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.     

Genetic and radiation hybrid mapping of the hyperekplexia region on chromosome 5q.

Hyperekplexia, or startle disease (STHE), is an autosomal dominant neurologic disorder characterized by muscular rigidity of central nervous system origin, particularly in the neonatal period, and by an exaggerated startle response to sudden, unexpected acoustic or tactile stimuli. STHE responds dramatically to the benzodiazepine drug clonazepam, which acts at gamma-aminobutyric acid type A (GABA-A) receptors. The STHE locus (STHE) was recently assigned to chromosome 5q, on the basis of tight linkage to the colony-stimulating factor 1-receptor (CSF1-R) locus in a single large family. We performed linkage analysis in the original and three additional STHE pedigrees with eight chromosome 5q microsatellite markers and placed several of the most closely linked markers on an existing radiation hybrid (RH) map of the region. The results provide strong evidence for genetic locus homogeneity and assign STHE to a 5.9-cM interval defined by CSF1-R and D5S379, which are separated by an RH map distance of 74 centirays (roughly 2.2-3.7 Mb). Two polymorphic markers (D5S119 and D5S209) lie within this region, but they could not be ordered with respect to STHE. RH mapping eliminated the candidate genes GABRA1 and GABRG2, which encode GABA-A receptor components, by showing that they are telomeric to the target region.     

Diurnal variation of corticotropin-releasing factor binding sites in the rat brain and pituitary

Summary 1. Corticotropin-releasing factor (CRF) is thought to be involved in the regulation of the diurnal activity of the hypothalamus-pituitary-adrenal (HPA) axis and to act as a neurotransmitter in the brain. To date it is unknown whether the binding sites of the central CRF system are subject to diurnal variations. 2. We measured the number of CRF binding sites over the course of a complete 24-hr light-dark cycle in the pituitary, amygdala, bed nucleus of the stria terminalis (BNST), cingulate cortex, visceral cortex, paraventricular nucleus of the hypothalamus, hippocampus, and locus ceruleus of rats byin vitro receptor autoradiography with iodinated ovine CRF. A 24-hr time course was also established for plasma CRF and corticosterone. 3. The diurnal pattern of plasma CRF does not correlate with the pattern of plasma corticosterone. Within the brain, CRF binding in the basolateral nucleus of the amygdala showed a U-shaped curve with maximum levels in the morning and a wide hallow between 1500 and 0100. A biphasic profile with a small depression in the afternoon and a more pronounced depression in the second half of the activity period is characteristic for the other brain areas and the pituitary. The profile for the pituitary correlates with those for the BNST and the area of the locus ceruleus. Furthermore, the diurnal pattern of CRF binding sites in the BNST correlates with that of the hippocampus, and the daytime pattern of the visceral cortex is similar to that of both the hippocampus and the BNST. 4. Since the CRF-binding profiles in the brain and the pituitary clearly differ from the profiles of both plasma CRF and corticosterone, one may assume that the diurnal pattern of central CRF binding sites is not directly coupled to the activity of the HPA axis.     

A YAC contig encompassing the Treacher Collins syndrome critical region at 5q31.3-32.

Treacher Collins syndrome (TCOF1) is an autosomal dominant disorder of craniofacial development the features of which include conductive hearing loss and cleft palate. Previous studies have localized the TCOF1 locus between D5S519 (proximal) and SPARC (distal), a region of 22 centirays as estimated by radiation hybrid mapping. In the current investigation we have created a contig across the TCOF1 critical region, using YAC clones. Isolation of a novel short tandem repeat polymorphism corresponding to the end of one of the YACs has allowed us to reduce the size of the critical region to approximately 840 kb, which has been covered with three nonchimeric YACs. Restriction mapping has revealed that the region contains a high density of clustered rare-cutter restriction sites, suggesting that it may contain a number of different genes. The results of the present investigation have further allowed us to confirm that the RPS14 locus lies proximal to the critical region and can thereby be excluded from a role in the pathogenesis of TCOF1, while ANX6 lies within the TCOF1 critical region and remains a potential candidate for the mutated gene.     

