Transposition pattern of a modified Ds element in tomato

Several aspects of transposition of an in vitro modified Ds element are described. This Ds element, designated ds-r, is equipped with bacterial plasmid sequences and can, therefore, be rescued from the plant genome. Our results indicate that the Ds-r element has a late timing of transposition from T-DNAs. This feature of the element might be advantageous for tagging experiments because it leads to independently transposed germinally transmitted elements. Furthermore, it is shown that Ds-r transposition generates clusters of insertions, indicating that genes to be tagged should be located in genomic regions covered by insertions.     

Structural and mutational analysis of the SBDS protein family. Insight into the leukemia-associated Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure with significant predisposition to the development of poor prognosis myelodysplasia and leukemia, exocrine pancreatic failure and metaphyseal chondrodysplasia. Although the SBDS gene mutated in this disorder is highly conserved in Archaea and all eukaryotes, the function is unknown. To interpret the molecular consequences of SDS-associated mutations, we have solved the crystal structure of the Archaeoglobus fulgidus SBDS protein orthologue at a resolution of 1.9 angstroms, revealing a three domain architecture. The N-terminal (FYSH) domain is the most frequent target for disease mutations and contains a novel mixed alpha/beta-fold identical to the single domain yeast protein Yhr087wp that is implicated in RNA metabolism. The central domain consists of a three-helical bundle, whereas the C-terminal domain has a ferredoxin-like fold. By genetic complementation analysis of the essential Saccharomyces cerevisiae SBDS orthologue YLR022C, we demonstrate an essential role in vivo for the FYSH domain and the central three-helical bundle. We further show that the common SDS-related K62X truncation is non-functional. Most SDS-related missense mutations that alter surface epitopes do not impair YLR022C function, but mutations affecting residues buried in the hydrophobic core of the FYSH domain severely impair or abrogate complementation. These data are consistent with absence of homozygosity for the common K62X truncation mutation in individuals with SDS, indicating that the SDS disease phenotype is a consequence of expression of hypomorphic SBDS alleles and that complete loss of SBDS function is likely to be lethal.     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

Genomic selection of purebred animals for crossbred performance in the presence of dominant gene action

Background

Genomic selection is an appealing method to select purebreds for crossbred performance. In the case of crossbred records, single nucleotide polymorphism (SNP) effects can be estimated using an additive model or a breed-specific allele model. In most studies, additive gene action is assumed. However, dominance is the likely genetic basis of heterosis. Advantages of incorporating dominance in genomic selection were investigated in a two-way crossbreeding program for a trait with different magnitudes of dominance. Training was carried out only once in the simulation.

Results

When the dominance variance and heterosis were large and overdominance was present, a dominance model including both additive and dominance SNP effects gave substantially greater cumulative response to selection than the additive model. Extra response was the result of an increase in heterosis but at a cost of reduced purebred performance. When the dominance variance and heterosis were realistic but with overdominance, the advantage of the dominance model decreased but was still significant. When overdominance was absent, the dominance model was slightly favored over the additive model, but the difference in response between the models increased as the number of quantitative trait loci increased. This reveals the importance of exploiting dominance even in the absence of overdominance. When there was no dominance, response to selection for the dominance model was as high as for the additive model, indicating robustness of the dominance model. The breed-specific allele model was inferior to the dominance model in all cases and to the additive model except when the dominance variance and heterosis were large and with overdominance. However, the advantage of the dominance model over the breed-specific allele model may decrease as differences in linkage disequilibrium between the breeds increase. Retraining is expected to reduce the advantage of the dominance model over the alternatives, because in general, the advantage becomes important only after five or six generations post-training.

Conclusion

Under dominance and without retraining, genomic selection based on the dominance model is superior to the additive model and the breed-specific allele model to maximize crossbred performance through purebred selection.