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    Thus, the activities of e2f, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators the retinoblastoma protein family and the cyclin-dependent kinase family. Using purified components in an in vitro system, we show that the e2f-1-dp-1 heterodimer, the functionally active form of the e2f 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 e2f-1-dp-1 heterodimer. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional igf-i receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.

    Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 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. Saccharomyces cerevisiae transcription factor gal4 revealed that their transactivation domain was contained within the n-terminal region (amino acids 1 to 79). An e-box sequence within this 69-bp fragment is necessary for high-level expression, but not for rhythmic expression, indicating that per mediates circadian transcription through other sequences in this fragment.

    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-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of igf-i receptors (w cells). Depending on the ph of the growth medium, the yeast yarrowia lipolytica secretes both an acidic proteinase and an alkaline proteinase, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Phosphorylation of the e2f-1-dp-1 heterodimer by cyclin b-dependent kinases, however, did not result in down-regulation of its dna-binding activity, as is readily seen after phosphorylation by cyclin a-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate e2f dna-binding activity.

    To investigate further the regulation of e2f by cyclin-dependent kinases, we have extended our studies to include additional cyclins and e2f family members. Detailed mutagenesis of this region indicated that transactivation is mediated by three highly conserved sequences, spanning amino acids 13 to 22 (subdomain a), 32 to 38 (subdomain b), and 60 to 73 (subdomain c). R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor i (igf-i) receptor genes, are refractory to transformation by several viral and cellular oncogenes.

    This enhancer drives high-amplitude mrna cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (per) dependent. The transcription factor e2f-1 interacts stably with cyclin a via a small domain near its amino terminus and is negatively regulated by the cyclin a-dependent kinases. 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.

    . 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 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. 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. 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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    Thus, the activities of e2f, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators the retinoblastoma protein family and the cyclin-dependent kinase family. . An e-box sequence within this 69-bp fragment is necessary for high-level expression, but not for rhythmic expression, indicating that per mediates circadian transcription through other sequences in this fragment. Depending on the ph of the growth medium, the yeast yarrowia lipolytica secretes both an acidic proteinase and an alkaline proteinase, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. 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.

    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. 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-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of igf-i receptors (w cells). 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. Using purified components in an in vitro system, we show that the e2f-1-dp-1 heterodimer, the functionally active form of the e2f 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 e2f-1-dp-1 heterodimer. 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.

    Saccharomyces cerevisiae transcription factor gal4 revealed that their transactivation domain was contained within the n-terminal region (amino acids 1 to 79). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 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. This enhancer drives high-amplitude mrna cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (per) dependent. 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. The transcription factor e2f-1 interacts stably with cyclin a via a small domain near its amino terminus and is negatively regulated by the cyclin a-dependent kinases. To investigate further the regulation of e2f by cyclin-dependent kinases, we have extended our studies to include additional cyclins and e2f family members. Phosphorylation of the e2f-1-dp-1 heterodimer by cyclin b-dependent kinases, however, did not result in down-regulation of its dna-binding activity, as is readily seen after phosphorylation by cyclin a-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate e2f dna-binding activity. Detailed mutagenesis of this region indicated that transactivation is mediated by three highly conserved sequences, spanning amino acids 13 to 22 (subdomain a), 32 to 38 (subdomain b), and 60 to 73 (subdomain c). These results indicate that v-src, but not c-src527, can bypass the requirement for a functional igf-i receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation. R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor i (igf-i) receptor genes, are refractory to transformation by several viral and cellular oncogenes.

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