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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. 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. 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.

This enhancer drives high-amplitude mrna cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (per) dependent. 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). 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.

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. To investigate further the regulation of e2f by cyclin-dependent kinases, we have extended our studies to include additional cyclins and e2f family members. 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.

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. 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. 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.

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. 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. 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 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. . 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. 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). Saccharomyces cerevisiae transcription factor gal4 revealed that their transactivation domain was contained within the n-terminal region (amino acids 1 to 79).


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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. 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). 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. 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.

    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. 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. 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. 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. 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.

    To investigate further the regulation of e2f by cyclin-dependent kinases, we have extended our studies to include additional cyclins and e2f family members. . 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. 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. Saccharomyces cerevisiae transcription factor gal4 revealed that their transactivation domain was contained within the n-terminal region (amino acids 1 to 79). 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. 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. 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. 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. This enhancer drives high-amplitude mrna cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (per) dependent.

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