Gene mapping as a method for verifying sequence localization based on interspecific chromosome painting (ZOO-FISH).

The results obtained in the present study made it possible to place selected markers on the physical map of the arctic fox genome. With the use of fluorescence in situ hybridization (FISH) the GHR (3q24) and 1110 (1q21.1-21.2) genes and the FH2537 (5q11.3) microsatellite were localized on arctic fox chromosomes. The results confirmed previously proposed homologies using the ZOO-FISH technique, except for the 1110 gene. This suggests that the gene underwent a rearrangement (an inversion) that changed its localization compared to the dog.

The use of primed in situ synthesis (PRINS) to analyze nucleolar organizer regions (NORs) and telomeric DNA sequences in the domestic chicken genome.

One of the most often analyzed avian genomes is the domestic chicken genome (Gallus domesticus) whose diploid number is 2n = 78. In the chicken karyotype, similarly to other birds, there is a group of microchromosomes for which the determination of morphology and banding pattern is impossible using classic cytogenetics methods. The aim of this study was to evaluate telomeric and rDNA repetitive sequences in the chicken genome by the PRINS technique as an alternative method to fluorescence in situ hybridization. This is the first report on the application of the PRINS method to locate these repetitive sequences in the chicken nuclei and metaphase chromosomes.

Polymorphism of cytogenetic markers in wild and farm red fox (Vulpes vulpes) populations.

Analysis of the origin of domestic animals is of wide interest and has many practical applications in areas such as agriculture and evolutionary biology. Identification of an ancestor and comparison with the domesticated form allows for an analysis of genetic, physiological, morphological and behavioral effects of domestication. Because fox breeding has been an ongoing process for over a century, differences are expected between farm and wild populations at the chromosomal level. The aim of this work was to analyse polymorphisms at the chromosomal level in foxes raised on farms and those living in the wild. Blood samples and lung tissue served as the experimental material and were obtained after slaughter of 35 foxes, including 28 breeding animals and 7 wild animals. The classical cytogenetic method was used including AgNOR technique, as well as molecular methods such as fluorescence in situ hybridization (FISH), and primed in situ labeling (PRINS). Analysis of the number of B chromosomes showed the presence of polymorphisms in foxes from both studied populations, but there was no correlation between the number of B chromosomes and the origin and gender of particular animals. An analysis ofactive nucleolar organizers showed the presence of a large number of polymorphisms and a tendency towards reduction of the number of NORs in the captive-raised population.

Polymorphism of the prion protein gene (PRNP) in Polish cattle affected by classical bovine spongiform encephalopathy.

Recent attempts to discover genetic factors affecting cattle resistance/susceptibility to bovine spongiform encephalopathy (BSE) have led to the identification of two insertion/deletion (indel) polymorphisms, located within the promoter and intron 1 of the prion protein gene PRNP, showing a significant association with the occurrence of classical form of the disease. Because the effect of the polymorphisms was studied only in few populations, in this study we investigated whether previously described association of PRNP indel polymorphisms with BSE susceptibility in cattle is also present in Polish cattle population. We found a significant relation between the investigated PRNP indel polymorphisms (23 and 12 bp indels), and susceptibility of Polish Holstein-Friesian cattle to classical BSE (P < 0.05). The deletion variants of both polymorphisms were related to increased susceptibility, whereas insertion variants were protective against BSE.

Characteristics of X- and Y-chromosome specific regions of the amelogenin gene and a PCR-based method for sex identification in red deer (Cervus elaphus).

The present study attempts to analyse sequences of the X- and Y-chromosome specific regions of the amelogenin (AMEL) gene in red deer. To this end, primers specific for each form of the gene (AMELX and AMELY) were designed based on bovine genomic sequences and the homologous regions of the genes were sequenced. The obtained sequence of AMELX gene showed high similarity with the corresponding region in cattle (91%) and humans (77%), but this similarity was slightly lower among AMELY genes and showed 87 and 73% of identical nucleotides, respectively. In addition, three single nucleotide polymorphisms (SNPs) were found in the AMELX gene of the female red deer investigated. Comparative analysis of the homologous fragments of the red deer AMELX and AMELY genes confirmed the deletion of an AMELY gene fragment in relation to AMELX. Homology of both sequences was 82% of identical nucleotides in the coding region and 74% in 3' non-coding sequence. The sequences studied showed considerable similarity to homologous fragments of the human and bovine gene, but the structural differences observed lead us to design PCR-based method for sex identification in red deer, based on the presented sequences.

