Mechanism of transformation in Pichia methanolica yeast: transforming and nontransforming genes

Two types of genes were found in the study of transformation in yeast Pichia methanolica: transforming (Trg) and nontransforming (Ntg) genes. Transforming genes (P-ADE7,4 and S-LEU2), as linear DNA molecules, can transform competent cells with high efficiency inversely proportional to the molecule size. Nontransforming genes (P-ADE5 and H-LEU2) transform P. methanolica cells at an extremely low rate even when they are combined with transforming genes. The analysis showed that linear DNA molecules with Trg and Ntg can be either rearranged and integrated in random sites of the recipient genome or form circular plasmids, which are capable of autonomous replication irrespective of the presence of specific replicative elements.     

Genetic Control of Purine Biosynthesis in Yeast Pichia methanolica. The ADE5 (PUR4) Gene Controlling 5"-Phosphoribosylformyl Glycinamidine Synthetase

By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system from red to white, it was shown that the PUR4 gene encoding 5"-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeastSaccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe(the ade3 gene), andPichia methanolica (theADE5 gene). This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group. Study of characteristics of spontaneous mutations in early BPN genes of P. methanolica demonstrated that the vast majority of unstable ade5s ^U alleles (mutations with a high reversion frequency ranging from 0.2 × 10^–6 to 2 × 10^–6) appeared solely among mutants for the ADE5 gene. Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene. The DNA sequence that can compensate for theP. methanolica ade5mutation and probably is the structural P-ADE5gene, was cloned from a genomic library of P. methanolicaby the ade6 mutation complementation in the recipient S. cerevisiae strain.     

Homology-facilitated plasmid transfer in Haemophilus influenzae

Summary The 8 kbp plasmid pAT4 transformed Haemophilus influenzae Rd cells at low frequencies. Transformation was increased up to 100 times, however, when the recipient cells carried a DNA segment in either their chromosome or in a resident plasmid that was homologous to at least part of plasmid pAT4. Linearized plasmid DNA molecules did not transform cells without DNA homology; they efficiently transformed homology recipients, but only when the cuts had been made in the region of shared homology. In most cases examined the circular donor plasmid had been reconstituted from the transforming DNA; in some cases the reconstituted plasmid carried a mutation initially present in the recipient chromosome, provided the transforming plasmid had been linearized in the region of shared homology. Plasmid reconstitution was not observed in recA1 cells. We conclude that homology-facilitated plasmid transformation (transfer) is similar to that reported for Bacillus subtilis and Streptococcus pneumoniae.     

Different specific activities of the monomeric and oligomeric forms of plasmid DNA in transformation of B. subtilis and E. coli.

Summary (1) The low residual transforming activity in preparations of monomeric, supercoiled, circular (CCC) forms of the plasmids pC194 and pHV14 could be attributed to the presence in such isolates of a small number of contaminating multimeric molecules. (2) E. coli derived preparations of pHV14, an in vitro recombinant plasmid capable of replication in both E. coli and B. subtilis, contain oligomeric forms of plasmid DNA in addition to the prevalent monomeric CCC form. The specific transforming activity of pHV14 DNA for E. coli is independent of the degree of oligomerization, whereas in transformation of B. subtilis the specific activity of the purified monomeric CCC molecules is at least four orders of magnitude less than that of the unfractionated preparation. (3) Oligomerization of linearized pHV14 DNA by T4 ligase results in a substantial increase of specific transforming activity when assayed with B. subtilis and causes a decrease when used to transform E. coli.     

Metabolic regulation adapting to high methanol environment in the methylotrophic yeast Ogataea methanolica

Since methylotrophic yeasts such as Ogataea methanolica can use methanol as a sole carbon feedstock, they could be applied to produce valuable products from methanol, a next-generation energy source synthesized from natural gases, using genetic engineering tools. In this study, metabolite profiling of O. methanolica was conducted under glucose (Glc) and low and high methanol (L- and H-MeOH) conditions to show the adaptation mechanism to a H-MeOH environment. The yeast strain responded not only to the presence of methanol but also to its concentration based on the growth condition. Under H-MeOH conditions, O. methanolica downregulated the methanol utilization, glycolytic pathway and alcohol oxidase (AOD) isozymes and dihydroxyacetone synthase (DAS) expression compared with L-MeOH-grown cells. However, levels of energy carriers, such as ATP, were maintained to support cell survival. In H-MeOH-grown cells, reactive oxygen species (ROS) levels were significantly elevated. Along with increasing ROS levels, ROS scavenging system expression was significantly increased in H-MeOH-grown cells. Thus, we concluded that formaldehyde and H₂O₂, which are products of methanol oxidation by AOD isozymes in the peroxisome, are overproduced in H-MeOH-grown cells, and excessive ROS derived from these cells is generated in the cytosol, resulting in upregulation of the antioxidant system and downregulation of the methanol-utilizing pathway to suppress overproduction of toxic intermediates.     

Substrate specificities of the ntg1 and ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical.

