Elevated apoptosis in pre-mature neurons differentiated from mouse ES cells containing a single human chromosome 21

A decrease in the number and density of neurons is the most common phenotype in the brains of Down syndrome (DS) patients, causing mental retardation. Studies using primary cultured neurons from DS patients or from model mice have suggested that a defect in metabolism of reactive oxygen species, or diminished levels of glutathione, causes mitochondrial and caspase-mediated neuronal apoptosis in vitro. However, it is not well documented whether neuronal apoptosis also occurs in immature DS neurons, owing to the difficulty in isolating or identifying neuronal stem cells in human or mouse fetuses. Here we utilized an in vitro model system for neuronal differentiation, with mouse embryonic stem cells containing human chromosome 21 (TT2F/hChr.21) to examine the effect of an additional hChr.21 on the early phases of neurogenesis. The differentiation profile of TT2F/hChr.21 cells was essentially the same as those of parental TT2F ES cells. In differentiations of both TT2F and TT2F/hChr.21 cells, high level of apoptosis was observed in neuronal stem cells, but the rate of apoptosis in TT2F/hChr.21 cells was significantly higher than that of parental cells. These results suggest that quantitative changes in the level of apoptosis in DS neuronal stem cells may account for the reduction of neuronal number and density in the DS brain.     

Defective expression of mouse adenovirus Fl in human cells

HeLa cells infected with mouse adenovirus strain Fl ( AdFl ) produce at least 2000 times less virus than permissive mouse 3T3 cells. Viral DNA synthesis, however, proceeds unimpaired. The defect in virion production was linked to a dramatic reduction in the synthesis of AdFl structural proteins, in particular the hexon. The identity of the AdFl hexon gene product was recognized through its immunogenic reactivity towards an antiserum raised against the human adenovirus type 2 (Ad2) hexon gene product. This cross-reactivity is reflected in an extensive DNA sequence homology between AdFl and Ad2 DNA at position 53-60, the locus of the hexon gene, on the Ad2 physical map. Through hybridization at different formamide concentrations, the present study identifies one additional, highly conserved region at map positions 14-15 on the Ad2 genome.     

A radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 locus.

We describe a high-resolution radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 (NF2) gene. Eighty-five hamster-human somatic cell hybrids generated by X-irradiation and cell fusion were used to generate the radiation hybrid map. The presence or absence of 18 human chromosome 22-specific markers was determined in each hybrid by using Southern blot hybridization. Sixteen of the 18 markers were distinguishable by X-ray breakage in the radiation hybrids. Analysis of these data using two different mathematical models and two different statistical methods resulted in a single framework map consisting of 8 markers ordered with odds greater than 1000:1. The remaining nonframework markers were all localized to regions consisting of two adjoining intervals on the framework map with odds greater than 1000:1. Based on the RH map, the NF2 region of chromosome 22, defined by the flanking markers D22S1 and D22S28, is estimated to span a physical distance of approximately 6 Mb and is the most likely location for 9 of the 18 markers studied: D22S33, D22S41, D22S42, D22S46, D22S56, LIF, D22S37, D22S44, and D22S15.     

Specific impairment of cardiogenesis in mouse ES cells containing a human chromosome 21

Down syndrome (DS) leads to cardiac defects which are common and significant in babies with DS. We recently generated chimeric mice carrying a human chromosome (hChr) 21. The contribution ratio of embryonic stem (ES) cells containing a hChr 21 was specifically low in the heart, compared to other organs, and cardiovascular malformations were observed, suggesting that an additional copy of hChr 21 also disrupts the normal development of heart in mice. Here we describe that the presence of hChr 21 in ES cells delays the appearance of beating cardiomyocyte during differentiation, whereas differentiation into other cell types is not disrupted. Furthermore, the defect in cardiogenesis was restored following the deletion of a specific region of hChr 21. Therefore, we conclude that the imbalance of specific gene(s) on hChr 21 may lead to the disturbance of cardiogenesis and that this may be a useful system to model and investigate the cardiac defects of human DS.     

Comparative mapping of mouse chromosome 2 and human chromosome 9q: the genes for gelsolin and dopamine beta-hydroxylase map to mouse chromosome 2.

The mapping of human chromosome 9 (HSA9) and mouse chromosome 2 (MMU2) has revealed a conserved syntenic region between the distal end of the long arm of chromosome 9 and proximal mouse chromosome 2. Two genes that map to human chromosome 9q34, gelsolin (GSN) and dopamine beta-hydroxylase (DBH), have not previously been located in the mouse. We have used an interspecific backcross to map each of these genes, by Southern blot analysis, to mouse chromosome 2. Gelsolin (Gsn) is tightly linked to the gene for complement component C5 (Hc), and dopamine beta-hydroxylase (Dbh) is just proximal to the Abelson leukemia virus oncogene (Abl) and alpha-spectrin 2 (Spna-2). The loci for gelsolin and dopamine beta-hydroxylase therefore form part of the conserved synteny between HSA9q and MMU2.     

Assignment of the locus for Waardenburg syndrome type I to human chromosome 2q37 and possible homology to the Splotch mouse.