FISH mapping of six genes responsible for development of the nervous and skeletal systems on donkey (Equus asinus) chromosomes.

The results obtained in the present study made it possible to place selected markers responsible for development of the nervous and skeletal systems on the physical map of the donkey genome. Fluorescence in situ hybridization (FISH) was used to localize genes such as GDF5 (15q13), FRZB (4q23.1), TWIST (1q31), PAX6 (20q25), SALL1 (24q15) and SHH (1q35) on donkey chromosomes. The identification of their localization confirmed previously proposed homologies using ZOO-FISH technique, except for FRZB and SALL1 genes. This suggests that they were affected by rearrangements that changed their localization compared to horse, and in the case of the SALL1 gene also compared to human.

PRINS detection of 18S rDNA in pig, red fox and Chinese raccoon dog, and centromere DNA in horse.

The fluorescence in situ hybridization (FISH) technique is widely used in animal cytogenetics. Contrary to FISH procedure, primed in situ DNA synthesis (PRINS) does not require the DNA probe preparation (design, synthesis, gel purification of PCR products and labeling). The PRINS method with primers used as 'DNA probes' is both PCR-sensitive and allows for chromosomal localization of DNA sequences. Here, we show the application of PRINS reaction with one unlabeled oligonucleotide pair to identify 18S rDNA loci in three different animal species: domestic pig (Sus scrofa), red fox (Vulpes vulpes) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides). We present the data of indirect labeling with the digoxigenin-PRINS using two different pairs of primers complementary to centromeric region of horse (Equus caballus) chromosomes. Our new PRINS application may be considered as a useful tool for chromosome investigation in the field of domestic and wild animal genetics and evolution.

Use of cytochrome b polymorphism for species identification of biological material derived from cattle, sheep, goats, roe deer and red deer.

The objective of this study was species identification of the following biological trace material: skin, blood stains, meat samples and jawbone with a tooth, which were the subject of expert opinion ordered by a court. The expert appraisement was conducted by an analysis of a cytochrome b fragment. The choice of mtDNA fragment for analysis was based on its conservation in mammals which enabled several farm and wild species to be identified with one pair of primers. The PCR product was differentiated by Tsp509I and Alulenzymes. Due to problems with amplification of roe deer DNA, primers specific to this species only, flanking a cytochrome b fragment (Y1495 1.1), were designed. On the basis of this analysis, it was concluded that the skin sample was derived from a goat, dried blood from a roe deer, the jawbone from cattle, and two meat samples from a roe deer and red deer. This method allowed rapid and efficient identification of several species of mammals using diverse biological material.

Cytogenetic analysis of meiotic cells obtained from stallion testes.

A normal course of meiosis and the associated course of spermatogenesis in males are very significant from the viewpoint of animal breeding, in particular animal reproduction. This takes on special significance when studying late-maturing animals such as horses. The aim of the study was to analyse meiotic cells, with particular consideration of synaptonemal complexes obtained from the testes of young stallions and cryptorchids, based on observations of the X-Y bivalent. The analysis was performed in successive stages of meiotic division using the FISH technique. The greatest diversity and most advanced meiotic stages were observed in the normal testis of a unilateral cryptorchid. No abnormalities were observed that could have caused cryptorchidism in the analysed horses.

Rapid detection of yeast rRNA genes with primed in situ (PRINS) labeling.

In yeast, rRNA genes can be detected with the FISH technique using rRNA gene probes. This technique yields reliable, reproducible and precise results, but is time-consuming. Here, the primed in situ DNA synthesis (PRINS) procedure has been optimized for rapid detection of yeast rRNA genes. PRINS, which is as sensitive as PCR and allows cytological localization of analyzed sequences, can be adapted for various screening tests requiring fast labeling of rRNA genes.

Evaluation of the cyto- and genotoxic activity of yerba mate (Ilex paraguariensis) in human lymphocytes in vitro.

