Roger J. Watson

B-MYB : A KEY REGULATOR OF THE CELL CYCLE

Publisher Summary The chapter describes that B-Myb gene is subjected to two putative cyclin-dependent regulatory mechanisms during the cell cycle that is upregulation of mRNA abundance by derepression of E2F/DRS-regulated transcription at the G1/S boundary, and phosphorylation of the protein during S phase. This dual mechanism has several features. First, it ensures that in quiescent cells, there is effectively no active B-Myb because transcription is greatly down regulated and the little protein that may be produced is not activated by cyclin-dependent kinases (Cdk)-mediated phosphorylation. Second, it allows for a huge increase in active B-Myb as cells go from the G1 phase into S phase. Third, in cycling cells, the B-Myb produced in S phase may be partially inactivated by dephosphorylation at later stages and only hyperactivated again on entry into the subsequent S phase. The precise role of B-myb in cell cycle regulation is still unknown. B-Myb as a transcriptional activator is foreseen to have function in regulation of other genes whose products are involved in synthesis of the components of nucleotide biosynthesis and DNA replication. As a transcriptional repressor, B-Myb is considered in extinguishing expression of negative regulators of cell proliferation. BMyb has the potential to activate transcription from promoters that lack Myb binding site (MBS), and these genes cannot be predicted from database searches. The realization that B-Myb is subject to controls on both its abundance and activity, that this directs maximal activity to S phase, and that B-Myb function appears to be necessary for cell proliferation leads to conclude that it plays a central role downstream of the cyclins in controlling the basic mechanisms that coordinate passage through the cell cycle.

Tandem repeated DNA in an intergenic region of Herpes simplex virus type 1 (Patton)

When the entire US region of HSV-1 (Patton) was cloned as an EcoRI fragment in bacteriophage lambda gtWES, the BamHI B6B5 fragment was observed to vary in size among independent isolates [Umene and Enquist, Gene 13 (1981) 251-268]. This fragment polymorphism also occurred in DNA of HSV-1 single plaque isolates. We report here that this heterogeneity is due to variation in copy number of a 15-bp tandem repeat of sequence 5-CCACTCCCCACCCAC-3, which apparently lies in an intergenic region of the HSV-1 DNA.

Interference of Myb transactivation activity by a conditional dominant negative protein: functional interference in a cytotoxic T-cell line results in G1 arrest

The ability to ablate the activity of specific transcription factors in vivo is a potentially important tool to study their roles in cellular processes such as the cell cycle. Previously, production of a dominant interfering c-Myb protein (comprising a fusion of the c-Myb DNA binding domain with the Drosophila Engrailed transrepressor) was found to inhibit the proliferation of immature thymocytes in the developing thymus of transgenic mice. We report here the further development of this stratagem by rendering the c-Myb/Engrailed protein conditionally active by fusion to a modified estrogen receptor hormone binding domain, ER. Co-transfection experiments in NIH 3T3 fibroblasts showed that the resulting chimeric protein, Myb/En/ER, repressed transactivation of a c-Myb-responsive reporter only in the presence of the synthetic steroid, 4-hydroxytamoxifen (OHT). Additionally, we found that Myb/En/ER could counteract transactivation by C/EBP-beta of the mim-1 promoter, which contains juxtaposed Myb and C/EBP binding sites. Cytotoxic T-cells stably producing the inactive Myb/En/ER protein were readily obtained by gene transfection. The addition of OHT to these cells resulted in inhibition of proliferation and arrest in G1. The utility of this experimental system to study Myb and other transcription factors is discussed.