Gnrh mRNA expression in the brain of cooperatively breeding female Damaraland mole-rats

The Damaraland mole-rat (Fukomys damarensis) is a eusocial, subterranean rodent, in which breeding is limited to a single reproductive pair within each colony. Non-reproductive females, while in the confines of the colony, exhibit socially induced infertility. Anovulation is thought to be caused by a disruption in the normal gonadotropin-releasing hormone (GNRH) secretion from the hypothalamus. To assess whether social suppression is associated with alteredGnrhmRNA expression in the brain, we investigated the distribution and gene expression levels by means ofin situhybridization in female breeders and non-breeders from field captured colonies of the Damaraland mole-rat. We found expression ofGnrhmRNA as a loose network in several forebrain areas of female Damaraland mole-rats with the majority of labelling in the preoptic and anterior hypothalamus. The distribution matched previous findings using immunocytochemistry in this and other social mole-rat species. Quantification of the hybridisation signal revealed no difference between breeding and non-breeding females in the average optical density of the hybridization signal and the size of the total area covered byGnrhmRNA. However, analysis along the rostro-caudal axis revealed significantly elevatedGnrhmRNA expression in the rostral preoptic region of breeders compared to non-breeders, whereas the latter had increasedGnrhmRNA expression at the caudal level of the anterior hypothalamus. This study indicates that social suppression affects the expression ofGnrhmRNA in female Damaraland mole-rats. Furthermore, differential regulation occurs within different neuron subpopulations.

Quantum Dot Based Duplex In Situ Hybridisation for Gene Expression Profiling.

Quantum dots (QDs) are fluorescent semiconductor nanocrystals (2–10-nm core diameter) possessing the unique properties of extremely high fluorescence efficiency, lack of photobleaching and long fluorescence lifetime, making them an ideal tool for bioimaging. We have developed a novel technique for in situ hybridisation (ISH) using biotinylated oligonucleotides conjugated to streptavidin coated QD, and used them in this study to label bone marrow trephine samples. 50-mer long oligonucleotide probes were conjugated to QDs prior to ISH and conjugation efficiency was demonstrated by gel electrophopresis. ISH conditions and molar ratio of QDs to probe were optimised using a polyT probe. Images were captured using a CRI Nuance spectral imaging system and signal intensity was semi-quantitated using IPLab software. Specific oligonucleotide hybridisation was demonstrated using a probe for myeloperoxidase (MPO) in 40 bone marrow sections infiltrated by acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) as well as reactive bone marrow. In each case hybridisation signal was consistent with the distribution of MPO by standard immunohistochemistry - MPO was strongly expressed by myeloid blasts and absent in lymphoid blasts; in CML, most, but not all, cells were positive for MPO, in comparison to many fewer positive cells in reactive marrow. Duplex ISH was demonstrated using a probe for bcl-2 together with MPO in 5 bone marrow sections infiltrated by follicular lymphoma (FL). Strong hybridisation signal for bcl-2 was detected in all cells of the paratrabecular aggregates of FL but showed only scattered positivity in the remainder of the bone marrow. Conversely, MPO was absent in the paratrabecular aggregates and present in the myeloid cells in the remainder of the marrow. This pattern was present in both single and duplex ISH for MPO and bcl-2 in the marrow infiltrated by FL. Duplex ISH was performed both by sequential hybridisation with bcl-2 followed by MPO, and simultaneously with a mix of bcl-2 and MPO probes. As negative controls, scrambled oligonucleotide probes for the corresponding genes were used in each case and did not show hybridisation. In summary, we have developed a generic method for QD labelling and semi-quantitative detection of oligonucleotide ISH in routinely processed clinical tissue samples. Although, in this study we primarily used bone marrow trephine samples, this technique can be applied to any tissue. It has the potential to facilitate transfer of microarray-identified gene signatures to clinical research and diagnostics, whilst the ability of spectral imaging to resolve multiple signals offers the possibility of multiplexed probe detection.