Use of Genetic and Physical Mapping to Locate the Spinal Muscular Atrophy Locus between Two New Highly Polymorphic DNA Markers

The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5ql3, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers—namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)—which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen–D5S6–D5S125/D5S465–D5S435–D5S629–SMA–D5S637–D5S351–MAP–1B/D5S112–D5S357–D5S39–tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene.     

The human skeletal muscle adenine nucleotide translocator gene maps to chromosome 4q35 in the region of the facioscapulohumeral muscular dystrophy locus

Facioscapulohumeral muscular dystrophy (FSHD) is a relatively common autosomal dominant neuromuscular disorder. The gene for FSHD has recently been assigned to chromosome 4q35. Although abnormal mitochondrial and biochemical changes have been observed in FSHD, the molecular defect is unknown. In addition to the FSHD gene, the human muscle adenine nucleotide translocator gene (ANT1) is located on chromosome 4. Interestingly, biochemical studies recently showed a possible defect of ANT1. In order to evaluate the potential role of ANT1 in the etiology of FSHD, the human ANT1 gene was isolated by cosmid cloning and localized to 4q35, in the region containing the FSHD gene. However, in situ hybridization and physical mapping of somatic cell hybrids localized the ANT1 gene proximal to the FSHD gene. In addition, a polymorphic CA-repeat 5 kb upsstream of the ANT1 gene was used as a marker in FSHD and Centre d'Etude du Polymorphisme Humain families to perform linkage analysis. These data together exclude ANT1 as the primary candidate gene for FSHD. The most likely order of the loci on chromosome 4q35 is cen-ANT1-D4S171-F11-D4S187-D4S163-D4S139-FSHD-tel.     

Genetic demonstration of mitotic recombination in cultured Chinese hamster cell hybrids

Chinese hamster ovary cell hybrids were constructed that are heterozygous for two markers, leuS and emtB, linked to the long arm of chromosome 2. In addition, the chromosome 2 carrying the wild-type leuS and emtB alleles contains, on its short arm, a homogeneously staining region (hsr) in which the gene encoding dihydrofolate reductase (dhfr) is amplified approximately 50-fold. This provides a convenient cytogenetic and biochemical means to distinguish the chromosome 2s from the different parents. Analysis of emetine-resistant segregants isolated from such hybrids identified three distinct classes of segregants. One rare class of segregants loses the wild-type leuS and emtB gene functions on the long arm of the hsr chromosome 2 (H-2) but retains the amplified dhfr genes on the opposite arm. Detailed genetic analysis of two such segregants that did not arise by chromosome loss or deletion revealed that new gene linkage relationships had been established on the H-2 chromosome in each, demonstrating that the segregation events in these cell lines involved mitotic recombination.     

Identification and characterization of a third complementation group of emetine-resistant Chinese hamster cell mutants.

We have isolated emetine-resistant cell lines from Chinese hamster peritoneal fibroblasts and have shown that they represent a third distinct class or complementation group of emetine-resistant mutants, as determined by three different criteria. These mutants, like those belonging to the two other complementation groups we have previously defined, which were isolated from Chinese hamster lung and Chinese hamster ovary cells, have alterations that directly affect the protein biosynthetic machinery. So far, there is absolute cell line specificity with respect to the three complementation groups, in that all the emetine-resistant mutants we have isolated from Chinese hamster lung cells belong to one complementation group, all those we have isolated from Chinese hamster ovary cells belong to a second complementation group, and all those isolated from Chinese hamster peritoneal cells belong to a third complementation group. Thus, in cultured Chinese hamster cells, mutations in at least three different loci, designated emtA, emtB, and emtC, encoding for different components of the protein biosynthetic machinery, can give rise to the emetine-resistant phenotype.