cis-acting sequences involved in exon selection in the chicken beta-tropomyosin gene.

The chicken beta-tropomyosin gene contains an internal pair of mutually exclusive exons (6A and 6B) that are selected in a tissue-specific manner. Exon 6A is incorporated in fibroblasts and smooth muscle cells, whereas exon 6B is skeletal muscle specific. In this study we show that two different regions in the intron between the two mutually exclusive exons are important for this specific selection in nonmuscle cells. Sequences in the 3' end of the intron have a negative effect in the recognition of the 3' splice site, while sequences in the 5' end of the intron have a positive effect in the recognition of the 5' splice site. First, sequences in exon 6B as well as in the intron upstream of exon 6B are both able to inhibit splicing when placed in a heterologous gene. The sequences in the polypyrimidine stretch region contribute to splicing inhibition of exons 5 or 6A to 6B through a mechanism independent of their implication in the previously described secondary structure around exon 6B. Second, we have identified a sequence of 30 nucleotides in the intron just downstream of exon 6A that is essential for the recognition of the 5' splice site of exon 6A. This is so even after introduction of a consensus sequence into the 5' splice site of this exon. Deletion of this sequence blocks splicing of exon 6A to 6B after formation of the presplicing complex. Taken together, these results suggest that both the mutually exclusive behavior and the choice between exons 6A and 6B of the chicken beta-tropomyosin gene are trans regulated.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Identification of cis-acting sequences required for translational autoregulation of the ermC methylase.

ermC methylase gene expression has been shown to be limited by translational autorepression, presumably due to methylase binding to ermC mRNA. It was found that this repression occurs in trans, yielding a 50% reduction in translation of an ermC-lacZ fusion mRNA. We investigated the ermC mRNA sequences required for translational repression in vivo. A series of deletions identified sequences in the 5' regulatory region that were required for translational repression. These included sequences of the 5' stem-loop structure that were not required for induction, as well as some that were required. The implications of these results for regulation are discussed.