Comparison of karyotype and rDNA-distribution in somatic chromosomes of Bowenia species (Stangeriaceae, Cycadales)

Somatic chromosomes at mitotic metaphase of Bowenia serrulata, B. spectabilis and B. sp. ‘Tinaroo’ is investigated by the standard aceto-orcein staining method and the fluorescent in situ hybridisation method (FISH) with ribosomal DNA (rDNA) probe. Bowenia serrulata, B. spectabilis and B. sp. ‘Tinaroo’ each have a chromosome number of 2n = 18. The karyotype of B. serrulata exhibits 10 median-centromeric chromosomes, while B. spectabilis and B. sp. ‘Tinaroo’ exhibit eight median-centromeric chromosomes. By using FISH, B. serrulata, B. spectabilis and B. sp. ‘Tinaroo’ show a hybridisation signal on the satellite of the short arm of two submedian-centromeric chromosomes. However, the other hybridisation signal pattern is different among B. serrulata, B. spectabilis and B. sp. ‘Tinaroo’.

Characterisation and physical localisation of Ty1-copia-like retrotransposons in four Alstroemeria species

The genus Alstroemeria contains species with large genomes (2C = 36.5-78.9 pg (17 600 - 38 000 Mb) in those species with 2n = 2x = 16). We investigated the diversity and genomic and chromosomal organisation of Ty1-copia-like retrotransposons in four Alstroemeria species. Analysis of 33 PCR-amplified sequences corresponding to a conserved domain of the Ty1-copia reverse transcriptase (rt) gene showed high heterogeneity among predicted amino acid sequences; no two sequences were identical, but most fell into one of five subgroups. Levels of inter- and intra-specific heterogeneity of sequences were similar. HaeIII-digested genomic DNA of various Alstroemeria species contained distinct bands upon hybridisation with individual rt gene fragments. Hybridisation with the heterogeneous PCR pool of rt fragments (retrotransposon pool) revealed additional bands; some minor bands were characteristic of either Brazilian or Chilean species. In situ hybridisation of the retrotransposon pool from three species to metaphase chromosomes from the same species showed a dispersed distribution of the retrotransposon pool with exclusion from rDNA and other chromosomal sites.Alstroemeria pelegrina, which is without major heterochromatic sites, showed some clustering and small negative bands. The retrotransposon pool was excluded from major DAPI-staining bands in Alstroemeria aurea, but in contrast, the sites of the major tandemly repeated sequences in Alstroemeria inodora showed a hybridisation signal similar to that in the rest of the chromosomes. The data are discussed in the context of the contribution of Ty1-copia-like retrotransposons to plant genome size, their evolution, and their value for phylogenetic and biodiversity studies.Key words: Alstroemeria, in situ hybridisation, genome organisation, retrotransposable elements, Ty1-copia.

Time-Resolved Fluorometry Based Sandwich Hybridisation Assay for HLA-DQA1 Typing

A microtitration plate based time-resolved fluorescence (TRF) hybridisation assay was developed for HLA typing utilising biotinylated sequence-specific catching probes and europium (Eu) labelled gene locus-specific detection probe to allow time-resolved fluorometer reading of the reaction. In an application for HLA-DQA typing a 228 base pair long region of the polymorphic exon 2 of DQA1 gene was amplified and the denatured PCR product distributed into streptavidin-coated microtitration wells together with the detection probe and one of the catching probes. After incubation and washes, the enhancement solution was added and specific hybridisation signal detected by measuring the emitted light. A series of 100 isolated genomic DNA samples were studied using biotinylated probes specific for DQA1*01, *0101/0104, *0103/0201/0601, *0201, *03, *0401/0601, *05 and *0502 alleles with results demonstrating the capacity of the test to detect aimed alleles. A series of whole blood spot samples were also studied and the results confirmed the applicability of this modification of the test.

EXPRESSION OF THE RAT AMYLIN (IAPP / DAP) GENE.

ABSTRACT We have used the polymerase chain reaction with mixed sequence primers to generate a probe for rat amylin and have used this to detect expression in various rat tissues. Amylin mRNA is found in greatest concentrations in the pancreas where a single mRNA species can be detected giving a hybridisation signal intensity approximately 10% that of insulin mRNA. When the beta cell population was depleted with streptozotocin, both amylin and insulin mRNAs were reduced to a similar extent. Consistent with its supposed role in the control of carbohydrate metabolism, amylin mRNA was also found in the stomach. Unlike the related peptide, CGRP, amylin mRNA is not present in the thyroid and is not widely distributed in the central nervous system. The only nervous tissue in which it could be detected was the dorsal root ganglion. Surprisingly, amylin mRNA was also found in the lung though only at very low levels.