The recent discovery of sequences at the site of the Duchenne muscular dystrophy (DMD) gene in humans has opened up the possibility of a detailed molecular analysis of the genes in humans and in related mammalian species. Until relatively recently, there was no obvious mouse model of this genetic disease for the development of therapeutic strategies. The identification of a mouse X-linked mutant showing muscular dystrophy, mdx, has provided a candidate mouse genetic homologue to the DMD locus; the relatively mild pathological features of mdx suggest it may have more in common with mutations of the Becker muscular dystrophy type at the same human locus, however. But the close genetic linkage of mdx to G6PD and Hprt on the mouse X chromosome, coupled with its comparatively mild pathology, have suggested that the mdx mutation may instead correspond to Emery Dreifuss muscular dystrophy which itself is closely linked to DNA markers at Xq28-qter in the region of G6PD on the human X chromosome. Using an interspecific mouse domesticus/spretus cross, segregating for a variety of markers on the mouse X chromosome, we have positioned on the mouse X chromosome sequences homologous to a DMD cDNA clone. These sequences map provocatively close to the mdx mutation and unexpectedly distant from sparse fur, spf, the mouse homologue of OTC (ornithine transcarbamylase) which is closely linked to DMD on the human X chromosome.

A large number of microclones obtained by microdissection of the mouse X chromosome have been mapped using an interspecific Mus domesticus/Mus spretus cross. Clones displaying close linkage to a number of loci of known phenotype but unknown gene product, such as mdx (X‐linked muscular dystrophy), have been obtained. Over a central 30 cM span of the mouse X chromosome, 17 clones have been mapped and ordered at a sufficient density to contemplate the complete physical mapping of this region that will aid in the isolation of a number of unidentified genes. Some of the mapped microclones detect moderately repetitive sequences that were clustered in several discrete regions of the mouse X chromosome.

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A wild mouse (CD) karyotype in which all the chromosomes bar the X, 19, and Y, are fused as metacentrics has been used for the microdissection and microcloning of a specific mouse X chromosome region. Dissection of a proximal region of the X chromosome encompassing the genetic loci Hprt to Tfm and including mdx has yielded 650 clones. A number of the recovered clones containing sizable inserts have been confirmed as X chromosome specific. This X chromosome bank ofclones provides a start point for the isolation ofthe mdx locus.It is now clear that microdissection and microcloning can be applied to all mouse chromosomes, including the X chromosome, yielding premapped banks of clones that will greatly aid in the isolation and characterization of important genetic loci.The mouse X chromosome has become a paradigm for the study of a variety of biological phenomena of molecular interest: X chromosome inactivation (1, 2), sex determination (3-6), and the comparative evolution of mammalian chromosomes (7,8 methanol/acetic acid (3:1) and spread by air-drying. Metaphase spreads were stained with 1% Giemsa for photography.Microdissection and Microcloning. Unstained metaphase spreads of the CD strain were prepared on coverslips according to the methods described above. Coverslips were inverted over an oil chamber and chromosomes were dissected by using glass microneedles with the aid of a de Fonbrunne micromanipulator as described (11,12). All manipulations were observed under a phase-contrast microscope with a long working distance. Specially fine microneedles were drawn on a de Fonbrunne microforge and were used for the microdissection of the proximal region of X chromosomes well separated in the metaphase plate. After dissection, DNA was prepared and cloned essentially according to published micromethods (11, 12) but with some modifications. Briefly, fragments were pooled in a drop of proteinase K/0.5% NaDodSO4 residing on a separate coverslip in the oil chamber. After dissection was completed, the oil chamber was incubated at 37TC for 2 hr. After incubation, the nanoliter drop was extracted several times with phenol. After chloroform extraction to remove residual phenol the nanoliter drop was transferred to a fresh oil chamber and a nanoliter drop of restriction enzyme (EcoRI, 50 units/hd) was added and digestion proceeded by incubation at 370C for 2 hr. Phenol extraction to inactivate enzyme and chloroform treatment were repeated with subsequent transferance of the microdrop to a fresh oil chamber. A nanoliter microdrop ('400 pg) of EcoRI-digested vector, XgtlO, was added, followed by a nanoliter microdrop (0.4 unit) of ligase. Ligation proceeded overnight at 4°C. After ligation, the microdrop was taken up in several microliters of buffer for in vitro packaging. Recombinants were selected by plating on an Escherichia coli C600hflA-host (14, 15). Individual plaques were picked and plate stocks were prepared by propagation on SM32 (16) using standard procedures.Analysis of Microclones. A plate lysate method was u...

While increasing access to medical information is desirable, this information can be harmful if it is inaccurate, does not discuss risks or encourages people to use drugs in ways that were not intended. In the UK misoprostol is most commonly prescribed in the combination agent misoprostol/diclofenac (Arthrotec, Searle). There are, however, no reports of the combination being used as an abortifacient10.