Synthetic oligonucleotides containing GC-rich triplet sequences were used in a scanning strategy to identify unstable genetic sequences at the myotonic dystrophy (DM) locus. A highly polymorphic GCT repeat was identified and found to be unstable, with an increased number of repeats occurring in DM patients. In the case of severe congenital DM, the paternal triplet allele was inherited unaltered while the maternal, DM-associated allele was unstable. These studies suggest that the mutational mechanism leading to DM is triplet amplification, similar to that occurring in the fragile X syndrome. The triplet repeat sequence is within a gene (to be referred to as myotonin-protein kinase), which has a sequence similar to protein kinases.

An unstable expansion of the CTG repeat in the 3' untranslated region of the myotonin protein kinase (MT-PK) gene is the mutation specific for myotonic dystrophy (DM). To examine somatic stability of the repeat, we studied tissue variability of the repeat size. In five DM patients, the restriction fragment containing the repeat region was substantially larger in skeletal muscle than in peripheral blood leukocytes (PBL). In addition, one normal subject showed a size discrepancy in one of the normal alleles by one repeat on the polymerase chain reaction analysis. In most DM patients, the repeat size of native PBL differed from the transformed lymphoblastoid cells after passages. In contrast, various tissues from a congenital DM patient showed a similar size of the expanded repeat, including the transformed lymphoblastoid cells. We conclude that somatic instability of the CTG repeat may cause substantial tissue variability of the CTG repeat size in adult-onset DM, providing a potential mechanism for the variable pleiotropism.

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L-form (L-phase) cultures of Clostridium perfringens were tested for their transformability with plasmid DNA. Three L-form strains were transformable, but one, strain L-13, was superior to the others. This strain was easily and reproducibly transformed with previously described shuttle vectors which were derived from either C. perfringens or Escherichia coli. Strain L-13 was transformable by a variety of methods, and a new micromethod worked well under both aerobic and anaerobic conditions. The maximal number of transformants was attained after strain L-13 was exposed for 4 h to the transforming DNA and polyethylene glycol. Viable counts determined in tubes of semisolid brain heart infusion medium containing 10% sucrose, with or * Corresponding author.

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A method for detection of the M and Z alleles of the alpha 1-antitrypsin deficiency (AAT) gene has been developed using denaturing gradient gel electrophoresis (DGGE) of DNA samples amplified in vitro by the polymerase chain reaction. Amplification of the 90 nucleotides surrounding the Z mutation site with concurrent attachment of a 40 bp GC-rich region yields DNA fragments that are easily and quickly separated by DGGE. Results are consistently attained in 1-2 days, making this one of the most rapid method of diagnosis of AAT deficiency to date. Additionally, the analyses are completed entirely without the use of radioactive probes, thus eliminating the problems and precautions that are inherent with the use of 32P. The simplicity and reliability of this technique make it well suited for routine use in diagnostic laboratories.