Dual-Color High-Resolution Fiber-FISH Analysis on Lethal White Syndrome Carriers in Sheep

Abstract

Molecular defects occurring in the endothelin receptor type-B (EDNRB) gene are known to be associated with pigmentary anomalies and intestinal aganglionosis in humans, rodents and horses. We carried out a cytogenetic investigation in 2 ewes heterozygous for the deletion of the EDNRB gene and in 2 more females as control. The RBA-banding showed that all 4 ewes were karyologically normal. EDNRB gene-specific probes were produced by PCR and cloning. The application of the R-banding and propidium iodide-staining fluorescent in situ hybridization allowed mapping the gene to OAR 10q22 and confirmed the heterozygous status of the ewes investigated for the EDNRB gene deletion. For the fine estimation of the gene length in sheep and for the correct sizing of the chromosomal gap, a dual-color FISH was applied to high-resolution DNA fibers in combination with digital imaging microscopy. The comparison of the DNA fiber barcodes indicated a chromosomal deletion larger than the EDNRB gene itself. The length of the gene, not known for sheep until now, was estimated to be ∼21 kb, whereas the microchromosomal deletion was ∼100 kb. EDNRB is located in a chromosomal region previously shown to be a fragile site. The applied method allowed locating the potential breakpoints, thus permitting further interesting prospective investigations also in the field of the fragile sites in sheep.

Casein Gene Cluster in Camelids: Comparative Genome Analysis and New Findings on Haplotype Variability and Physical Mapping

“…The presence of repetitive DNA within casein genes in dromedary was already evidenced in CSN2 (Pauciullo et al, 2014) and CSN3 genes (Pauciullo et al, 2013b). In these studies, LINEs belonging to L1MA family were found to be species specific in comparison to cattle.…”

“…The casein gene probes were prepared by PCR amplification and cloning of five DNA fragments spread over the casein genes (primers are provided in Table 1 ) according to the method described by Pauciullo et al (2013b). Labeling was carried out by standard nick translation reactions (Roche, Germany) using biotin-16-dUTP (Roche) as modified nucleotide.…”

“…RPBI-FISH was performed according to Pauciullo et al (2013b) and Pauciullo et al (2016) with minor modifications. Briefly, 500 ng of labeled DNA from each of the nick translation reactions were combined and mixed together with competitor DNA.…”

“…A total of 30 randomly selected metaphase cells were examined per each alpaca to ensure the reliability of the probe signals by FISH. The hybridization efficiency was calculated as follows: FISH efficiency (%) is equal to the number of cells with hybridization signals present at the 2q21 region of both chromosomes 2 divided by the number of cells examined (Pauciullo et al, 2013b).…”

A de novo reciprocal chromosomal translocation t(3;6)(p14;q26) in the black Lucano pig

“…However, the variability of chromosome preparations, and limited banding resolution, makes the clarification of certain genetic events difficult, including the precise localization of rcp breakpoints. In this respect, the molecular methods show a higher resolution level and their combination with classic methods are more accurate (Pauciullo, Fleck, Lühken, Berardino, & Erhardt, 2013). In light of these considerations, a dual‐colour FISH on conventional metaphases and the use of species‐specific painting probes confirmed the chromosome involved in the translocation (Figure 3a).…”