The application of recombinant DNA technology to prenatal diagnosis of many recessively inherited X-linked diseases is complicated by a high frequency of heterogeneous, new mutations (1). Partial gene deletions account for more than 50% of Duchenne muscular dystrophy (DMD) lesions, and approximately one-third of all cases result from a new mutation (2-5). We report the isolation and DNA sequence of several deletion prone exons from the human DMD gene. We also describe a rapid method capable of detecting the majority of deletions in the DMD gene. This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus. We demonstrate the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.

The trb operon of broad-host-range plasmid RK2 encodes most of the genes required for formation of mating-pair apparatus and is thus essential for the promiscuous spread of this plasmid. Only two promoters, lying upstream of trbA and trbB, have been identified for this operon. trbB encodes a protein belonging to a large family of proteins which function in the assembly of apparatuses associated with the cell surface. trbA encodes a repressor protein, one of whose targets is the trbB promoter. trbAp is arranged as a face-to-face divergent promoter with trfAp, the strongest of the three promoters in this region. trfAp completely inhibits trbAp unless it is repressed by the KorA protein, a key regulator encoded in the plasmid's central control operon. We show that when trfAp is firing constitutively, it also appears to interfere with trbBp, but that trbBp activity increases when trfAp activity is decreased by repression or mutation. A second global regulator encoded in the central control operon, KorB, represses trbBp, trfAp, and trbAp. The results presented here show that both KorB and TrbA are necessary for full repression of trbBp. The region between trbA and trbB encodes a large inverted repeat which has been proposed to modulate translation of trbB on transcripts which are initiated at trbAp but not trbBp. Using translational fusions to lacZ, we show that translation of trbB is completely blocked when transcripts incorporate the inverted repeat upstream of trbB but proceeds with reasonable efficiency when deletions remove the sequences predicted to sequester the ribosome binding site. Results from both transcriptional fusion and direct measurement of transcript size and intensity by Northern blot analysis show that most trbA transcripts are monocistronic and serve to express only trbA, although some transcription continues into trbB. The monocistronic trbA transcript appears to be the result of transcription termination downstream of trbA. Thus, trbAp and trbA appear to form an operon distinct from the trbB-trbP operon. Consequently, trbA and the switch that controls its expression help to provide the sequential steps which allow efficient expression of transfer genes during plasmid establishment but tight repression once the plasmid is established.

The complete nucleotide sequence of a naturally occurring Staphylococcus aureus plasmid, pT48 (from S. aureus strain T48), has been determined. The 2475 bp plasmid confers inducible resistance to macrolide-lincosamide-streptogramin B (MLS) type antibiotics. It is similar to the constitutive MLS resistance plasmid, pNE131, from Staphylococcus epidermidis and shows homology with S. aureus plasmids pSN2 and pE194. It contains a palA structure homologous to that on S. aureus plasmid pT181. The open reading frame, ORF B, within the pSN2 homologous region has a frameshifted C-terminus, relative to pNE131, resulting in a smaller, 158 amino acid putative polypeptide. The pE194 homologous region has the ermC resistance determinant and retains the leader region, deleted in pNE131, required for inducible expression of an adenine methylase. Another naturally occurring S. aureus strain, J74, shows constitutive resistance to erythromycin and contains a small plasmid, pJ74, which is similar to pNE131 but with a different deletion in the leader sequence. The results are consistent with the translational attenuation model for ermC expression.