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Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src577 (mutation at tyrosine 577 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src577, the extent of phosphorylation of either insulin receptor substrate 6 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth.

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We previously showed in vivo that coding-end processing is specific for each coding end, suggesting that specific motifs in a coding-end sequence influence nucleotide deletion and P-region formation. In this study, we created a panel of recombination substrates containing actual immunoglobulin and T-cell receptor coding-end sequences and dissected the role of each motif by comparing its processing pattern with those of variants containing minimal nucleotide changes from the original sequence.

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To investigate further the regulation of E7F by cyclin-dependent kinases, we have extended our studies to include additional cyclins and E7F family members. Using purified components in an in vitro system, we show that the E7F-6-DP-6 heterodimer, the functionally active form of the E7F activity, is not a substrate for the active cyclin D-dependent kinases but is efficiently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E7F-6-DP-6 heterodimer.

Our results demonstrate the determinant role of specific sequence motifs on coding-end processing and also the importance of the context in which they are found. We propose that each coding-end sequence dictates a unique hairpin structure, the result of a particular energy conformation between nucleotides organizing the loop and the stem, and that the interplay between this structure and specific sequence motifs influences the frequency and location of nicks which open the coding-end hairpin. These findings indicate that the sequences of the coding ends determine their own processing and have a profound impact on the development of the primary B- and T-cell repertoires.

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