Ts65Dn -- localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome

Fluorescent in situ hybridization (FISH) -- using mouse chromosome paints, probes for the mouse major centromeric satellite DNA, and probes for genes on chromosomes (Chr) 16 and 17 -- was employed to locate the breakpoint in a translocation used to produce a mouse model for Down syndrome. The Ts65Dn trisomy is derived from the reciprocal translocation T(16;17)65Dn. The Ts65Dn mouse carries a marker chromosome containing the distal segment of Chr 16, a region that shows linkage conservation with human Chr 21, and the proximal end of Chr 17. This chromosome confers trisomy for most of the genes in the Chr 16 segment and Ts65Dn mice show many of the phenotypic features characteristic of Down syndrome. We used FISH on metaphase chromosomes from translocation T65Dn/+ heterozygotes and Ts65Dn mice to show that the Chr 17 breakpoint is distal to the heterochromatin of Chr 17, that the Ts65Dn marker chromosome contains a small portion of Chr 17 euchromatin, that the Chr 16 breakpoint lies between the Ncam2 and Gabpa/App genes, and that the Ts65Dn chromosome contains >80% of the human Chr 21 homologs. The significance of this finding is discussed in terms of the utility of this mouse model.     

The purification and steady-state kinetic behaviour of rabbit heart mitochondrial NAD(P)+ malic enzyme.

The mitochondrial NAD(P)+ malic enzyme [EC 1.1.1.39, L-malate:NAD+ oxidoreductase (decarboxylating)] was purified from rabbit heart to a specific activity of 7 units (mumol/min)/mg at 23 degrees C. A study of the reductive carboxylation reaction indicates that this enzymic reaction is reversible. The rate of the reductive carboxylation reaction appears to be completely inhibited at an NADH concentration of 0.92 mM. A substrate saturation curve of this reaction with NADH as the varied substrate describes this inhibition. The apparent kinetic parameters for this reaction are Ka(NADH) = 239 microM and Vr = 1.1 mumol/min per mg at 23 degrees C. The steady-state product-inhibition patterns for pyruvate and NADH indicate a sequential binding of the substrates: NAD+ followed by L-malate. These data also indicate that NADH is the last product released. A steady-state kinetic model is proposed that incorporates NADH-enzyme dead-end complexes.     

The mouse polycystic kidney disease mutation (cpk) is located on proximal chromosome 12

The mouse congenital polycystic kidney (cpk) mutation produces a condition that resembles human autosomal recessive polycystic kidney disease (ARPKD) in its pattern of inheritance, clinical progression, and histopathology. Inheritance of this mouse mutation in crosses segregating the Rb(12.14)8Rma translocation chromosome and various DNA markers of Chromosome 12 have localized cpk to a site near D12Nyu2, approximately 7 cM from the centromere of Chromosome 12. This result suggests that the homologous PKD2 gene should be localized to either human chromosome 2p23-p25 or chromosome 7q22-q31.     

