Deletion of Drosophila Nopp140 induces subcellular ribosomopathies.

The nucleolar and Cajal body phosphoprotein of 140 kDa (Nopp140) is considered a ribosome assembly factor, but its precise functions remain unknown. To approach this problem, we deleted the Nopp140 gene in Drosophila using FLP-FRT recombination. Genomic PCR, reverse transcriptase-PCR (RT-PCR), and immunofluorescence microscopy confirmed the loss of Nopp140, its messenger RNA (mRNA), and protein products from all tissues examined. Nopp140-/- larvae arrested in the second instar stage and most died within 8 days. While nucleoli appeared intact in Nopp140-/- cells, the C/D small nucleolar ribonucleoprotein (snoRNP) methyltransferase, fibrillarin, redistributed to the nucleoplasm in variable amounts depending on the cell type; RT-PCRs showed that 2'-O-methylation of ribosomal RNA (rRNA) in Nopp140-/- cells was reduced at select sites within both the 18S and 28S rRNAs. Ultrastructural analysis showed that Nopp140-/- cells were deficient in cytoplasmic ribosomes, but instead contained abnormal electron-dense cytoplasmic granules. Immunoblot analysis showed a loss of RpL34, and metabolic labeling showed a significant drop in protein translation, supporting the loss of functional ribosomes. Northern blots showed that pre-RNA cleavage pathways were generally unaffected by the loss of Nopp140, but that R2 retrotransposons that naturally reside within the 28S region of normally silent heterochromatic Drosophila ribosomal DNA (rDNA) genes were selectively expressed in Nopp140-/- larvae. Unlike copia elements and the related R1 retrotransposon, R2 expression appeared to be preferentially dependent on the loss of Nopp140 and not on environmental stresses. We believe the phenotypes described here define novel intracellular ribosomopathies resulting from the loss of Nopp140.

A Hoxb13-driven reverse tetracycline transactivator system for conditional gene expression in the prostate.

BACKGROUND: Genetically engineered mouse models play important roles in analyses of prostate development and pathobiology. While constitutive genetic gain- and loss-of-function models have contributed significantly to our understanding of molecular events driving these processes, the availability of a tightly regulated inducible expression system could extend the utility of transgenic approaches. Here, we describe the development of a Tet-regulatory system that employs Hoxb13 transcriptional control elements to direct reverse tetracycline transactivator (rtTA) expression in the prostate. METHODS: Using recombineering technology, the rtTA gene was placed under Hoxb13 cis-regulatory transcriptional control in the context of a 218-kb bacterial artificial chromosome. F(1) offspring carrying the Hoxb13-rtTA transgene were bred to a Tetracycline operator-Histone 2B-Green Fluorescent Protein (TetO-H2BGFP) responder line. Detailed reporter gene expression analyses, including doxycycline (Dox) induction and withdrawal kinetics, were performed in Hoxb13-rtTA|TetO-H2BGFP double transgenic adult mice and embryos. RESULTS: Dox-dependent GFP expression was observed exclusively in the prostate and distal colon epithelia of double transgenic mice. Reporter gene mRNA was detected in the prostate within 6 hr of Dox exposure, and was extinguished within 24 hr after Dox withdrawal. Furthermore, Dox-induced reporter gene expression persisted after castration. CONCLUSIONS: The Hoxb13-rtTA transgenic system provides a powerful tool for conditional Tet operator-driven transgene expression in the normal prostate and during disease progression. Used in conjunction with other prostate pathology models, these mice will enable precise, temporally controlled analyses of gene function and can provide opportunities for detailed analyses of molecular events underlying prostate diseases.

Mapping of the domestic cat "SILVER" coat color locus identifies a unique genomic location for silver in mammals.

The SILVER locus has been mapped in the domestic cat, identifying a unique genomic location distinct from that of any known reported gene associated with silver or hypopigmentation in mammals. A demonstrated lack of linkage to SILV, the strong candidate gene for silver, led to the initiation of a genome scan utilizing 2 pedigrees segregating for silver coat color. Linkage mapping defined a genomic region for SILVER as a 3.3-Mb region, (95.87-99.21 Mb) on chromosome D2, (peak logarithm of the odds = 10.5, = 0), which displays conserved synteny to a genomic interval between 118.58 and 121.85 Mb on chromosome 10 in the human genome. In the domestic cat, mutations at the SILVER locus suppress the development of pigment in the hair, but in contrast to other mammalian silver variants, there is an apparently greater influence on the production of pheomelanin than eumelanin pigment. The mapping of a novel locus for SILVER offers much promise in identifying a gene that may help elucidate aspects of pheomelanogenesis, a pathway that has been very elusive, and illustrates the promise of the cat genome project in increasing our understanding of basic biological processes of general relevance for mammals.