Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, −3 and +4, are usually such that support initiation (A₋₃ = 42%, G₋₃ = 36% and G₊₄ = 47%), only 37.4% of the genes adhere to the purine (R)₋₃/G₊₄ rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R₋₃ and G₊₄, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A₊₁T₊₂G₊₃ initiation codons adhered much more strongly to the R₋₃/G₊₄ rule. Among the intron-less histone genes, only the H3 genes adhere to the R₋₃/G₊₄ rule, while the H1, H2A, H2B and H4 genes usually lack either R₋₃ or G₊₄. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A₊₁→T₊₁ mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.     

Rejecting strictly allopatric speciation on a continental island: prolonged postdivergence gene flow between Taiwan (Leucodioptron taewanus,Passeriformes Timaliidae) and Chinese (L. canorum canorum) hwameis

Allopatry is conventionally considered the geographical mode of speciation for continental island organisms. However, strictly allopatric speciation models that assume the lack of postdivergence gene flow seem oversimplified given the recurrence of land bridges during glacial periods since the late Pliocene. Here, to evaluate whether a continental island endemic, the Taiwan hwamei (Leucodioptron taewanus, Passeriformes Timaliidae) speciated in strict allopatry, we used weighted‐regression‐based approximate Bayesian computation (ABC) to analyse the genetic polymorphism of 18 neutral nuclear loci (total length: 8500 bp) in Taiwan hwamei and its continental sister species, the Chinese hwamei (L. canorum canorum). The nonallopatry model was found to fit better with observed genetic polymorphism of the two hwamei species (posterior possibility = 0.82). We also recovered unambiguous signals of nontrivial bidirectional postdivergence gene flow (N_em » 1) between Chinese hwamei and Taiwan hwamei until 0.5 Ma. Divergence time was estimated to be 3.5 to 2 million years earlier than that estimated from mitochondrial cytochrome b sequences. Finally, using the inferred nonallopatry model to simulate genetic variation at 24 nuclear genes examined showed that the adiponectin receptor 1 gene may be under divergent adaptation. Our findings imply that the role of geographical barrier may be less prominent for the speciation of continental island endemics, and suggest a shift in speciation studies from simply correlating geographical barrier and genetic divergence to examining factors that facilitate and maintain divergence, e.g. differential selection and sexual selection, especially in the face of interpopulation gene flow.     

Isolation and characterization of 12 tetranucleotide repeat microsatellite loci from the green‐backed tit (Parus monticolus)

From a partial genomic library enriched for GATA short tandem repeats, we developed 12 polymorphic microsatellite loci from the green‐backed tit (Parus monticolus). We characterized these loci by genotyping 30 adult individuals with unknown relationship. The number of alleles ranged from four to 17 per locus (mean = 9.3 alleles) and the observed heterozygosity for each locus ranged from 0.633 to 0.933 (mean = 0.789). All loci conformed to Hardy–Weinberg expectations. Four of 66 possible pairwise comparisons between loci showed significant gametic disequilibrium.     

Prevention of recurrence and prolonged survival in primary central nervous system lymphoma (PCNSL) patients treated with adjuvant high-dose methylprednisolone

Five patients at risk for primary central nervous system lymphoma (PCNSL) recurrence were treated with high-dose methylprednisolone, (HDMP) to prevent ‘trafficking’ of malignant lymphocytes into the central nervous system (CNS). HDMP was chosen because of its ability to stabilize the ‘blood brain barrier (BBB)’. Three men with newly diagnosed PCNSL, ages 62, 76 and 78 y, whose survival was projected to be 6.6 months, began treatment after achieving complete response (CR) to initial radiation therapy alone and survived 27, 37 and 59 months after treatment. In none was death from recurrent disease in CNS but one patient did die of systemic non-Hodgkin’s lymphoma (NHL) five years after PCNSL diagnosis. A 20 y old man was treated with HDMP after successful combined modality therapy and is alive 75+months after initial diagnosis without evidence of disease recurrence. A 34 y old man relapsed after combined modality initial treatment and failed to respond to HDMP when treatment was begun after unsuccessful salvage therapy; he died of disease 12 months after initial diagnosis. There were no treatment complications. The promising results in this pilot study from the basis for a North Central Cancer Treatment Group (NCCTG) 96-73-51, a Phase 2 clinical trial of brain radiotherapy and HDMP for PCNSL patients 70 y of age and older, a group of patients at high risk for toxicity from intensive combined modality therapy.