The papillomavirus E2 gene product plays a pivotal role in viral replication. E2 has multiple functions, including (i) transcriptional activation and repression of viral promoters and (ii) the enhancement of viral DNA replication. It was previously reported that E2 suppressed the growth of papillomavirus-positive cervical carcinoma cell lines. In the present study, we investigated the mechanisms of E2 growth inhibition. We found that the transcriptional activation function of E2 is required for inhibition of the growth of HeLa cells as well as for transcriptional repression of the viral E6/E7 promoter. It had been previously postulated that transcriptional repression of the E6/E7 promoter results from E2 binding its cognate sites proximal to the E6/E7 promoter and displacing other cellular transcriptional factors. In this study, we report a requirement for the transcription activation function for the binding of E2 to transcriptionally active templates.The papillomavirus replication cycle is regulated by the viral E2 protein, a sequence-specific DNA binding protein (1,35,53). Depending on the promoter context, E2 can act either as a transcriptional activator or as a repressor of viral gene expression. The promoters for E6/E7 gene expression of human papillomavirus type 16 (HPV16) and HPV18 are negatively regulated by E2. This repression is thought to be mediated by the binding of E2 to its recognition sites within the promoter and the displacement of cellular transcriptional factors from the promoter (3,12,14,15,20,23,28,41,42,54,55,58,59,61,62). E2 is also involved in the regulation of viral DNA replication through its association with E1, the viral replication factor (36,50,63,65,66,67,68). The conserved N-terminal domain of E2 is required for transactivation (TA), E1 binding, and DNA replication functions. The conserved C-terminal domain forms a dimer and functions as a DNA binding domain. Both conserved domains are linked by a hinged region (reviewed in reference 24).The loss of E2 expression has been also implicated in the development of HPV-induced carcinoma. Most human cervical carcinoma cells contain integrated HPV DNA and actively express E6/E7 genes (2, 52, 69). The E2 gene is frequently disrupted as a consequence of the integration of the viral genome, and it has been postulated that the loss of E2 somehow contributes to carcinogenic progression (9,40,47,64). E6/E7 genes are invariably expressed in HPV-positive cancers and are considered to be involved in the development of HPVassociated cancers. E6 targets the ubiquitination and proteolysis of p53 through its association with the ubiquitin protein ligase, E6AP (25,45,46). E7 binds pRB and inactivates its tumor suppressor function (17,38). Although E6 and E7 may have additional functions and cellular targets, it is believed that their inactivation of these important tumor suppressor proteins is critical for HPV-associated carcinogenesis. As mentioned above, E2 has the ability to suppress E6/E7 expression; thus, disruption of the E2 gene results in the...

We made a library of Saccharomyces cerevisiae F 1 hybrids from all possible crosses of 16 wild-type strains, including two common laboratory strains and two commercial winemaking varieties. Fourteen of the starting strains have been sequenced. Thus, the sequences of both genomes are known in 182 novel hybrids, and the sequence of one genome is known in 56. All tested strains sporulated. Fertilities were in the range 0-100%. Hybrids showed no more variation than parental strains for ethanol production, ethanol tolerance or growth at temperature extremes, but some F 1 s appeared to display hybrid vigour (heterosis). We tested four tetrads from one hybrid for their ability to grow at low temperature or in the presence of an inhibitory concentration of ethanol. Only one F 2 was as tolerant as the most tolerant F 0 parent. A few showed intermediate tolerance, but most were less tolerant than either parent or the F 1 hybrid, consistent with uncoupling of genes contributing to an optimized quantitative trait. The diversity and structure of the library should make it useful for analysis of genetic interactions among diverse strains, quantitative inheritance and heterosis, and for breeding.

The budding yeast Saccharomyces cerevisiae has many traits that make it useful for studies of quantitative inheritance. Genome-wide association studies and bulk segregant analyses often serve as first steps toward the identification of quantitative trait loci. These approaches benefit from having large numbers of ascospores pooled by mating type without contamination by vegetative cells. To this end, we inserted a gene encoding red fluorescent protein into the MATa locus. Red fluorescent protein expression caused MATa and a/α diploid vegetative cells and MATa ascospores to fluoresce; MATα cells without the gene did not fluoresce. Heterozygous diploids segregated fluorescent and nonfluorescent ascospores 2:2 in tetrads and bulk populations. The two populations of spores were separable by fluorescence-activated cell sorting with little cross contamination or contamination with diploid vegetative cells. This approach, which we call Fluorescent Ascospore Technique for Efficient Recovery of Mating Type (FASTER MT), should be applicable to laboratory, industrial, and undomesticated, strains.

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