Breakpoint analysis of human chromosome 3 inversions during hominoid evolution

Comparative fluorescence in situ hybridization (FISH) mapping revealed four large DNA segments which have been conserved in their entirety between human chromosome 3 and Bornean orangutan chromosome 2 as well as three evolutionary breakpoints which distinguish between the human and Bornean orangutan chromosome forms. Examination of the structural and functional features of evolutionary breakpoints provides new insights into the possible effects of evolutionary rearrangements on genome function and the relationship between human chromosome pathology and evolution. FISH of human BAC clones which were assesssed in human genomic sequence to primate chromosomes, combined with precise breakpoint localizations by polymerase chain reaction (PCR) analysis of flow-sorted chromosomes and in silico analysis, were used to characterize the evolutionary breakpoints. None of the three breakpoints studied disrupts a validated gene(s), however they are all associated with segmental duplications. At least eleven DNA segments (&a

In-vitro synthesis and reconstitution of cytochrome bo 3 ubiquinol oxidase in artificial membranes

Forschung über Membranenproteine stellt strenge Hindernisse, seit ruhigem gerade wenige Beispiele der Membranenproteinsorten sind gekennzeichnet worden in den verwendbaren experimentellen Plattformen gegenüber. Die Hauptherausforderung ist, ihre ausgezeichnete entworfene strukturelle Vollständigkeit zu konservieren, während die Ausdruck-, Lokalisierungs- und Wiederherstellungprozesse auftreten. In-vitro übersetzungssysteme können Vorteile über auf Zellenbasisgenausdruck zum Beispiel haben, wenn das über-ausgedrückte Produkt zur Wirtszelle giftig ist oder wenn fehlende Pfosten-Übersetzungsänderung in den bakteriellen Ausdrucksystemen die Funktionalität der Säugetier- Proteine oder Mangel an vorhandenem Membranenraum verdirbt, Funktionsausdruck verbieten.rn Der Nachahmer von biologische Membranen wie feste gestützte Lipidmembranen sind als Plattform am meisten benutzt, Proteinmembraneninteraktionen nachzuforschen. Wir sind in der Lage, Membranenproteinsorte, da wir eine Plattform für Membranenproteinsynthese vorstellen, nämlich die in-vitrosynthese der Membranenproteine in ein Peptid gestütztes Membranensystem zu adressieren. Die Wiederherstellung der Membranenproteine in den Lipid bilayers resultiert im Allgemeinen mit verschiedenen Proteinanpassungen. Als Alternative erforschen wir dieses System zum ersten Mal, um genaueres Modell zu den zellularen Membranen zu verursachen und ihre Funktion, wie Proteineinfügung, Proteinfunktion und Ligandinteraktionen nachzuahmen.rn In dieser Arbeit ist unser Ziel, komplizierte Transmembraneproteine, wie des Cytochrome bo3-ubiquinol Oxydase (Cyt-bo3) direkt innerhalb der biomimetic vorbildlichen Membrane zu synthetisieren. In unserem System wird festes gestütztes tBLM wie, P19/DMPE/PC als Plattform benutzt. Dieses künstliche Membranensystem mimiks die amphiphile Architektur eines Zelle-abgeleiteten Membranensystems.rn

A high-resolution comparative radiation hybrid map of equine chromosome 4q12-q22

In this study, we present a comprehensive 5000-rad radiation hybrid map of a 40-cM region on equine chromosome 4 (ECA4) that contains quantitative trait loci for equine osteochondrosis. We mapped 29 gene-associated sequence tagged site markers using primers designed from equine expressed sequence tags or BAC clones in the ECA4q12-q22 region. Three blocks of conserved synteny, showing two chromosomal breakpoints, were identified in the segment of ECA4q12-q22. Markers from other segments of HSA7q mapped to ECA13p and ECA4p, and a region of HSA7p was homologous to ECA13p. Therefore, we have improved the resolution of the human-equine comparative map, which allows the identification of candidate genes underlying traits of interest.

