Aggregation of truncated GST-HD exon 1 fusion proteins containing normal range and expanded glutamine repeats.

We have shown previously by electron microscopy that the purified glutathione S-transferase (GST)-Huntington's disease (HD) exon 1 fusion protein with 51 glutamine residues (GST-HD51) is an oligomer, and that site-specific proteolytic cleavage of this fusion protein results in the formation of insoluble more highly ordered protein aggregates with a fibrillar or ribbon-like morphology (E. Scherzinger et al. (1997) Cell 90, 549-558). Here we report that a truncated GST HD exon 1 fusion protein with 51 glutamine residues, which lacks the proline-rich region C-terminal to the polyglutamine (polyQ) tract (GST-HD51 delta P) self-aggregates into high-molecular-mass protein aggregates without prior proteolytic cleavage. Electron micrographs of these protein aggregates revealed thread-like fibrils with a uniform diameter of ca. 25 nm. In contrast, proteolytic cleavage of GST-HD51 delta P resulted in the formation of numerous clusters of high-molecular-mass fibrils with a different, ribbon-like morphology. These structures were reminiscent of prion rods and beta-amyloid fibrils in Alzheimer's disease. In agreement with our previous results with full-length GST-HD exon 1, the truncated fusion proteins GST-HD20 delta P and GST-HD30 delta P did not show any tendency to form more highly ordered structures, either with or without protease treatment.     

The alpha-A-crystallin and cystathionine beta-synthase genes are physically very closely linked in proximal mouse chromosome 17

Murine genes homologous to those contributing to the Down syndrome (DS) phenotype in man are currently of interest because of their potential for providing animal models for the study of specific DS symptoms. Most of the genes mapping to human chromosome 21q22, where the DS genes are concentrated, are related to sequences located on mouse chromosome 16. Others, however, are known to map to mouse chromosome 10, and two genes, cystathionine beta-synthase (Cbs) and alpha-A-crystallin (Crya-1), have been localized to the proximal portion of mouse chromosome 17. In this paper, we show that the two genes mapping to human chromosome 21q22 and mouse chromosome 17 are very tightly linked in mouse, being separated by at least 70 kb, but not more than 130 kb. The very close physical linkage of mouse Cbs and Crya-1, combined with data that localize homologs of the closely flanking markers H2k and Pim-1 to human chromosome 6, suggests that the human 21q22/mouse chromosome 17 conserved segment is of a very limited total physical size and is likely to contain a relatively small number of genes.     

A 10-megabase physical map of human Xp21, including the Duchenne muscular dystrophy gene

Using pulsed-field gel electrophoresis and 12 Xp21-derived DNA probes, we have constructed a continuous restriction map spanning more than 4 million base pairs (4 Mbp), including the Duchenne muscular dystrophy gene of more than 2 Mbp. This detailed map is part of a less detailed map spanning 10 Mbp, also spanning the genes for glycerol kinase and congenital adrenal hypoplasia, constructed under electrophoresis conditions which separated DNA fragments in the range 200 to 4000 kbp. DNA from three different tissues was analyzed, and differential methylation was observed.     

Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene

The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.     

Large-scale mapping and chromosome jumping in the q27 region of the human X chromosome

We have used pulsed field gel electrophoresis for further physical mapping studies in the q27 region of the human X chromosome. We show that the DXS 102 locus and the F9 gene are separated by only 300 kb despite a genetic distance of 1.4 cM; this linkage orients our large-scale map and shows that the mcf.2 transforming sequence is telomeric to F9. A BssHII complete-digest jumping library was used to jump toward the DXS 105 locus; a 130-kb jump was achieved and the corresponding "linking clone" was obtained.     

A new DNA marker (D4S90) is located terminally on the short arm of chromosome 4, close to the Huntington disease gene

Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5), which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a θ of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene.     

Labelling oligonucleotides to high specific activity (I).

The normal procedure for labelling oligonucleotides radioactively is the use of polynucleotide kinase and gamma 32P-ATP. However, this has the disadvantage of only introducing one labelled base per molecule of the oligonucleotide. In this paper we describe an approach based on primer/template combinations using conventional fill-in conditions followed by the release of the labelled sequence by digestion with uracil-DNA glycosylase.