Hybridization and introgression can impact the evolution of natural populations. Several wild canid species hybridize in nature, sometimes originating new taxa. However, hybridization with free-ranging dogs is threatening the genetic integrity of grey wolf populations (Canis lupus), or even the survival of endangered species (e.g., the Ethiopian wolf C. simensis). Efficient molecular tools to assess hybridization rates are essential in wolf conservation strategies. We evaluated the power of biparental and uniparental markers (39 autosomal and 4 Y-linked microsatellites, a melanistic deletion at the β-defensin CBD103 gene, the hypervariable domain of the mtDNA control-region) to identify the multilocus admixture patterns in wolf x dog hybrids. We used empirical data from 2 hybrid groups with different histories: 30 presumptive natural hybrids from Italy and 73 Czechoslovakian wolfdogs of known hybrid origin, as well as simulated data. We assessed the efficiency of various marker combinations and reference samples in admixture analyses using 69 dogs of different breeds and 99 wolves from Italy, Balkans and Carpathian Mountains. Results confirmed the occurrence of hybrids in Italy, some of them showing anomalous phenotypic traits and exogenous mtDNA or Y-chromosome introgression. Hybridization was mostly attributable to village dogs and not strictly patrilineal. The melanistic β-defensin deletion was found only in Italian dogs and in putative hybrids. The 24 most divergent microsatellites (largest wolf-dog FST values) were equally or more informative than the entire panel of 39 loci. A smaller panel of 12 microsatellites increased risks to identify false admixed individuals. The frequency of F1 and F2 was lower than backcrosses or introgressed individuals, suggesting hybridization already occurred some generations in the past, during early phases of wolf expansion from their historical core areas. Empirical and simulated data indicated the identification of the past generation backcrosses is always uncertain, and a larger number of ancestry-informative markers is needed.

• MeSH
• beta-defensiny genetika   MeSH
• chromozom Y MeSH
• genetická variace MeSH
• genetické markery * MeSH
• genotyp MeSH
• hybridizace genetická * MeSH
• mikrosatelitní repetice MeSH
• mitochondriální DNA MeSH
• molekulární evoluce MeSH
• multilokusová sekvenční typizace * MeSH
• populační genetika MeSH
• psi MeSH
• shluková analýza MeSH
• vlci MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Itálie MeSH
• Názvy látek
• beta-defensiny MeSH
• genetické markery * MeSH
• mitochondriální DNA MeSH

BACKGROUND: There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and β-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories. FINDINGS: In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech) of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations. CONCLUSIONS: QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number.

• MeSH
• beta-defensiny genetika   MeSH
• genetická predispozice k nemoci MeSH
• genom lidský genetika   MeSH
• genová dávka * MeSH
• kohortové studie MeSH
• lidé MeSH
• mapování chromozomů metody   MeSH
• molekulární sekvence - údaje MeSH
• populace MeSH
• populační genetika metody   MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Geografické názvy
• Česká republika MeSH
• Dánsko MeSH
• Ghana MeSH
• Portugalsko MeSH
• Spojené království MeSH
• Názvy látek
• beta-defensiny MeSH

Studies of the human defensins have been hampered by the lack of a simple expression system allowing for rapid production of functional peptide forms. Here, we describe a Saccharomyces cerevisiae AH22 expression system that meets that condition. The 42 amino acid form of human beta-defensin-1 was expressed under the control of the ADH1 promoter. The optimum conditions for expression were determined and the stable maintenance of the pVT103L-hBD-1 chimeric vector in the yeast population was confirmed. Expressed hBD-1 was secreted into the medium (approximately 55 microg l(-1)) and purified using cation-exchange chromatography. Isolated defensin exhibited strong bactericidal effect on Escherichia coli ML-35p. We conclude that the expression system described here will be a useful tool where readily prepared and active forms of the human defensins are needed.

• MeSH
• antibakteriální látky biosyntéza   izolace a purifikace   farmakologie   MeSH
• beta-defensiny biosyntéza   chemie   genetika   farmakologie   MeSH
• Escherichia coli cytologie   účinky léků   MeSH
• genetické vektory genetika   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• proliferace buněk účinky léků   MeSH
• rekombinantní proteiny biosyntéza   chemie   izolace a purifikace   farmakologie   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-defensiny MeSH
• rekombinantní proteiny MeSH

In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with isolation procedures and toxicity caused by rhBD-1 to the host organism. As a result, the majority of fusion protein (LMM-rhBD-1(43)) was obtained in the soluble state, isolated by a low salt-high salt treatment of total cell protein. The rhBD-1(43) was cleaved from the fusion with Protease 4 and purified on CM Sepharose Fast Flow column with the yield of approximately 1 mg rhBD-1(43) from 6 g of wet weight cells. Purified rhBD-1(43) showed antimicrobial activity against E. coli ML-35p at a concentration of 129 microM. The procedure of rhBD-1 expression and purification we present can provide a reliable and simple method for production of different cationic peptides for biological studies.

• MeSH
• antiinfekční látky * izolace a purifikace   metabolismus   MeSH
• beta-defensiny * genetika   izolace a purifikace   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• králíci MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární sekvence - údaje MeSH
• myosinové subfragmenty * genetika   izolace a purifikace   metabolismus   MeSH
• peptidové fragmenty genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny * genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiinfekční látky * MeSH
• beta-defensiny * MeSH
• DEFB1 protein, human MeSH Prohlížeč
• myosinové subfragmenty * MeSH
• peptidové fragmenty MeSH
• rekombinantní fúzní proteiny * MeSH