RFC5

Abstract: These experiments identify an Epstein-Barr virus-encoded gene product, called ZEBRA (BamHI fragment Z Epstein-Barr replication activator) protein, which activates a switch between the latent and replicative life cycle of the virus. Our previous work had shown that the 2.7-kilobase-pair WZhet piece of rearranged Epstein-Barr virus DNA from a defective virus activated replication when introduced into cells with a latent genome, but it was not clear whether a protein product was required for the phenomenon. We now use deletional, site-directed, and chimeric mutagenesis, together with gene transfer, to show that a 43-kilodalton protein, encoded in the BZLF1 open reading frame of het DNA, is responsible for this process. The rearrangement in defective DNA does not contribute to the structural gene for the protein. Similar proteins with variable electrophoretic mobility (37 to 39 kilodaltons) were encoded by BamHI Z fragments from standard, nondefective Epstein-Barr virus genomes. Plasmids expressing the ZEBRA proteins from B95-8 and HR-1 viruses were less efficient at activating replication in D98/HR-1 cells than those which contained the ZEBRA gene from the defective virus. It is not yet known whether these functional differences are due to variations in expression of the plasmids or to intrinsic differences in the activity of these polymorphic polypeptides.

Abstract: Replication factor-A (RF-A) is a three-subunit protein complex originally purified from human cells as an essential component for SV40 DNA replication in vitro. We have previously identified a functionally homologous three-subunit protein complex from the yeast Saccharomyces cerevisiae. Here we report the cloning and characterization of the genes encoding RF-A from S. cerevisiae. Each of the three subunits is encoded by a single essential gene. Cells carrying null mutations in any of the three genes arrest as budded and multiply budded cells. All three genes are expressed in a cell-cycle-dependent manner; the mRNA for each subunit peaks at the G1/S-phase boundary. A comparison of protein sequences indicates that the human p34 subunit is 29% identical to the corresponding RFA2 gene product. However, expression of the human protein fails to rescue the rfa2::TRP1 disruption.

Abstract: The DNA binding subunit of nuclear factor kappa B (NF-kappa B), a B-cell protein that interacts with the immunoglobulin kappa light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor alpha (TNF alpha), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-kappa B protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-kappa B binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNF alpha or phorbol ester. Thus, both factors not only activate NF-kappa B protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-kappa B.