Ty insertions upstream and downstream of native DUR1,2 promoter elements generate different patterns of DUR1,2 expression in Saccharomyces cerevisiae.

Expression of allantoin pathway genes is subject to induction and nitrogen catabolite repression. Two classes of cis-dominant mutations (DUR80 and DUR1,2-Oh) result in overproduction of DUR1,2 mRNA. In DUR80 mutants, DUR1,2 expression remained inducible, nitrogen catabolite repression sensitive, and unresponsive to cell ploidy, i.e., overproduction was superimposed on normal gene regulation. DUR1,2-Oh mutations, in contrast, generated a pattern of DUR1,2 expression similar to that often reported when a Ty element inserts upstream of a gene, the ROAM phenotype. We analyzed four independent DUR80 and DUR1,2-Oh alleles. The DUR1,2-Oh mutation was, as expected, a Ty insertion at -445 3' of the native DUR1,2 upstream activation sequences (UASs). All three DUR80 alleles were also Ty insertions between -644 and -653 immediately 5' of the native DUR1,2 USASs. We suggest that the difference in DUR1,2-Oh and DUR80 phenotypes depends on whether the native cis-acting elements and transcription factors associated with them can operate. If they can, enhancement of normally regulated DUR1,2 expression is observed. This is a novel phenotype for Ty insertions. If the native DUR1,2 cis-acting elements are not present, the case when Ty insertion occurs 3' of them, a ROAM phenotype is generated. Nitrogen-regulated upstream activation sequence (UASNTR)-homologous sequences present in the Ty delta elements rather than cis-acting elements required for Ty transcription are the most likely candidates to serve as the cis-acting elements mediating the DUR80 phenotype.

Multiple early transcripts and splicing of the Autographa californica nuclear polyhedrosis virus IE-1 gene.

The immediate-early IE-1 gene of Autographa californica nuclear polyhedrosis virus was cloned, and its nucleotide sequence was determined. Sequence analysis indicated that this gene would encode a protein of 582 amino acids with a predicted molecular weight of 66,822. Analysis of IE-1 gene expression during baculovirus infection identified two transcripts. One, 1.9 kilobases (kb), was expressed at constant steady-state levels throughout infection, whereas the other, 2.1 kb, was expressed only early in infection. Analysis of IE-1 cDNA clones demonstrated that the 2.1-kb transcript contained the entire 1.9-kb transcript (exon 1) plus an additional 5' end (exon 0). Genomic Southern analysis placed the exon 0 sequences on the EcoRI B fragment, 4 kilobase pairs upstream of exon 1. Sequencing of the upstream region identified an open reading frame whose 5' end was identical to the exon 0 sequences in the cDNAs. Examination of the genomic DNA sequences around the exon-exon junction revealed sequences similar to published consensus splice acceptor and donor sequences. This is the first example of splicing of any viral transcript during baculovirus infection.

DNA mismatch repair detected in human cell extracts.

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.

The promoter for the late gene encoding Vp5 of herpes simplex virus type 1 is recognized by cell extracts derived from uninfected cells.

The ability of whole-cell extracts from uninfected HeLa cells to recognize the promoter for the herpes simplex virus type 1 late gene encoding the major capsid protein Vp5 was investigated by using both in vitro transcriptional and S1 nuclease protection analysis. This gene promoter was recognized by the cell extracts and produced abundant amounts of transcript in the absence of any other virus-encoded factors. This transcript was shown to arise, in vitro, from specific initiation at or very near the physiological mRNA start site. Thus, it appears that cell extracts from uninfected HeLa cells can efficiently recognize both early- and late-gene promoters.

Tau, sigma, and delta. A family of repeated elements in yeast.

We report here the isolation and structure of a new repeated DNA element, tau. This element, from Saccharomyces cerevisiae, is 371 base pairs long and is flanked on either end by the same invertedly repeated sequence found at the ends of some Ty and sigma elements in yeast, copia elements in Drosophila and spleen necrosis virus. The tau inverted repeats are themselves flanked by a 5-base pair directly repeated genomic sequence that is present only once in a cognate tau-allele. These structural characteristics, the presence of multiple copies of tau in the genome, and the isolation of tau+ and tau- allelic pairs suggest that tau may be capable of transposition either alone or in association with some larger element. Detailed sequence analysis of the tau, sigma, and delta elements revealed that all three contain significant regions of homology, suggesting that they are probably members of a single family derived from a common progenitor.

tau, a repeated DNA sequence in yeast.

We have found a 371-base-pair (bp) repeated DNA element, tau, in Saccharomyces cerevisiae. The ends of tau are composed of a 5-bp inverted repeat, similar in sequence to those reported for the Ty, sigma, copia, and spleen necrosis virus elements. These inverted repeats are flanked by 5-bp direct repeats of a target sequence that occurs only once in an allele that lacks the tau element. This overall structure is characteristic of transposable elements. Like sigma, tau elements have been found (in both orientations) closely associated with tRNA genes (409 and 198 bp from the 5' end, respectively). It is noteworthy that one representative of tau was isolated in a concentric insertion of tau, delta, and sigma.

Isolation and characterization of a cycloheximide-resistant mutant of Acanthamoeba castellanii Neff.

A cycloheximide-resistant mutant was isolated from the amoeba Acanthamoeba castellanii Neff. Drug resistance was found to be due to a ribosomal modification.