Complete nucleotide sequence of a mouse VL30 retro-element.

The complete nucleotide sequence of a mouse retro-element is presented. The cloned element is composed of 4,834 base pairs (bp) with long terminal repeats of 568 bp separated by an internal region of 3,698 bp. The element did not appear to have any open reading frames that would be capable of encoding the functional proteins that are normally produced by retro-elements. However, some regions of the genome showed some homology to retroviral gag and pol open reading frames. There was no region in VL30 corresponding to a retroviral env gene. This implies that VL30 is related to retrotransposons rather than to retroviruses. The sequence also contained regions that were homologous to known reverse transcriptase priming sites and viral packaging sites. These observations, combined with the known transcriptional capacity of the VL30 promoter, suggest that VL30 relies on protein functions of other retro-elements, such as murine leukemia virus, while maintaining highly conserved cis-active promoter, packaging, and priming sites necessary for its replication and cell-to-cell transmission.

Expression of the FOX1 gene of Saccharomyces cerevisiae is regulated by carbon source, but not by the known glucose repression genes.

We have investigated the regulation of expression of the FOX1 gene of Saccharomyces cerevisiae which encodes acyl-CoA oxidase, the first enzyme in the peroxisomal beta oxidation of fatty acids. We have found that the FOX1 steady state mRNA level is repressed by glucose, partially induced by ethanol (but not by raffinose) and fully induced by oleic acid as a carbon source. Glucose repression was observed even if cultures were grown to stationary phase; however, if the glucose supply was limited initially then partial induction of FOX1 mRNA occurred upon growth to high cell density. A variety of mutants are known to affect the glucose repression of many genes, including the FOX3 gene which encodes the thiolase activity in peroxisomal beta oxidation. However, upon examination none of these mutants showed de-repression of FOX1 expression. Similarly we investigated the role of two inducers of genes encoding peroxisomal enzymes (namely SNF1 and ADR1). No evidence was found to suggest that either of these plays a significant role in the induction of FOX1 mRNA levels. These observations indicate that the regulation of FOX1 is under the control of as yet unidentified genes involved in catabolite repression and suggest that the regulatory circuit influencing acyl CoA oxidase activity, and hence beta oxidation and peroxisome function, is significantly different than that which might have been assumed from other studies.

Cloning and characterization of the 5'-flanking region of the human topoisomerase II alpha gene.

Topoisomerases are essential enzymes for DNA metabolism in prokaryotes and eukaryotes. In human cells, DNA topoisomerase II enzyme activity can be modulated by both viral transformation and changes in proliferation status. To identify elements important for regulation of topoisomerase II alpha gene expression, genomic DNA clones covering the 5'-end of the gene were isolated. The intron/exon structure of a 2.5-kilobase region encompassing the translation start site was determined. Transcription was found to initiate at multiple sites clustered around 90 base pairs 5' to the ATG initiation codon. Transient expression of chimeric topoisomerase II-reporter gene constructs in HeLa cells revealed that the 5'-flanking region exhibited promoter activity. The region -90 to -1 upstream of the major transcription start site was shown by deletion analysis to include a promoter. This minimal promoter lacks a TATA box, is moderately GC-rich, and contains a high frequency of CpG dinucleotides; characteristic of a "housekeeping" gene promoter. Maximal promoter activity was observed using a fragment extending to position -562. Putative regulatory elements are contained within and immediately upstream of the minimal promoter region. The regulatory region of the topoisomerase II alpha gene identified here is similar in basic structure to those of the human thymidine kinase and DNA polymerase alpha genes, which are also controlled by proliferation-specific factors.

Characterization of the transcriptional potency of sub-elements of the UAS of the yeast PGK gene in a PGK mini-promoter.

The upstream activator (UAS) of the yeast PGK gene comprises three different sequence elements. These are 1) a region of strong protein binding called the YFP, 2) three repeats of the motif CTTCC and 3) an essential activator core (AC) sequence that binds the protein RAP1. To assess the function of each of these elements in transcriptional activation we have inserted them individually and in various combinations into a PGK mini-promoter. This comprises only the transcription initiation elements from the PGK promoter and is inactive in the absence of activator sequences. None of the individual sequence elements was capable of activating the mini-promoter. However either the YFP or the CTTCC boxes in conjunction with the AC box resulted in efficient expression. Transcription levels were not however as high as when all three elements were inserted. These data suggest that the efficiency of PGK transcription depends upon the interactions between three different sequences. Furthermore while RAP1 per se is not a transcriptional activator it can associate promiscuously with other factors to create a functional transcription complex.

Efficient activation of transcription in yeast by the BPV1 E2 protein.

