Characterization of dystrophin and utrophin diversity in the mouse.
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Utrophin is a 400 kDa autosomal homolog of dystrophin and a component of the submembranous cytoskeleton. While multiple dystrophin isoforms have been identified along with alternatively spliced products, to date only two different mRNA species of utrophin have been identified. To determine the degree of evolutionary conservation between dystrophin and utrophin isoforms, we have compared their expression patterns in adult mice. Northern blot analysis of multiple adult tissues confirmed that only two major sizes of transcripts are produced from each gene: 13 and 5.5 kb from utrophin and 14 and 4.8 kb from dystrophin. However, western blot analysis detected several putative short utrophin isoforms that may be homologs of the dystrophin isoforms Dp140, Dp116 and Dp71. We also identified an alternatively spliced utrophin transcript that lacks the equivalent of the alternatively spliced dystrophin exon 71. Finally, we demonstrated that the C-terminal domain of utrophin targeted to neuromuscular junctions in normal mice, but localized to the sarcolemma efficiently only in the absence of dystrophin. Our results provide further evidence for a common evolutionary origin of the utrophin and dystrophin genes. (+info)
Inner ear and kidney anomalies caused by IAP insertion in an intron of the Eya1 gene in a mouse model of BOR syndrome.
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A spontaneous mutation causing deafness and circling behavior was discovered in a C3H/HeJ colony of mice at the Jackson Laboratory. Pathological analysis of mutant mice revealed gross morphological abnormalities of the inner ear, and also dysmorphic or missing kidneys. The deafness and abnormal behavior were shown to be inherited as an autosomal recessive trait and mapped to mouse chromosome 1 near the position of the Eya1 gene. The human homolog of this gene, EYA1, has been shown to underly branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by hearing loss with associated branchial and renal anomalies. Molecular analysis of the Eya1 gene in mutant mice revealed the insertion of an intracisternal A particle (IAP) element in intron 7. The presence of the IAP insertion was associated with reduced expression of the normal Eya1 message and formation of additional aberrant transcripts. The hypomorphic nature of the mutation may explain its recessive inheritance, if protein levels in homozygotes, but not heterozygotes, are below a critical threshold needed for normal developmental function. The new mouse mutation is designated Eya1(bor) to denote its similarity to human BOR syndrome, and will provide a valuable model for studying mutant gene expression and etiology. (+info)
Two novel genes in the center of the 11p15 imprinted domain escape genomic imprinting.
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We previously reported the isolation of a 2.5 Mb tumor-suppressing subchromosomal transferable fragment (STF) from human chromosome 11p15 and the identification of nine known genes and four novel genes within this STF. We now report the isolation of two novel cDNAs, designated here as TSSC4 and TSSC6 (tumor-suppressing STF cDNA 4 and 6), located within the STF. TSSC4 and TSSC6 encode predicted proteins of 329 and 290 amino acids, respectively, with no close similarity to previously reported proteins. TSSC4 and TSSC6 are both located in the center of a 1 Mb imprinted domain, which contains the imprinted genes TSSC3, TSSC5, p57(KIP2), KVLQT1, ASCL2, IGF2 and H19. However, we found that neither TSSC4 nor TSSC6 was significantly imprinted in any of the fetal or extra-embryonic tissues examined. Based on this result, the imprinted gene domain of 11p15 appears to contain at least two imprinted subdomains, between which TSSC4 and TSSC6 substantially escape imprinting, due either to lack of initial silencing or to an early developmental relaxation of imprinting. (+info)
Structural and functional characterization of the mouse Sox9 promoter: implications for campomelic dysplasia.
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Mutations in SOX9 cause campomelic dysplasia (CD), a dominant skeletal dysmorphology and XY sex reversal syndrome. The CD phenotype is sensitive to dosage and expression levels of SOX9. Sox9 is expressed during chondrocyte differentiation and is up-regulated in male and down-regulated in female genital ridges during sex differentiation. In order to study the sex- and tissue-specific regulation of Sox9, we have defined the transcription start site and characterized the mouse Sox9 promoter region. The Sox9 proximal promoter shows moderately high nucleotide similarity between mouse and human. Transient transfection experiments using various deletion constructs of the 6.8 kb upstream region of mouse Sox9 fused to a luciferase reporter showed that the interval between 193 and 73 bp from the transcription start site is essential for maximal promoter activity in cell lines and in primary male and female gonadal somatic cells and liver cells isolated from 13.5 d.p.c. mouse embryos. This minimal promoter region was shown by DNase I hypersensitive site assay to be in an 'open' state of chromatin structure in gonads of both sexes, but not in the liver. Promoter activity was higher in testis than in ovary and liver, but deletion of the region from -193 to -73 bp abolished this difference. We conclude that the proximal promoter region is in part responsible for the sex- and tissue-specific expression of the Sox9 gene and that more distal positive and negative elements contribute to its regulation in vivo, consistent with the observation that translocations upstream from SOX9 can result in campomelic dysplasia. (+info)
Isolation and embryonic expression of the novel mouse gene Hic1, the homologue of HIC1, a candidate gene for the Miller-Dieker syndrome.
