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PubMed2025

Comparative Pharmacological Analysis of Mitotic Inhibitors Using Isogenic Ploidy Series of HAP1 Cells.

Drastic changes in chromosome number and cellular contents upon ploidy alterations profoundly affect the stability of mitotic regulation in different biological and pathological processes. Isogenic ploidy series of somatic cell lines are useful for studying the effects of ploidy differences on mitotic regulation at cellular and molecular levels. This chapter describes experimental procedures using isogenic human HAP1 cell lines that cover haploid, diploid, and tetraploid states. We first describe methods to establish and maintain these isogenic HAP1 ploidy series using a flow cytometer. We then describe a procedure of comparative pharmacological assay for analyzing ploidy-dependent changes in the functionality of the mitotic spindle components.

Humans↗

PubMed1991

Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants.

Apurinic/apyrimidinic (AP) sites in cellular DNA are considered to be both cytotoxic and mutagenic, and can arise spontaneously or following exposure to DNA damaging agents. We have isolated cDNA clones which encode an endonuclease, designated HAP1 (human AP endonuclease 1), that catalyses the initial step in AP site repair in human cells. The predicted HAP1 protein has an Mr of 35,500 and shows striking sequence similarity (93% identity) to BAP 1, a bovine AP endonuclease enzyme. Significant sequence homology to two bacterial DNA repair enzymes, E. coli exonuclease III and S. pneumoniae ExoA proteins, and to Drosophila Rrp1 protein is also apparent. We have expressed the HAP1 cDNA in E. coli mutants lacking exonuclease III (xth), endonuclease IV (nfo), or both AP endonucleases. The HAP1 protein can substitute for exonuclease III, but not for endonuclease IV, in respect of some, but not all, DNA repair and mutagenesis functions. Moreover, a dut xth (ts) double mutant, which is nonviable at 42 degrees C due to an accumulation of unrepaired AP sites following excision of uracil from DNA, was rescued by expression of the HAP1 cDNA. These results indicate that AP endonucleases show remarkable conservation of both primary sequence and function. We would predict that the HAP1 protein is important in human cells for protection against the toxic and mutagenic effects of DNA damaging agents.

Amino Acid Sequence↗

PubMed1992

Structure of the human DNA repair gene HAP1 and its localisation to chromosome 14q 11.2-12.

Apurinic/apyrimidinic (AP) sites are pre-mutagenic DNA lesions which occur spontaneously and following exposure of cells to ionising radiation or chemical mutagens. HAP1 (Human AP endonuclease 1), the major enzyme in human cells initiating repair of AP sites, shows strong sequence homology to DNA repair enzymes from bacteria, Drosophila and other mammalian species. We have cloned the HAP1 gene and determined its complete nucleotide sequence. The site of transcription initiation has been mapped to 452 bp upstream of the ATG initiation codon in the genomic DNA. The HAP1 gene consists of five exons and is unusually small (less than 2.6 kb from transcription initiation site to polyadenylation sequence) with 54% of the protein coding region and the entire 3' untranslated region contained within a single exon. The first exon is non-coding. Regions of three exons show sequence homology to the E.coli xth (exonuclease III) gene. Using in situ hybridisation, the HAP1 gene has been localised to human chromosome 14q 11.2-12.

Base Sequence↗

PubMed1989

Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene.

The COX6 gene encodes subunit VI of cytochrome c oxidase. Previously, this gene and its mRNAs were characterized, and its expression has been shown to be subject to glucose repression/derepression. In this study we have examined the effects of heme and the HAP1 (CYP1) and HAP2 genes on the expression of COX6. By quantitating COX6 RNA levels and assaying beta-galactosidase activity in yeast cells carrying COX6-lacZ fusion genes, we have found that COX6 is regulated positively by heme and HAP2, but is unaffected by HAP1. Through 5' deletion analysis we have also found that the effects of heme and HAP2 on COX6 are mediated by sequences between 135 and 590 base pairs upstream of its initiation codon. These findings identify COX6 as the fourth respiratory protein gene that is known to be regulated positively by heme and HAP2. The other three, CYC1, COX4, and COX5a, encode iso-1-cytochrome c, cytochrome c oxidase subunit IV, and an isolog, Va, of cytochrome c oxidase subunit V, respectively. Thus, it appears that the biogenesis of two interacting proteins, cytochrome c and cytochrome c oxidase, in the mitochondrial respiratory chain, are under the control of common factors.

Blotting, Northern↗

PubMed1992

Inducibility of the response of yeast cells to peroxide stress.

Exponential phase cells of the yeast, Saccharomyces cerevisiae when treated with a non-lethal concentration of hydrogen peroxide (H2O2; 0.2mM) for 60 min adapted to become resistant to the lethal effects of a higher dose of H2O2 (2mM). From studies using cycloheximide to inhibit protein synthesis it appears that protein synthesis is required for maximal induction of resistance but that some degree of protection from the lethal effects of peroxide can be acquired in the absence of protein synthesis. Treatment of cells with 50 micrograms cycloheximide ml-1 alone lead to them acquiring some protection from peroxide. Cells subjected to heat shock became more resistant to 2mM-H2O2; however, peroxide pretreatment did not confer thermotolerance. L-[35S]Methionine labelling of cells subjected to 0.2 mM-H2O2 stress showed that synthesis of at least ten polypeptides was induced by peroxide treatment. Some of these were also induced in cells subjected to heat shock (23 to 37 degrees C shift) but the synthesis of at least four polypeptides (45, 39.5, 38 and 24 kDa) was unique to peroxide-stressed cells. Resistance to peroxide was also inducible in an isogenic petite and an isogenic strain with a mutation in the HAP1 gene, indicating that the adaptive response does not require functional mitochondria.