The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities

An autosomal recessive mutation, termed fatty liver dystrophy (fld), can be identified in neonatal mice by their enlarged and fatty liver (Sweet, H. O., Birkenmeier, E. H., and Davisson, M. T. (1988) Mouse News Letter 81, 69). We have examined the underlying metabolic abnormalities in fld/fld mice from postnatal days 3-40. Serum and hepatic triglyceride levels were elevated 5-fold in suckling fld/fld mice compared to their +/? littermates but abruptly resolved at the suckling/weaning transition. Blot hybridization analysis of liver and intestinal RNAs revealed a liver-specific increase in apolipoprotein (apo) A-IV and C-II mRNA concentrations (100- and 6-fold, respectively) that was limited to the suckling and early weaning stages in fld/fld mice. Resolution of these differences during the weaning period could not be delayed by prolonging suckling to the 20th postnatal day nor could the mutant phenotype be elicited in young adult animals with a high fat diet. Lipoprotein lipase (LPL) activity was reduced 16-fold in the white adipose tissue of fld/fld mice until the onset of weaning. Heart activity was decreased less than 2-fold, but there were no deficits in brown adipose tissue or liver. Hepatic lipase (HL) mRNA levels and activity were significantly reduced in fld/fld livers and sera, respectively, during the suckling period. Mapping studies show the fld locus to be distinct from loci encoding LPL, HL, and apoA-IV, and those responsible for the combined lipase deficiencies in cld/cld and W/Wv mice. These data suggest that the fld mutation is associated with developmentally programmed tissue-specific defects in the neonatal expression of LPL and HL activities and provide evidence for a new regulatory locus which affects these lipase activities. This mutation could serve as a useful model for (i) analyzing the homeostatic mechanisms controlling lipid metabolism in newborn mice and (ii) understanding and treating certain inborn errors in human triglyceride metabolism.     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Two paracentric inversions in the mouse, In(1)1 Rk and In(2)5 Rk, have been studied in surface microspreads of spermatocytes from heterozygotes. At zytogene, synaptic initiation occurs independently in three regions: within the inversion, and without, on either side. Synaptonemal complex (SC) formation is restricted to homologous regions, resulting in inversion loops in all early pachytene spermatocytes. An adjusting phase then occurs during pachytene in which the inversion loop is reduced by desynapsis of homologously synapsed SC, followed immediately by non-homologous synapsis with the alternate pairing partner, progressing from the ends toward the middle. Adjustment occurs during the first half of pachytene, but is not closely synchronized with sub-stage. It is complete by late pachytene, the loop having been eliminated in all cases and replaced by straight SCs in which the inverted region is heterosynapsed. Synapsis in the adjustment phase is evidently permitted only after the homosynaptic phase, and is indifferent to homology. It may lead to heterosynapsis, as in the inversion region, or to synapsis of homologous regions not synapsed at zytogene. The anaphase bridge frequency, a measure of crossing over within the inversion, is about 34% for both inversions studied, indicating that such crossovers do not block adjustment, that crossing over probably occurs before or during the adjustment period, and that there is some crossover suppression. The last could be the consequence of blocking by desynapsis/heterosynapsis. Synaptic adjustment appears to be a general phenomenon that occurs to varying extents in different forms. A hypothetical scheme for two phases of synapsis is proposed: at zytogene, a basic propensity for indifferent SC formation is limited by a restricting condition to synapsis between homologous regions. Subsequently, the restriction is lifted, whereupon synaptic instability is resolved by desynapsis, followed by resynapsis that is indifferent to homology, but that results in a topologically more stable structure.     

IGF-1 modulates gene expression of proteins involved in inflammation,cytoskeleton, and liver architecture

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1’s effects on liver by comparing wild-type controls, heterozygous igf1^+/?, and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.     

Evolutionary breakpoints on human chromosome 21.

Segments of the long arm of human chromosome 21 are conserved, centromere to telomere, in mouse chromosomes 16, 17, and 10. There have been 28 genes identified in human chromosome 21 between TMPRSS2, whose orthologue is the most distal gene mapped to mouse chromosome 16, and PDXK, whose orthologue is the most proximal gene mapped to mouse chromosome 10. Only 6 of these 28 genes have been mapped in mouse, and all are located on chromosome 17. To better define the chromosome 17 segment and the 16 to 17 transition, we used a combination of mouse radiation hybrid panel mapping and physical mapping by mouse: human genomic sequence comparison. We have determined the mouse chromosomal location of an additional 12 genes, predicted the location of 7 more,and defined the endpoints of the mouse chromosome 17 region. The mouse chromosome 16/chromosome 17 evolutionary breakpoint is between human genes ZNF295 and UMODL1, showing there are seven genes in the chromosome 16 segment distal to Tmprss2. The chromosome 17/chromosome 10 breakpoint seems to have involved a duplication of the gene PDXK, which on chromosome 21 lies immediately distal to the KIAA0179 gene. These data suggest that there may be as few as 21 functional genes in the mouse chromosome 17 segment. This information is important for defining existing and constructing more complete mouse models of Down syndrome.