The full-length gene product encoded by the E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV1) is a transcriptional transactivator. It is believed to mediate its effect on the BPV1 long control region (LCR) by binding to motifs with the consensus sequence ACCN6GGT. The minimal functional cis active site, called the E2 response element (E2RE), in mammalian cells comprises two copies of this motif. Here we have shown that E2 can function in Saccharomyces cerevisiae by placing an E2RE upstream of a synthetic yeast assay promoter which consists of a TATA motif and an mRNA initiation site, spaced correctly. This E2RE-minimal promoter is only transcriptionally active in the presence of E2 protein and the resulting mRNA is initiated at the authentic start site. This is the first report of a mammalian viral transactivator functioning in yeast. The level of activation by E2 via the E2RE was the same as observed with the highly efficient authentic PGK promoter where the upstream activation sequence is composed of three distinct elements. Furthermore a single E2 motif which is insufficient in mammalian cells as an activation site was as efficiently utilized in yeast as the E2RE (2 motifs). Previous studies have shown that mammalian cellular activators can function in yeast and our data now extend this to viral-specific activators. Our data indicate however that while the mechanism of transactivation is broadly conserved there may be significant differences at the detailed level.

The yeast co-activator GAL11 positively influences transcription of the phosphoglycerate kinase gene, but only when RAP1 is bound to its upstream activation sequence.

Transcription of the yeast phosphoglycerate kinase gene (PGK) is activated by an array of nuclear factors including the multifunctional protein RAP1. We have demonstrated that the transcriptional co-activator GAL11, which was identified as an auxiliary factor to GAL4 and which is believed to interact with the zinc finger of the trans-activator, positively influences the level of PGK transcription on both fermentable and non-fermentable carbon sources. This positive effect is only observed when the RAP1 site in the upstream activation sequence (UAS) is present, implying that GAL11 acts through RAP1. Expression of the RAP1 gene is not reduced in the gal11 background, and in vivo footprinting shows that GAL11 does not influence RAP1 DNA-binding activity. Therefore the effect of GAL11 on PGK transcription must be mediated at the PGK UAS, presumably as part of the activation complex. It has been proposed that RAP1 may act as a facilitator of GCR1 binding at the PGK UAS and therefore it is conceivable that the target for GAL11 may in fact be GCR1. A further implication of this study is that GAL11 can interact with proteins such as RAP1 or GCR1 that are apparently structurally dissimilar from GAL4 and other zinc finger DNA-binding proteins.

A heat shock element in the phosphoglycerate kinase gene promoter of yeast.

The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. Its activity in unstressed cells has been shown to be due to an upstream activator site (UASPGK) at -402 to -479. Since levels of PGK mRNA can sometimes be elevated by heat shock of yeast cultures this investigation determined how specific deletions of PGK promoter sequences effect levels of PGK mRNA both before and after heat shock. A series of PGK promoter deletions was inserted on a high copy plasmid into cells having a TRP1 gene disruption of the solitary chromosomal PGK locus. This enabled PGK transcripts of plasmid and chromosomal origin to be distinguished by virtue of their different sizes. Certain deletions lacking UASPGK displayed activities that were very low in unstressed cells, but which increased fifty to one-hundred fold after heat shock. With UASPGK present heat shock had only a relatively small or negligible effect on PGK mRNA levels. Heat shock activation was abolished when the -256 to -377 region with homology to the heat shock element consensus of eukaryotes was deleted in addition to UASPGK, but was unaffected by the deletion of regions further downstream containing TATA- and CAAT- sequence motifs. This is the first demonstration of a heat shock element, an activator site normally found upstream of eukaryotic heat shock protein genes, as a natural constituent of a high efficiency glycolytic promoter. It is proposed that PGK may be one member of a small subset of yeast genes that are highly expressed in unstressed cells yet possess a heat shock element to ensure their continued transcription after heat shock.

The yeast protein Gcr1p binds to the PGK UAS and contributes to the activation of transcription of the PGK gene.

Analysis of the upstream activation sequence (UAS) of the yeast phosphoglycerate kinase gene (PGK) has demonstrated that a number of sequence elements are involved in its activity and two of these sequences are bound by the multifunctional factors Rap1p and Abf1p. In this report we show by in vivo footprinting that the regulatory factor encoded by GCR1 binds to two elements in the 3' half of the PGK UAS. These elements contain the sequence CTTCC, which was previously suggested to be important for the activity of the PGK UAS and has been shown to be able to bind Gcr1p in vitro. Furthermore, we find that Gcr1p positively influences PGK transcription, although it is not responsible for the carbon source dependent regulation of PGK mRNA synthesis. In order to mediate its transcriptional influence we find that Gcr1p requires the Rap1p binding site, in addition to its own, but not the Abf1p site. As neither a Rap1p nor a Gcr1p binding site alone is able to activate transcription, we propose that Gcr1p and Rap1p interact in an interdependent fashion to activate PGK transcription.