Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, which is a structural homolog of the RAD2 and RAD27 DNA repair genes

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.?cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression.     

A wat1 mutant of fission yeast is defective in cell morphology

The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing of wat1 ⁺ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1 ⁺ are slow growing but viable at 25° C and temperature-sensitive for growth above 33° C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1 ⁺ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This analysis suggests that wat1 ⁺ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology. Received: 18 September 1996 / Accepted: 22 October 1996     

Cloning and molecular analysis of the Arabidopsis gene Terminal Flower 1

The Arabidopsis gene Terminal Flower 1 (TFL1) controls inflorescence meristem identity. A terminal flower (tfl1) mutant, which develops a terminal flower at the apex of the inflorescence, was induced by transformation with T-DNA. Using a plant DNA fragment flanking the integrated T-DNA as a probe, a clone was selected from a wild-type genomic library. Comparative sequence analysis of this clone with an EST clone (129D7T7) suggested the existence of a gene encoding a protein similar to that encoded by the cen gene which controls inflorescence meristem identity in Antirrhinum. Nucleotide sequences of the region homologous to this putative TFL1 gene were compared between five chemically induced tfl1 mutants and their parental wild-type ecotypes. Every mutant was found to have a nucleotide substitution which could be responsible for the tfl1 phenotype. This result confirmed that the cloned gene is TFL1 itself. In our tfl1 mutant, no nucleotide substitution was found in the transcribed region of the gene, and the T-DNA-insertion site was located at 458 bp downstream of the putative polyadenylation signal, suggesting that an element important for expression of the TFL1 gene exists in this area. Received: 14 November 1996 / Accepted: 29 November 1996     

The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase δ

The cdc6 mutants of Schizosaccharomyces pombe have been classified as being defective in progression through the G2 phase of the cell cycle. We cloned an S. pombe gene that could complement the temperature-sensitive growth of the cdc6-23 mutant. Unexpectedly, the cloned gene was allelic to pol3, which encodes the catalytic subunit of DNA polymerase δ. Integration mapping confirmed that cdc6 and pol3 are identical. The cdc6-23 mutant carries one amino acid substitution in the conserved N3 region of Pol3. Received: 17 October 1996 / Accepted: 19 November 1996     

A novel fission yeast gene, kms1 +, is required for the formation of meiotic prophase-specific nuclear architecture

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1 ⁺ gene is involved in SPB function. However, the kms1 ⁺ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins. Received: 5 September 1996 / Accepted: 21 November 1996     

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell. Received: 15 July 1996 / Accepted: 24 October 1996     

A novel cytoplasmic structure containing DNA networks in Entamoeba histolytica trophozoites

We report here the presence of cytoplasmic DNA arranged in networks in the trophozoites of the human parasite Entamoeba histolytica. Cytoplasmic DNA was detected in live trophozoites in a structure that we called EhkO, using the fluorescent dye acridine orange, and by in situ hybridization to trophozoites with a rDNA probe. The EhkO was found in the axenically grown clones A, L6 (strain HM1:IMSS) and MAVax (strain MAV) and in the polyxenically grown clone MAVpx (strain MAV). Bacteria present in MAVpx did not cross hybridize with the DNA probe neither in in situ hybridization or in Southern blot experiments. Autoradiography of metabolically [³H]thymidine-labeled trophozoites showed the presence of EhkO, and an EhkO-enriched fraction, purified from a nuclei-free extract and examined by light microscopy, exhibited [³H]thymidine incorporation into this structure. DNA was purified from the EhkO and enriched nuclear fractions and analyzed by transmission electron microscopy. The EhkO fraction contained DNA networks resembling those of trypanosome kDNA, whereas nuclear DNA was present mainly as linear molecules and some circles. Our findings imply that E. histolytica may be taxonomically more closely related to the Trypanosomatidae than previously suspected. Received: 7 August 1996 / Accepted: 7 October 1996     

An Aspergillus nidulans nuclear protein, AnCP, involved in enhancement of Taka-amylase A gene expression, binds to the CCAAT-containing taaG2 , amdS , and gatA promoters

Using AnCP (Aspergillus nidulans CCAAT-binding protein) as a CCAAT-specific binding factor model, the possibility that one factor is able to recognize CCAAT sequences in several different genes in A.␣nidulans was examined. DNase I protection analysis showed that AnCP specifically bound to CCAAT sequence-containing regions comprising 21 to 36 bp of the taa, amdS and gatA genes. Furthermore, replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. This clearly demonstrates a positive function of the CCAAT element. However, amylase was induced by starch and repressed by glucose in a CCAAT-box disruptant, as in wild-type cells. Received: 28 June 1996 / Accepted: 7 October 1996     

Analyses of Saccharomyces cerevisiae Cdc7 kinase point mutants: dominant-negative inhibition of DNA replication on overexpression of kinase-negative Cdc7 proteins