Despite its antioxidant capacity and well-known health benefits, yerba mate tea (Ilex paraguariensis) has been shown to possess some genotoxic and mutagenic activities and to increase incidence of some types of cancer. The aim of this study was to estimate the cyto- and genotoxicity of mate tea in human peripheral lymphocytes in vitro. We found that yerba mate extract induced a concentration-dependent, statistically significant increase in the level of apoptotic and necrotic cells and a decrease in the nuclear division index (NDI). Mate-exposed lymphocytes had a reduced transcriptional rDNA activity, which may be due to the stress conditions, and showed an elevated production of micronuclei. The FISH technique revealed the appearance of an acrocentric signal in mate-induced micronuclei, which suggests that under these conditions yerba mate extract may display aneugenic activity. Since caffeine is one of the most abundant compounds found in the dry mass of mate, we conducted additional experiments with caffeine alone. We showed that caffeine used at the same concentrations manifests a more potent cyto- and genotoxic effect that may account, at least in part, for the disadvantageous effects observed for yerba mate extract.

Identification of chromosome abnormalities in the horse using a panel of chromosome-specific painting probes generated by microdissection.

Fluorescent in situ hybridisation (FISH) using a panel of molecular probes for all chromosome pairs obtained by chromosome microdissection of the domestic horse ( Equus caballus ) was used to diagnose karyotype abnormalities in 35 horses (32 mares, 2 stallions and 1 intersex), which were selected for the study due to infertility (23 horses), reduced fertility (10 horses) and developmental anomalies (2 horses). The use of the FISH technique with probes for each horse chromosome pair enabled the diagnosis of many different chromosome aberrations in this population. Among the horses analysed, 21 animals had normal karyotype - 64,XX (19 mares) and 64,XY (2 stallions). Fourteen animals, constituting 40% of the population studied, showed the following chromosome abnormalities: 63,X (1 mare); 63,X/64,XX (6 mares); 63,X/64,XX/65,XXX (3 mares); 63,X/65,XXX (1 mare); 64,XX/65,XX+Xp (1 mare); 63,X/64,XX/65,XX+Xq (1 mare), and 63,X/64,XX/65,XX+delY (1 intersex). When only the mares studied because of complete infertility were taken into consideration, this proportion exceeded 56%. Due to the increased frequency of the above-mentioned aberrations in the mosaic form of two or more lines, it was necessary to analyse a large number (100-300) of metaphase spreads. The use of specific molecular probes obtained by chromosome microdissection made these diagnoses much easier.

Optimalization of fluorescence in situ hybridization conditions in mare oocytes and mouse embryos.

The aim of the study was to optimize hybridization conditions of molecular probes specific for X sex chromosomes of the domestic horse in mare oocyte chromosomes. Mare oocytes, recovered from slaughterhouse ovaries by scraping the granulosa layer, were cultured in vitro. Metaphase II mature oocytes were treated with hypotonic solution and fixed, followed by hybridization of the molecular probe specific for the X chromosome ofthe domestic horse. Hybridization of probes specific for mouse heterosomes on mouse oocytes and early embryos was performed to verify the FISH technique. Of 438 oocytes analysed, 29% reached metaphase II. Despite many changes in the composition of hypotonic solutions and modification of the FISH protocol, the fluorescence signal was observed in mouse oocytes and embryos but not in mare oocytes.

Chromosomal homology between the human and the bovine DMRT1 genes.

Doublesex and mab-3 related transcription factor 1 (DMRT1) is considered to be the most conserved gene among loci involved in the molecular pathways of animal sexual development. In the majority of the extensively examined vertebrates, its function is limited to the upstream or downstream testis regulators acting during embryogenesis. Our present study demonstrated the structural homology between DMRT1 orthologos in human and cattle. A BAC clone with a specific bovine sequence of the gene was used in the FISH mapping experiments. The physical localization of DMRT1 in cattle (BTA 8q17) was determined and its homology to the human locus was shown (HSA 9p24.3). Furthermore, another BAC probe, containing the sequence of the human homologue (pBACe3.6), generated hybridisation signals on bovine metaphase chromosomes and indicated the physical location of the autosomal bovine DMRT1 locus. Further investigations of the gene in domestic animals might provide more support for its conservative status and may help in understanding the molecular mechanisms involved in the occurrence of sexual abnormalities often diagnosed in livestock.

The nitroxide antioxidant Tempol affects metal-induced cyto- and genotoxicity in human lymphocytes in vitro.