Adaptation, Physiological↗

PubMed2026

Identification of OsCsLF6 Gene Responsible for Rice Seed Submergence Germination Through Genome-Wide Association Analysis.

Flooding stress is a primary environmental barrier that severely limits the widespread adoption of direct-seeded rice systems. Under submerged conditions, rapid coleoptile elongation serves as a vital morphological strategy that facilitates anaerobic germination and successful seedling establishment, yet its underlying molecular mechanisms remain poorly understood. Through a genome-wide association study, we identified a critical locus governing anaerobic coleoptile elongation, in which OsCsLF6, encoding a mixed-linkage glucan (MLG) synthase, was characterized as the causal gene. Genetic and biochemical analyses demonstrated that OsCsLF6 positively regulated coleoptile elongation by directly mediating MLG deposition into the primary cell wall. Mechanistically, we identified OsERF74, an AP2/ERF transcription factor, as an upstream master repressor that directly binds to a conserved core cis-element within the OsCsLF6 promoter. Under submergence, OsERF74 deficiency (oserf74 mutants) completely releases this transcriptional suppression, triggering a substantial upregulation of OsCsLF6 expression and subsequent hyper-accumulation of cell wall MLG. In contrast, constitutive overexpression of OsERF74 persistently blocks MLG biosynthesis. Crucially, a natural single-nucleotide polymorphism located within the OsERF74 binding element in the promoter defines two distinct haplotypes. The elite haplotype (Hap1) effectively disrupts OsERF74 binding affinity, which in turn attenuates transcriptional repression and sustains high OsCsLF6 expression, ultimately driving accelerated MLG synthesis and coleoptile elongation. Our findings establish a condition-specific OsERF74-OsCsLF6 regulatory module that serves as a central biochemical hub orchestrating cell wall remodelling during anaerobic germination. This module thus represents a promising molecular target and elite genetic resource for molecular breeding of flood-tolerant and direct-seeded rice varieties.

OsCsLF6↗

PubMed1991

CYP1 (HAP1) is a determinant effector of alternative expression of heme-dependent transcribed genes in yeast [corrected].

The CYP1 (HAP1) gene of Saccharomyces cerevisiae is known to activate a number of target genes in response to the presence of heme. Several features of the protein, deduced from the sequence of the gene, suggest that CYP1 is a general sensor of the redox state of the cell. To investigate further the function of CYP1, we analysed its effects on the transcription of two genes, HEM13 and 14DM, which are preferentially expressed in anaerobiosis. HEM13 encodes coproporphyrinogen oxidase which catalyses the sixth enzymatic step in the heme biosynthetic pathway and 14DM encodes lanosterol-14-demethylase which is involved in sterol biosynthesis and is a member of the cytochrome P450 family. Isogenic CYP1+ and cyp1 degree deleted strains, either heme-sufficient or heme-deficient (HEM1 disrupted), were grown in aerobic or anaerobic conditions, and transcripts of HEM13 and 14DM were analysed on Northern blots. The results show that in anaerobic and in heme-deficient cells, CYP1 activates the transcription of HEM13 and inhibits that of 14DM. Opposite effects of CYP1 are observed in aerobic, heme-sufficient cells. We concluded that: (i) CYP1 is an efficient activator especially in heme-depleted cells; (ii) CYP1 exerts both positive and negative regulatory effects; (iii) the nature of the regulatory function of CYP1 depends on the target gene; and (iv) for a given gene, the presence or absence of heme or oxygen reverses the sense of CYP1-dependent regulation.

Anaerobiosis↗

PubMed1990

Expression of the gene encoding subunit II of yeast QH2: cytochrome c oxidoreductase is regulated by multiple factors.

In Saccharomyces cerevisiae, the COR2 gene codes for the 40 kDa subunit II of the QH2: cytochrome c oxidoreductase, an enzyme of the mitochondrial respiratory chain. Regions in the 5' flank of this gene important for regulated expression were identified by assaying beta-galactosidase activities in cells carrying different COR2-lacZ fusion genes. Sequences downstream of position -201 relative to the translational initiation codon are sufficient to confer regulation by carbon source, whereas sequences downstream of position -153 do not give rise to significant expression. A binding site for the abundant general transcription factor GFI is present in the region between -201 and -153 just upstream from sequences which resemble the consensus DNA recognition sequence of the regulatory protein complex HAP2/HAP3: 5'-TNATTGGT-3'. By quantitating RNA levels and assaying beta-galactosidase activities we show that synthesis of COR2, which is not a hemoprotein, is regulated by HAP1, HAP2/HAP3 and heme.

Base Sequence↗