An Integrated Map with Cosmid/PAC Contigs of a 4-Mb Down Syndrome Critical Region

The major phenotypic features of Down syndrome have been correlated with partial trisomies of chromosome 21, allowing us to define the candidate gene region to a 4-Mb segment on the 21q22.2 band. We present here a high-resolution physical map with megabase-sized cosmid/PAC contigs. This ordered clone library has provided unique material for the integration of a variety of mappable objects, including exons, cDNAs, restriction sites, etc. Furthermore, our results have exemplified a strategy for the completion of the chromosome 21 map to sequencing.     

Stay Focused! The Effects of Internal and External Focus of Attention on Movement Automaticity in Patients with Stroke

Dual-task performance is often impaired after stroke. This may be resolved by enhancing patients’ automaticity of movement. This study sets out to test the constrained action hypothesis, which holds that automaticity of movement is enhanced by triggering an external focus (on movement effects), rather than an internal focus (on movement execution). Thirty-nine individuals with chronic, unilateral stroke performed a one-leg-stepping task with both legs in single- and dual-task conditions. Attentional focus was manipulated with instructions. Motor performance (movement speed), movement automaticity (fluency of movement), and dual-task performance (dual-task costs) were assessed. The effects of focus on movement speed, single- and dual-task movement fluency, and dual-task costs were analysed with generalized estimating equations. Results showed that, overall, single-task performance was unaffected by focus (p = .341). Regarding movement fluency, no main effects of focus were found in single- or dual-task conditions (p’s ≥ .13). However, focus by leg interactions suggested that an external focus reduced movement fluency of the paretic leg compared to an internal focus (single-task conditions: p = .068; dual-task conditions: p = .084). An external focus also tended to result in inferior dual-task performance (β = -2.38, p = .065). Finally, a near-significant interaction (β = 2.36, p = .055) suggested that dual-task performance was more constrained by patients’ attentional capacity in external focus conditions. We conclude that, compared to an internal focus, an external focus did not result in more automated movements in chronic stroke patients. Contrary to expectations, trends were found for enhanced automaticity with an internal focus. These findings might be due to patients’ strong preference to use an internal focus in daily life. Future work needs to establish the more permanent effects of learning with different attentional foci on re-automating motor control after stroke.     

Two Sequence-Ready Contigs Spanning the Two Copies of a 200-kb Duplication on Human 21q: Partial Sequence and Polymorphisms

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications.     

An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model

Background

Down syndrome (DS), caused by trisomy of human chromosome 21 (HSA21), is the most common genetic cause of mental retardation in humans. Among complex phenotypes, it displays a number of neural pathologies including smaller brain size, reduced numbers of neurons, reduced dendritic spine density and plasticity, and early Alzheimer-like neurodegeneration. Mouse models for DS show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. Early postnatal development of both human and mouse-model DS shows the reduced capability of neuronal precursor cells to generate neurons. The exact molecular cause of this reduction, and the role played by increased dosage of individual HSA21 genes, remain unknown.

Results

We have subcutaneously injected mouse pluripotent ES cells containing a single freely segregating supernumerary human chromosome 21 (HSA21) into syngeneic mice, to generate transchromosomic teratomas. Transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. Tumours were grown for 30 days, a time-span equivalent to combined intra-uterine, and early post-natal mouse development. When paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mRNA levels for neuron specific (Tubb3) and glia specific (Gfap) genes, relative to euploid controls. Two thirds of transchromosomic tumours also showed a lack of PCR amplification with multiple primers specific for HSA21, which were present in the ES cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of HSA21 genes.

Conclusion

We demonstrate that a supernumerary chromosome 21 causes Inhibition of Neuroectodermal DIfferentiation (INDI) of pluripotent ES cells. The data suggest that trisomy of less than a third of HSA21 genes, in two chromosomal regions, might be sufficient to cause this effect.     

Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5′ deleted nptII gene can restore a Kmr phenotype in tobacco

Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Km^r) phenotype. The gene targeting frequency was 1×10^–5. In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35×10⁸ protoplasts were transformed with the repair construct. Out of nearly 221 000 transformed cells 477 Km^r calli were selected. Screening the Km^r calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Km^r calli in which gene targeting had not occurred we analysed plants regenerated from 24 Km^r calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.     

Transchromosomic cell model of Down syndrome shows aberrant migration, adhesion and proteome response to extracellular matrix

Background  

Down syndrome (DS), caused by trisomy of human chromosome 21 (HSA21), is the most common genetic birth defect. Congenital heart defects (CHD) are seen in 40% of DS children, and >50% of all atrioventricular canal defects in infancy are caused by trisomy 21, but the causative genes remain unknown.