Molecular characterization of the porcine deleted in malignant brain tumors 1 gene (DMBT1)

The human gene deleted in malignant brain tumors 1 (DMBT1) is considered to play a role in tumorigenesis and pathogen defense. It encodes a protein with multiple scavenger receptor cysteine-rich (SRCR) domains, which are involved in recognition and binding of a broad spectrum of bacterial pathogens. The SRCR domains are encoded by highly homologous repetitive exons, whose number in humans may vary from 8 to 13 due to genetic polymorphism. Here, we characterized the porcine DMBT1 gene on the mRNA and genomic level. We assembled a 4.5 kb porcine DMBT1 cDNA sequence from RT-PCR amplified seminal vesicle RNA. The porcine DMBT1 cDNA contains an open reading frame of 4050 nt. The transcript gives rise to a putative polypeptide of 1349 amino acids with a calculated mass of 147.9 kDa. Compared to human DMBT1, it contains only four N-terminal SRCR domains. Northern blotting revealed transcripts of approximately 4.7 kb in size in the tissues analyzed. Analysis of ESTs suggested the existence of secreted and transmembrane variants. The porcine DMBT1 gene spans about 54 kb on chromosome 14q28-q29. In contrast to the characterized cDNA, the genomic BAC clone only contained 3 exons coding for N-terminal SRCR domains. In different mammalian DMBT1 orthologs large interspecific differences in the number of SRCR exons and utilization of the transmembrane exon exist. Our data suggest that the porcine DMBT1 gene may share with the human DMBT1 gene additional intraspecific variations in the number of SRCR-coding exons.

A human-horse comparative map based on equine BAC end sequences

In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E BAC clones to the human genome, on average one corresponding equine BAC clone per megabase of human DNA. We used the BLASTN anchored BESs for an in silico prediction of the gene content and chromosome assignment of comparatively mapped equine BAC clones. As a first verification of our in silico mapping strategy we placed 19 equine BESs with matches to HSA6 onto the RH map. All markers were assigned to the predicted localizations on ECA10, ECA20, and ECA31, respectively.

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.

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.

Phylogeny of horse chromosome 5q in the genus Equus and centromere repositioning

Horses, asses and zebras belong to the genus Equus and are the only extant species of the family Equidae in the order Perissodactyla. In a previous work we demonstrated that a key factor in the rapid karyotypic evolution of this genus was evolutionary centromere repositioning, that is, the shift of the centromeric function to a new position without alteration of the order of markers along the chromosome. In search of previously undiscovered evolutionarily new centromeres, we traced the phylogeny of horse chromosome 5, analyzing the order of BAC markers, derived from a horse genomic library, in 7 Equus species (E. caballus, E. hemionus onager, E. kiang, E. asinus, E. grevyi, E. burchelli and E. zebra hartmannae). This analysis showed that repositioned centromeres are present in E. asinus (domestic donkey, EAS) chromosome 16 and in E. burchelli (Burchell's zebra, EBU) chromosome 17, confirming that centromere repositioning is a strikingly frequent phenomenon in this genus. The observation that the neocentromeres in EAS16 and EBU17 are in the same chromosomal position suggests that they may derive from the same event and therefore, E. asinus and E. burchelli may be more closely related than previously proposed; alternatively, 2 centromere repositioning events, involving the same chromosomal region, may have occurred independently in different lineages, pointing to the possible existence of hot spots for neocentromere formation. Our comparative analysis also showed that, while E. caballus chromosome 5 seems to represent the ancestral configuration, centric fission followed by independent fusion events gave rise to 3 different submetacentric chromosomes in other Equus lineages.

The bovine dilated cardiomyopathy locus maps to a 1.0-Mb interval on chromosome 18

Cardiomyopathies are myocardial diseases that lead to cardiac dysfunction, heart failure, arrhythmia, and sudden death. In human medicine, cardiomyopathies frequently warrant heart transplantation in children and adults. Bovine dilated cardiomyopathy (BDCMP) is a heart muscle disorder that has been observed during the last 30 years in cattle of Holstein-Friesian origin. In Switzerland BDCMP affects Swiss Fleckvieh and Red Holstein breeds. BDCMP is characterized by a cardiac enlargement with ventricular remodeling and chamber dilatation. The common symptoms in affected animals are subacute subcutaneous edema, congestion of the jugular veins, and tachycardia with gallop rhythm. A cardiomegaly with dilatation and hypertrophy of all heart chambers, myocardial degeneration, and fibrosis are typical postmortem findings. It was shown that all BDCMP cases reported worldwide traced back to a red factor-carrying Holstein-Friesian bull, ABC Reflection Sovereign. An autosomal recessive mode of inheritance was proposed for BDCMP. Recently, the disease locus was mapped to a 6.7-Mb interval MSBDCMP06-BMS2785 on bovine Chr 18 (BTA18). In the present study the BDCMP locus was fine mapped by using a combined strategy of homozygosity mapping and association study. A BAC contig of 2.9 Mb encompassing the crucial interval was constructed to establish the correct marker order on BTA18. We show that the disease locus is located in a gene-rich interval of 1.0 Mb and is flanked by the microsatellite markers DIK3006 and MSBDCMP51.

