in concert with many other proteins to control chromosome assembly and segregation during mitosis. Aurora dysfunction can cause aneuploidy, mitotic arrest, and cell death. Aurora kinases are strongly expressed in a broad range of cancer types. Aurora A expression in tumors is often associated with gene amplification, genetic instability, poor histologic differentiation, and poor prognosis. Aurora B is frequently expressed at high levels in a variety of tumors, often coincidently with aurora A, and expression level has also been associated with increased genetic instability and clinical outcome. Further, aurora kinase gene polymorphisms are associated with increased risk or early onset of cancer. The expression of aurora C in cancer is less well studied. In recent years, several small-molecule aurora kinase inhibitors have been developed that exhibit preclinical activity against a wide range of solid tumors. Preliminary clinical data from phase I trials have largely been consistent with cytostatic effects, with disease stabilization as the best response achieved in solid tumors. Objective responses have been noted in leukemia patients, although this might conceivably be due to inhibition of the Abl kinase. Current challenges include the optimization of drug administration, the identification of potential biomarkers of tumor sensitivity, and combination studies with cytotoxic drugs. Here, we summarize the most recent preclinical and clinical data and discuss new directions in the development of aurora kinase inhibitors as antineoplastic agents.

Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells

In this study, a time-course comparison of human articular chondrocytes (HAC) and bone marrow-derived mesenchymal stem cells (MSC) immunophenotype was performed in order to determine similarities/differences between both cell types during monolayer culture, and to identify HAC surface markers indicative of dedifferentiation. Our results show that dedifferentiated HAC can be distinguished from MSC by combining CD14, CD90, and CD105 expression, with dedifferentiated HAC being CD14+/CD90bright/CD105dim and MSC being CD14-/CD90dim/CD105bright. Surface markers on MSC showed little variation during the culture, whereas HAC showed upregulation of CD90, CD166, CD49c, CD44, CD10, CD26, CD49e, CD151, CD51/61, and CD81, and downregulation of CD49a, CD54, and CD14. Thus, dedifferentiated HAC appear as a bona fide cell population rather than a small population of MSC amplified during monolayer culture. While most of the HAC surface markers showed major changes at the beginning of the culture period (Passage 1-2), CD26 was upregulated and CD49a downregulated at later stages of the culture (Passage 3-4). To correlate changes in HAC surface markers with changes in extracellular matrix gene expression during monolayer culture, CD14 and CD90 mRNA levels were combined into a new differentiation index and compared with the established differentiation indices based on the ratios of mRNA levels of collagen type II to I (COL2/COL1) and of aggrecan to versican (AGG/VER). A correlation of CD14/CD90 ratio at the mRNA and protein level with the AGG/VER ratio during HAC dedifferentiation in monolayer culture validated CD14/CD90 as a new membrane and mRNA based HAC differentiation index.

Space trajectories optimization using variable-chromosome-length genetic algorithms

The problem of optimal design of a multi-gravity-assist space trajectories, with free number of deep space maneuvers (MGADSM) poses multi-modal cost functions. In the general form of the problem, the number of design variables is solution dependent. To handle global optimization problems where the number of design variables varies from one solution to another, two novel genetic-based techniques are introduced: hidden genes genetic algorithm (HGGA) and dynamic-size multiple population genetic algorithm (DSMPGA). In HGGA, a fixed length for the design variables is assigned for all solutions. Independent variables of each solution are divided into effective and ineffective (hidden) genes. Hidden genes are excluded in cost function evaluations. Full-length solutions undergo standard genetic operations. In DSMPGA, sub-populations of fixed size design spaces are randomly initialized. Standard genetic operations are carried out for a stage of generations. A new population is then created by reproduction from all members based on their relative fitness. The resulting sub-populations have different sizes from their initial sizes. The process repeats, leading to increasing the size of sub-populations of more fit solutions. Both techniques are applied to several MGADSM problems. They have the capability to determine the number of swing-bys, the planets to swing by, launch and arrival dates, and the number of deep space maneuvers as well as their locations, magnitudes, and directions in an optimal sense. The results show that solutions obtained using the developed tools match known solutions for complex case studies. The HGGA is also used to obtain the asteroids sequence and the mission structure in the global trajectory optimization competition (GTOC) problem. As an application of GA optimization to Earth orbits, the problem of visiting a set of ground sites within a constrained time frame is solved. The J2 perturbation and zonal coverage are considered to design repeated Sun-synchronous orbits. Finally, a new set of orbits, the repeated shadow track orbits (RSTO), is introduced. The orbit parameters are optimized such that the shadow of a spacecraft on the Earth visits the same locations periodically every desired number of days.

XIAP discriminates between type I and type II FAS-induced apoptosis

FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development. Distinct cell types differ in the mechanisms by which the 'death receptor' FAS triggers their apoptosis. In type I cells, such as lymphocytes, activation of 'effector caspases' by FAS-induced activation of caspase-8 suffices for cell killing, whereas in type II cells, including hepatocytes and pancreatic beta-cells, caspase cascade amplification through caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (BH3 interacting domain death agonist) is essential. Here we show that loss of XIAP (X-chromosome linked inhibitor of apoptosis protein) function by gene targeting or treatment with a second mitochondria-derived activator of caspases (SMAC, also called DIABLO; direct IAP-binding protein with low pI) mimetic drug in mice rendered hepatocytes and beta-cells independent of BID for FAS-induced apoptosis. These results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.