Extracellular calcium modulates vitamin D-dependent calbindin-D28K gene expression in chick kidney cells.

The effect of extracellular calcium ion (Ca2+) concentration on 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induction of vitamin D-dependent calcium-binding protein (calbindin-D28K) and its mRNA levels was examined in primary chick kidney cells in vitro. When exposed to normal medium Ca2+ (1.0 mM), 1,25-(OH)2D3 increased calbindin-D28K mRNA, as measured by Northern analysis, by 4-10 fold over basal levels by 12 to 24 h after addition of hormone. In the presence of 0.5 mM Ca2+, 1,25-(OH)2D3 induced calbindin-D28K mRNA by only 2 fold, whereas, when cells were exposed to 2 mM Ca2+, the induction was 10-15 fold. This calcium modulation of 1,25-(OH)2D3 induction was also observed at the level of calbindin-D28K protein concentrations as measured by radioimmunoassay. The alterations in medium Ca2+ were not associated with any change in the rate of total RNA or protein synthesis. These studies suggest that both Ca2+ and 1,25-(OH)2D3 participate in the regulation of calbindin-D28K gene expression in the kidney.

Tissue-specific regulation of avian vitamin D-dependent calcium-binding protein 28-kDa mRNA by 1,25-dihydroxyvitamin D3.

We have studied the regulation, by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), of vitamin D-dependent calcium-binding protein (28-kDa CaBP) mRNA in chick tissues in vivo. Northern analysis of poly(A)+ RNA was carried out using, as hybridization probes, synthetic oligonucleotides complementary to chick 28-kDa CaBP mRNA. In vitamin D-deficient chicks, 28-kDa CaBP mRNA was virtually undetectable in intestine, was clearly detectable in kidney, and present at the highest levels in cerebellum. After a single intravenous dose of 500 ng of 1,25-(OH)2D3, intestinal 28-kDa CaBP mRNA levels were increased 50-fold, kidney levels were increased 4-fold, and cerebellum levels were unchanged. Increased levels of 28-kDa CaBP mRNA were appreciated 2 h after induction and were maximal at 12 h. Pretreatment of vitamin D-deficient chicks with actinomycin D had little effect on the acute phase of the 1,25-(OH)2D3 induction of 28-kDa CaBP mRNA in intestine but blunted the induction in kidney. Pretreatment with cycloheximide caused a delayed response to 1,25-(OH)2D3 in the intestine, although control (noninhibition) levels of 28-kDa CaBP mRNA were present 12 h after hormone administration. By contrast, in the kidney, cycloheximide pretreatment resulted in an increased steady-state (vitamin D-deficient) level of 28-kDa CaBP mRNA, but completely abolished the induction of 1,25-(OH)2D3. Our studies indicate that, whereas 1,25-(OH)2D3 does not regulate 28-kDa CaBP mRNA levels in the brain, the hormone modulates 28-kDa CaBP gene expression in intestine and kidney in a tissue-specific manner, by acting through both transcriptional and post-transcriptional mechanisms.

Possibilities and problems in genomic diagnosis of Duchenne muscular dystrophy with molecular probes.

Selected families affected with Duchenne muscular dystrophy from an extensive pedigree analysis with linked restriction fragment length polymorphisms and creatine kinase estimations are compiled to demonstrate the various counselling situations for carrier determination and prenatal diagnosis. The creatine kinase determination suffers from approximately 30% false negative values in carrier determination. The DNA analysis is limited by the uninformativity of the DNA markers and the occurrence of meiotic crossovers between the particular restriction fragment length polymorphism pattern and the Duchenne muscular dystrophy locus. The use of markers bridging the Duchenne muscular dystrophy locus, such as 754 and C7, is presently best suited for this purpose but is applicable to only a relatively small number of cases. The use of the physically closer probe, pERT 87, is much more informative althrough it too recombines with the Duchenne muscular dystrophy locus. DNA analysis allows prenatal diagnosis for unaffected boys from the restriction fragment length polymorphism pattern confined to the healthy grandpaternal X-chromosome in cases where the carrier status of the mother is established or in doubt. As in the case of carrier determination, crossover events and uninformativity of restriction fragment length polymorphisms limit the feasibility of this approach.

Human X chromosome markers and Duchenne muscular dystrophy.

Two DNA markers, a random DNA fragment 754 and the cDNA sequence encoding the gene for ornithine transcarbamylase (OTC) have been studied in kindreds segregating for Duchenne muscular dystrophy. 754 and OTC are located close physically to the mutation in the region Xp21 below the breakpoints in two Duchenne females. The genetic distance was found to be approximately 10cM between 754 and DMD (two crossovers in 26 meioses) and to be approximately 10cM between OTC and DMD (two crossovers in 26 meioses). Physical data suggest the order DMD-754-OTC. The frequency of recombination compared to physical distance between these markers and DMD suggests that there may be a hot spot of recombination. The relevance of these observations for the isolation of the DMD mutation and clinical use of these probes is discussed.

Linkage studies of X-linked mental retardation: high frequency of recombination in the telomeric region of the human X chromosome (fragile site/linkage/recombination/X chromosome).

One of the commonest forms of X-linked mental retardation is associated with a fragile site at Xq27 on the human X chromosome which can be visualised structurally after culturing cells in folate-deficient media. Unusually, the mutation can be transmitted through a phenotypically normal male. There is already some evidence that the gene loci for G6PD and factor IX are linked to this mental retardation locus. We have followed the inheritance of a DNA sequence 52A, in fragile site families that are also informative for factor IX. We demonstrate that these probes are localised at Xq27/Xq28-Xqter, close physically to the fragile site. We did not find close linkage between 52A, factor IX, and the fragile site in the families studied despite 52A and factor IX showing linkage in normal families. We discuss the importance of these data for the genetic mapping of this region of the human X chromosome and the implication for the use of these DNA probes for clinical diagnosis.

Analysis of deletions in DNA from patients with Becker and Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder for which the biochemical defect is as yet unknown. Recently, two cloned segments of human X-chromosome DNA have been described which detect structural alterations within or near the genetic locus responsible for the disorder. Both of these cloned segments were described as tightly linked to the locus and were capable of detecting deletions in the DNA of boys affected with DMD. In an attempt to determine more precisely the occurrence of these deletions within a large population of DMD patients and the accuracy of one of the segments, DXS164 (pERT87), in determining the inheritance of the DMD X chromosome, the subclones 1, 8 and 15 were made available to many investigators throughout the world. Here we describe the combined results of more than 20 research laboratories with respect to the occurrence of deletions at the DXS164 locus in DNA samples isolated from patients with DMD and Becker muscular dystrophy (BMD). The results indicate that the DXS164 locus apparently recombines with DMD 5% of the time, but is probably located between independent sites of mutation which yield DMD. The breakpoints of some deletions are delineated within the DXS164 locus, and it is evident that the deletions at the DMD locus are frequent and extremely large.