Stable, membrane-permeating nitroxide radicals have been reported to possess antioxidant activity in various experimental systems while, in parallel, they have been considered to be evidently harmful oxidants. The aim of this study was to evaluate the role of the piperidine nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) in the modulation of cyto- and genotoxicity in human lymphocytes in vitro by cadmium and chromium, which depend, at least in part, on formation of reactive oxygen species. The cytokinesis-block micronucleus (CBMN) assay to measure micronucleus (MN) formation, the nuclear division index (NDI) and the percentage of apoptotic and necrotic cells were assessed after exposure human lymphocytes to Cd(II), Cr(III) and Cr(VI) or co-incubation with these metals and Tempol. We found a significant ability of 5-50 microM Tempol to diminish toxic effects of the agents tested. In every system studied, Tempol decreased the micronucleus frequency and the percentage of apoptotic and necrotic cells, while it increased the nuclear division index (p<0.05). We observed adverse effects when 0.1-1 mM Tempol alone was used: inhibition of cell growth, induction of apoptotic and necrotic cell death and chromosomal damage (p<0.05). Collectively, we demonstrated that Tempol can be considered as a potent anti-apoptotic and antigenotoxic agent, but also as a cytotoxic and clastogenic chemical, depending on the concentration applied.

Oxidant-induced decrease of the expression of nucleolar organizer regions in pig lymphocytes can be useful for monitoring the cellular effects of oxidative stress.

The technique of silver staining of nucleolar organizer regions (AgNORs) was chosen to estimate the transcriptionally active metaphase and interphase nucleolar organizer regions (NORs) in pig peripheral blood lymphocytes exposed to oxidative agents in vitro. The quantitative analysis of AgNORs was performed by using the counting method and the morphometric method. We found that hydrogen peroxide and 2,2'-azobis-(2-amidinopropane)-dihydrochloride (AAPH) induced a concentration-dependent decrease in NORs activity - in the case of metaphase the NORs activity was exclusively seen on chromosome pairs 8 - which can be considered as another estimate of cellular oxidative stress. Moreover, biomarkers of cyto- and genotoxicity, such as the percentage of apoptotic and necrotic cells, the nuclear division index (NDI) and formation of micronuclei (MN) were used to measure harmful effects provoked by the agents tested.

Microdissected bovine X chromosome segment delineates homologous chromosomal regions in sheep and goat.

In this study we microdissected the proximal part band Xq24 of the bovine X chromosome long arm, in which the Xist gene is located in cattle. The obtained DNA fragment was PCR amplified, labelled and used as a probe for cattle, sheep and goat chromosomes. In cattle, as expected, distinct hybridization signals were observed on Xq24. The painting signals were also observed on Xq24 in sheep and goat. The chromosome painting probes can be used for comparative mapping and searching for internal X chromosome rearrangements in Bovidea and may contribute to the understanding of mammalian sex chromosome evolution.

A new case of reciprocal translocation t(10;13)(q16;q21) diagnosed in an AI boar.

A new case of reciprocal translocation t(10;13)(q16;q21) was detected in a hybrid boar (Large White x Pietrain x Duroc x Hampshire) from an artificial insemination (AI) station. Altogether, 258 sires of 4 pure breeds as well as hybrid lines and crossbreeds were investigated. The diagnosis was based on classical cytogenetic examination following the standard protocols of lymphocyte cultures, Giemsa staining and G-, C- and Ag-I banding techniques. The population screening performed was an initial part of a long-term karyotype control system of boars kept at AI stations, which was started by the National Research Institute of Animal Production in Poland in 2007.

Application of arm-specific painting probes of horse X chromosome for karyotype analysis in an infertile Hutsul mare with 64,XX/65,XX+Xp karyotype: case report.

A 5-year-old infertile Hutsul mare was subjected to cytogenetic analysis. Fluorescence in situ hybridisation (FISH) using the equine Xp and Xq chromosome painting probes was carried out on chromosome preparations obtained after blood lymphocyte culture. These probes were generated by chromosome microdissection and a large number of spreads was analysed (525). The karyotype formula of the analysed mare was 64,XX/65,XX+Xp with the ratio of the two lines being 99.4 and 0.6, respectively. The goal of the study was to apply chromosome microdissection and the FISH technique for cytogenetic diagnostics.

Detection of equine X chromosome mosaicism in a mare using an equine X whole chromosome painting probe (WCPP)--a case report.

An infertile mare with hypoplastic ovaries was subjected to cytogenetic analysis. Fluorescence in situ hybridisation (FISH) using the equine X whole chromosome painting probe (WCPP) was carried out on a chromosome preparation obtained from blood lymphocyte culture. The number of analysed spreads was high (235) and in the X chromosome aneuploidy in mosaic form was diagnosed. The karyotype formula was 63,X / 64,XX / 65,XXX. The ratio of the three lines was 15%, 82% and 3%, respectively. The application of the FISH technique with WCPP is discussed.