Saccharomyces cerevisiae Cdc7 kinase is required for initiation of S phase, and its kinase activity, which is positively regulated by Dbf4 protein, reaches maximum at the G1/S boundary. In this study, we constructed Cdc7 point mutants (T281E, T281A, D182N, D163N, and T167E) and examined the effect of each mutant on growth. All the mutants lost the ability to complement temperature-sensitive growth of cdc7(ts) mutants at a low protein level, whereas T281A (putative target of phosphorylation) and T167E (residue involved in substrate recognition) restored the growth of cdc7(ts) when overproduced to a high level. Three putative kinase-negative mutants (T281E, D182N, and D163N) inhibited growth when overexpressed in a wild-type strain. Analyses of DNA content and morphology revealed that most cells were arrested as dumbbells with 1C DNA, indicative of a block in the G1 to S transition. This growth inhibition was suppressed by co-overexpression of the wild-type Cdc7 or Dbf4 protein. Furthermore, deletion of the Dbf4 protein-binding region in each Cdc7 mutant resulted in loss of growth inhibitory effect. Thus, dominant-negative effects of T281E, D182N, and D163N on growth can be best explained by inactivation of the wild-type Cdc7 function through titration of Dbf4 by these inactive kinases. Our results are consistent with the notion that association of Dbf4 with Cdc7 is essential for the G1 to S transition in S. cerevisiae. Received: 17 September 1996 / Accepted: 6 January 1997     

Tolerance of low pH in Schizosaccharomyces pombe requires a functioning pub1 ubiquitin ligase

A strain of Schizosaccharomyces pombe carrying a disrupted Na⁺/H⁺ antiporter gene (sod2::sup3-5), in addition to the common auxotrophic mutations, ade6-216, ura4-D18 and leu1-32, is highly sensitive to media adjusted to pH 6.9. Reversion analysis of this strain yielded a group of revertants capable of growth at pH 6.9. Two of the revertants elongated and failed to form colonies at pH 3.5. Genetic characterization of one of the pH-sensitive elongated strains, J227, showed the presence of two independently segregating mutations. One, pub1 ( protein ubiquitin ligase 1), has recently been reported as an E3 protein ubiquitin ligase involved in cdc25 turnover. The second has been named elp3-1 (elongated at low pH). Genetic dissection of the original strain revealed that poor growth at high pH was due to the presence of the auxotrophic markers, suggesting a possible inhibitory effect of high pH on the function of permeases responsible for uptake of the necessary nutrients. Suppression of the high pH sensitivity required the presence of both the pub1-1 and elp3-1 mutations. While the pub1-1 mutation reduced the capacity of cells to tolerate relatively moderate concentrations of LiCl (3 mM) in liquid culture, it was capable of partially suppressing the extreme Li⁺ sensitivity caused by the sod2 disruption. Under these conditions, the growth of pub1-1 sod2::ura4 double mutant cells was improved over that of either pub1-1 or sod2::ura4 cells. The elp3-1 mutation had no effect on the Li⁺ tolerance in either wild-type or sod2::ura4 backgrounds. pub1-1 cells are elongated and incapable of colony formation at pH 3.5. In contrast, elp3-1 cells are elongated at pH 3.5 and pH␣5.5 (the normal pH of minimal medium) but can form colonies under both conditions. J227 cells are significantly longer than either single mutant at pH 3.5 and do not form colonies but are visually similar to elp3-1 cells at pH 5.5. Complementation cloning in the J227 background yielded a genomic clone of pub1, allowing us to define the intron-exon structure of the gene. Sequences with high homology to the predicted amino acid sequence of pub1 have been identified in Saccharomyces cerevisiae (RSP5/NPI1), human (hRPF1), mouse (mNedd4), and rat (rNedd4). Based on the nature of our mutant selection, the pH-sensitive phenotype of the strains selected, and the known involvement of RSP5/NPI1 in membrane permease turnover in S. cerevisiae, we hypothesize a role for pub1, either directly or indirectly, in regulating membrane transport processes. This is further supported by the broad range of effects that the pub1-1 mutation exerts on overall performance of cells at high and low external pH, and in the presence of toxic levels of Li⁺. Received: 12 September 1996 / Accepted: 19 December 1996     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress. Received: 17 April 1996 / Accepted: 21 October 1996     

Regulatory elements in the promoter of the vitelline membrane gene VM32E of Drosophila melanogaster direct gene expression in distinct domains of the follicular epithelium

The Drosophila vitelline membrane protein gene VM32E is expressed according to a precise temporal and spatial program in the follicle cells. Results from germ line transformation experiments using different fragments of the −465/−39 VM32E region fused to the hsp/lacZ reporter gene revealed that the region −348/−39 is sufficient to confer the wild-type expression pattern. Within this segment, distinct cis-regulatory elements control VM32E expression in ventral and dorsal follicle cells. The region between −135/−113 is essential for expression of the VM32E gene in the ventral columnar follicle cells. Expression in the dorsal domain requires the two regions −348/−254 and −118/−39. Furthermore, the region −253/−119 appears to contain a negative element that represses gene activity in anterior centripetal cells. We suggest that the expression of the VM32E gene throughout the follicular epithelium is controlled by specific cis-regulatory elements acting in distinct spatial domains and following a precise developmental program. Received: 22 October 1996 / Accepted: 14 November 1996     

Inactivation of OGG1 increases the incidence of G?·?C→T?·?A transversions in Saccharomyces cerevisiae : evidence for endogenous oxidative damage to DNA in eukaryotic cells

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254 nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (Can^R) and reversion to Lys⁺ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G · C→T · A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae. Received: 6 September 1996 / Accepted: 22 October 1996     

The regulatory protein MucR binds to a short DNA region located upstream of the muc R coding region in Rhizobium meliloti

The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan. The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17 kDa apparent molecular mass, corresponding to the size expected for MucR. As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene. Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site. Although binding of MucR to this site exhibited a moderate dissociation constant of M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding. Received: 9 October 1996 / Accepted: 20 December 1996