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The human gene HIC1 (hypermethylated in cancer) maps to chromosome 17p13.3 and is deleted in the contiguous gene disorder Miller-Dieker syndrome (MDS) [Makos-Wales et al. (1995) Nature Med., 1, 570-577; Chong et al. (1996) Genome Res., 6, 735-741]. We isolated the murine homologue Hic1, encoding a zinc-finger protein with a poxvirus and zinc-finger (POZ) domain and mapped it to mouse chromosome 11 in a region exhibiting conserved synteny to human chromosome 17. Comparison of genomic and cDNA sequences predicts two exons for the murine Hic1. The second exon exhibits 88% identity to the human HIC1 on DNA level. During embryonic development, Hic1 is expressed in mesenchymes of the sclerotomes, lateral body wall, limb and cranio-facial regions embedding the outgrowing peripheral nerves during their differentiation. During fetal development, Hic1 additionally is expressed in mesenchymes apposed to precartilaginous condensations, at many interfaces to budding epithelia of inner organs, and weakly in muscles. We observed activation of Hic1 expression in the embryonic anlagen of many tissues displaying anomalies in MDS patients. Besides lissencephaly, MDS patients exhibit facial dysmorphism and frequently additional birth defects, e.g. anomalies of the heart, kidney, gastrointestinal tract and the limbs (OMIM 247200). Thus, HIC1 activity may correlate with the defective development of the nose, jaws, extremities, gastrointestinal tract and kidney in MDS patients. (+info)
Pharmacokinetics of recombinant human granulocyte colony-stimulating factor in rabbits and mice.
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AIM: To study the pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhGCSF) in rabbits and mice. METHODS: 125I-rhGCSF was prepared by iodogen method and determined by size exclusive HPLC (SEHPLC). RESULTS: Concentration-time curves after i.v. 125I-rhGCSF in rabbits were best fitted with 2-compartment open model. The alpha and terminal elimination T1/2 were 0.25-0.33 and 3.2-4.6 h, respectively. AUC increased with doses, and Cls and K10 were similar. Tpeak was 0.59 +/- 0.25 h after s.c., and elimination T1/2 was similar to that after i.v. The bioavailability after sc was 1.0. In mice the highest level was found in renal system, the next was bile-enteric system. Levels in lymph nodes, bone marrow, and spleen were approximately equal to or slightly lower than that in plasma, while the levels in brain, fat, and muscles were the lowest. About 68%-86% were recovered in urine and feces. CONCLUSION: Pharamcokinetics of 125I-rhGCSF in rabbits and mice provided a useful index for clinical trial. (+info)
Activation in vivo of retroperitoneal fibromatosis-associated herpesvirus, a simian homologue of human herpesvirus-8.
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Retroperitoneal fibromatosis-associated herpesvirus of rhesus macaques (RFHVMm) is a gammaherpesvirus closely related to human herpesvirus-8 (HHV-8), which is thought to be a necessary cofactor for the development of Kaposi's sarcoma (KS) in humans. Here, RFHVMm infection of rhesus macaques exposed to the D-type retrovirus simian retrovirus-2 (SRV-2) is described. Development of SRV-2 viraemia, infection with simian immunodeficiency virus or administration of cyclosporin A could result in persistent RFHVMm viraemia. From this, it is concluded that productive retrovirus infection or otherwise-induced immune suppression has the ability to activate this herpesvirus in vivo. Elevated levels of circulating interleukin-6, a cytokine that plays a central role in KS, were found in RFHVMm-viraemic animals. In viraemic animals, RFHVMm was found in tissues that are common sites for the development of AIDS-associated KS, especially the oral cavity. Together, these data suggest a common biology between RFHVMm infection of macaques and HHV-8 infection and pathogenesis in humans. (+info)
Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR.
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The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of B. burgdorferi quantities to the mouse nidogen gene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples. (+info)