Effect of photoactivated disinfection with a light-emitting diode on bacterial species and biofilms associated with periodontitis and peri-implantitis

BACKGROUND To determine the effect of photoactivated disinfection (PAD) using toluidine blue and a light-emitting diode (LED) in the red spectrum (wave length at 625-635 nm) on species associated with periodontitis and peri-implantitis and bacteria within a periodontopathic biofilm. METHODS Sixteen single microbial species including 2 Porphyromonas gingivalis and 2 Aggregatibacter actinomycetemcomitans and a multispecies mixture consisting of 12 species suspended in saline without and with 25% human serum were exposed to PAD. Moreover, single-species biofilms consisting of 2 P. gingivalis and 2 A. actinomycetemcomitans strains and a multi-species biofilm on 24-well-plates, grown on titanium discs and in artificial periodontal pockets were exposed to PAD with and without pretreatment with 0.25% hydrogen peroxide. Changes in the viability were determined by counting the colony forming units (cfu). RESULTS PAD reduced the cfu counts in saline by 1.42 log₁₀ after LED application for 30s and by 1.99 log₁₀ after LED application for 60s compared with negative controls (each p<0.001). Serum did not inhibit the efficacy of PAD. PAD reduced statistically significantly (p<0.05) the cfu counts of the P. gingivalis biofilms. The viability of the A. actinomycetemcomitans biofilms and the multi-species biofilms was statistically significantly decreased when PAD was applied after a pretreatment with 0.25% hydrogen peroxide. The biofilm formed in artificial pockets was more sensitive to PAD with and without pretreatment with hydrogen peroxide compared with those formed on titanium discs. CONCLUSIONS PAD using a LED was effective against periodontopathic bacterial species and reduced viability in biofilms but was not able to completely destroy complex biofilms. The use of PAD following pretreatment with hydrogen peroxide resulted in an additional increase in the antimicrobial activity which may represent a new alternative to treat periodontal and peri-implant infections thus warranting further testing in clinical studies.

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance

SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

SNARE-fusion mediated inserton of membrane proteins into native and artificial membranes

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE-protein based method for incorporation of multiple membrane proteins into membranes, and for delivery of large water-soluble substrates into closed membrane vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0-ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

A comparative radiation hybrid map of sheep chromosome 10

Comparative radiation hybrid (RH) maps of individual ovine chromosomes are essential to identify genes governing traits of economic importance in sheep, a livestock species for which whole genome sequence data are not yet available. The USUoRH5000 radiation hybrid panel was used to generate a RH map of sheep chromosome 10 (OAR10) with 59 markers that span 1,422 cR over an estimated 92 Mb of the chromosome, thus providing markers every 2 Mb (equivalent to every 24 cR). The markers were derived from 46 BAC end sequences (BESs), a single EST, and 12 microsatellites. Comparative analysis showed that OAR10 shares remarkable conservation of gene order along the entire length of cattle chromosome 12 and that OAR10 contains four major homologous synteny blocks, each related to segments of the homologous human chromosome 13. Extending the comparison to the horse, dog, mouse, and chicken genome showed that these blocks share conserved synteny across species.

Defying death: Cellular survival strategies following plasmalemmal injury by bacterial toxins

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca(2+) and an efflux of cytoplasmic constituents. In order to ensure survival, the cell needs to identify, plug and remove lesions from its membrane. Quarantined by membrane folds and isolated by membrane fusion, the pores are removed from the plasmalemma and expelled into the extracellular space. Outward vesiculation and microparticle shedding seem to be the strategies of choice to eliminate toxin-perforated membrane regions from the plasmalemma of host cells. Depending on the cell type and the nature of injury, the membrane lesion can also be taken up by endocytosis and degraded internally. Host cells make excellent use of an initial, moderate rise in intracellular [Ca(2+)], which triggers containment of the toxin-inflicted damage and resealing of the damaged plasmalemma. Additional Ca(2+)-dependent defensive cellular actions range from the release of effector molecules in order to warn neighbouring cells, to the activation of caspases for the initiation of apoptosis in order to eliminate heavily damaged, dysregulated cells. Injury to the plasmalemma by bacterial toxins can be prevented by the early sequestration of bacterial toxins. Artificial liposomes can act as a decoy system preferentially binding and neutralizing bacterial toxins.