We have demonstrated close linkage between the locus for the autosomal dominant Waardenburg syndrome type I and the placental alkaline phosphatase locus on chromosome 2q37. In five families the peak lod score was 4.76 at a recombination fraction of .023. In the mouse the Splotch locus maps to near the homologous position. Splotch mice have white spotting and hearing defects, suggesting that Splotch may be the murine homologue of Waardenburg syndrome type I.     

beta2-microglobulin locus on human chromosome 15.

We have developed an autoradiographic/electrophoretic assay capable of distinguishing mouse and human beta2-microglobulin (beta2m) in spent culture media. The method is applicable to mouse and human lines and to hybrid cell lines made from them. With this technique, mouse/human hybrid cell lines were tested for the presence of human beta2m. Isozyme and karyotype analyses were also carried out with the hybrids. The combined results of these studies show that the structural gene for human beta2m is on the long arm of chromosome 15.     

Carboxyl ester lipase: a highly polymorphic locus on human chromosome 9qter.

Carboxyl ester lipase (CEL) is a major component of pancreatic juice and is responsible for the hydrolysis of cholesterol esters as well as a variety of other dietary esters. As part of an effort to elucidate the role of this enzyme in the genetic control of lipid metabolism, we report here the chromosomal mapping of the gene for CEL to the most distal part of the long arm of human chromosome 9 using analysis of mouse-human somatic cell hybrids and in situ hybridization to chromosomes. A chromosome 9 translocation was utilized to determine the position of the CEL gene relative to various genetic markers previously localized to this region. Finally, we report that the CEL locus exhibits a high degree of polymorphism and contains a hypervariable region of the insertion/deletion variety.     

Linkage of the mouse LDL receptor gene on chromosome 9

We identified restriction fragment length variants of the mouse low-density lipoprotein receptor gene and used these to map the gene, designated Ldlr, to the proximal region of chromosome 9. An interspecific backcross between strains MEV and CAST/Ei yielded the following gene order and distances in centimorgans: Ldlr-18.8 +/- 5.6-Apoa-4-7.3 +/- 3.5-Mpi-1-10.2 +/- 3.9-Emv-3 or dilute. Analysis of recombinant inbred strains also indicated that Ldlr is tightly linked to a previously unmapped retroviral marker, Xmmv-67.     

Esterase 13, a new mouse esterase locus with recessive expression and its genetic location on chromosome 9

A new esterase locus (Es-13) has been identified in Mus musculus. Strains AEJ/GnRk, LG/J, SJL/J, and SWR/J carry a recessive allele, Es-13 ^b, for a locus possibly involved in the posttranslational modification of a kidney esterase. All other strains observed carried the dominant Es-13 ^a allele. Es-13 was mapped on Chr 9 by recombinant inbred lines and by conventional backcrossing experiments. Backcross data produced the following gene order and map distances: Lap-1 (31.6±7.5 cM) Es-13 (2.6±2.6 cM) Mod-1.     

Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2.

The penton base gene from adenovirus type 12 (Ad12) was sequenced and encodes a 497-residue polypeptide, 74 residues shorter than the penton base from Ad2. The Ad2 and Ad12 proteins are highly conserved at the amino- and carboxy-terminal ends but diverge radically in the central region, where 63 residues are missing from the Ad12 sequence. Conserved within this variable region is the sequence Arg-Gly-Asp (RGD), which, in the Ad2 penton base, binds to integrins in the target cell membrane, enhancing the rate or the efficiency of infection. The Ad12 penton base was expressed in Escherichia coli, and the purified refolded protein assembled in vitro with Ad2 fibers. In contrast to the Ad2 penton base, the Ad12 protein failed to cause the rounding of adherent cells or to promote attachment of HeLa S3 suspension cells; however, A549 cells did attach to surfaces coated with either protein and pretreatment of the cells with an integrin alpha v beta 5 monoclonal antibody reduced attachment to background levels. Treatment of HeLa and A549 cells with integrin alpha v beta 3 or alpha v beta 5 monoclonal antibodies or with an RGD-containing fragment of the Ad2 penton base protein inhibited infection by Ad12 but had no effect on and in some cases enhanced infection by Ad2. Purified Ad2 fiber protein reduced the binding of radiolabeled Ad2 and Ad12 virions to HeLa and A549 cells nearly to background levels, but the concentrations of fiber that strongly inhibited infection by Ad2 only weakly inhibited Ad12 infection. These data suggest that alpha v-containing integrins alone may be sufficient to support infection by Ad12 and that this pathway is not efficiently used by Ad2.     

A single amino acid in the adenovirus type 37 fiber confers binding to human conjunctival cells

A 46-kDa receptor, coxsackievirus-adenovirus (Ad) receptor (CAR), mediates cell attachment of a number of different Ad serotypes; however, not all Ad serotypes utilize this receptor for infection. Moreover, the precise amino acid sequences in the Ad fiber protein that mediate cell attachment have yet to be identified. We investigated the interaction of subgroup D Ads with human ocular cells. Ad serotype 37 (Ad37), a virus associated with epidemic keratoconjunctivitis, but not a closely related virus serotype, Ad19p, exhibited preferential binding to and infection of human conjunctival cells. A single amino acid substitution in the Ad19p fiber distal domain (knob), Glu240 to Lys, conferred binding to conjunctival cells, while the reverse substitution in the Ad37 fiber abrogated cell binding. These findings provide new information on the fiber sequences that regulate Ad host cell tropism.