Two genes of Saccharomyces cerevisiae, NTG1 and NTG2, encode proteins with a significant sequence homology to the endonuclease III of Escherichia coli. The Ntg1 and Ntg2 proteins were overexpressed in E.coli and purified to apparent homogeneity. The substrate specificity of Ntg1 and Ntg2 proteins for modified bases in oxidatively damaged DNA was investigated using gas chromatography/isotope-dilution mass spectrometry. The substrate used was calf-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution. The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-derived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, and two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA. In contrast, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadenine from gamma-irradiated DNA. The Ntg1 and Ntg2 proteins also release 2, 6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine from damaged poly(dG-dC).poly(dG-dC). Excision was measured as a function of enzyme concentration and time. Furthermore, kinetic parameters were determined for each lesion. The results show that kinetic constants varied among the different lesions for the same enzyme. We also investigated the capacity of the Ntg1 and Ntg2 proteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mispaired with each of the four DNA bases. The results show that the Ntg1 protein preferentially cleaves a DNA duplex containing 8-OH-Gua mispaired with a guanine. Moreover, the Ntg1 protein releases free 8-OH-Gua from 8-OH-Gua/Gua duplex but not from duplexes containing 8-OH-Gua mispaired with adenine, thymine or cytosine. In contrast, the Ntg2 protein does not incise duplexes containing 8-OH-Gua mispaired with any of the four DNA bases. These results demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endonuclease III of E.coli and its homologues in Schizosaccharomyces pombe or human cells.     

Identification of SUMO modification sites in the base excision repair protein,Ntg1

DNA damaging agents are a constant threat to genomes in both the nucleus and the mitochondria. To combat this threat, a suite of DNA repair pathways cooperate to repair numerous types of DNA damage. If left unrepaired, these damages can result in the accumulation of mutations which can lead to deleterious consequences including cancer and neurodegenerative disorders. The base excision repair (BER) pathway is highly conserved from bacteria to humans and is primarily responsible for the removal and subsequent repair of toxic and mutagenic oxidative DNA lesions. Although the biochemical steps that occur in the BER pathway have been well defined, little is known about how the BER machinery is regulated. The budding yeast, Saccharomyces cerevisiae is a powerful model system to biochemically and genetically dissect BER. BER is initiated by DNA N-glycosylases, such as S. cerevisiae Ntg1. Previous work demonstrates that Ntg1 is post-translationally modified by SUMO in response to oxidative DNA damage suggesting that this modification could modulate the function of Ntg1. In this study, we mapped the specific sites of SUMO modification within Ntg1 and identified the enzymes responsible for sumoylating/desumoylating Ntg1. Using a non-sumoylatable version of Ntg1, ntg1ΔSUMO, we performed an initial assessment of the functional impact of Ntg1 SUMO modification in the cellular response to DNA damage. Finally, we demonstrate that, similar to Ntg1, the human homologue of Ntg1, NTHL1, can also be SUMO-modified in response to oxidative stress. Our results suggest that SUMO modification of BER proteins could be a conserved mechanism to coordinate cellular responses to DNA damage.     

Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress

DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.     

Mono- and Bifunctional DNA Glycosylases Involved in Repairing Oxidatively Damaged DNA

DNA repair is a basic biological process providing for the stability and integrity of the genome. Disturbed repair results in premature aging, autoimmune and cardiological disorders, tumorigenesis, etc. Data on enzymes which play key roles in repairing DNA with lesions generated by reactive oxygen species are reviewed. The substrate specificity, mechanism of catalysis, structure of the active center, and specific structural and functional features are described for Escherichia coli mono- and bifunctional DNA glycosylases (endonuclease III, Fpg, MutY, endonuclease VIII, AlkA, MutT) and their prokaryotic and eukaryotic homologs (Ntg1, Ntg2, yOgg1, yOgg2, hOgg1, hOgg2, mOgg1, rOgg1, hMTH, hMYH, MAG, ADPG, and ANPG) which are involved in base excision repair.     

A novel function for the Mre11-Rad50-Xrs2 complex in base excision repair

The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway. We studied the repair of methyl-methanesulfonate-induced heat-labile sites in chromosomal DNA in vivo and the in vitro BER capacity for the repair of uracil- and 8-oxoG-containing oligonucleotides in MRX-deficient cells. Both approaches show a clear BER deficiency for the xrs2 mutant as compared to wildtype cells. The in vitro analyses revealed that both subpathways, long-patch and short-patch BER, are affected and that all components of the MRX complex are similarly important for the new function in BER. The investigation of the epistatic relationship of XRS2 to other BER genes suggests a role of the MRX complex downstream of the AP-lyases Ntg1 and Ntg2. Analysis of individual steps in BER showed that base recognition and strand incision are not affected by the MRX complex. Reduced gap-filling activity and the missing effect of aphidicoline treatment, an inhibitor for polymerases, on the BER efficiency indicate an involvement of the MRX complex in providing efficient polymerase activity.     

Isolation of physarum DNA segments that support autonomous replication in yeast

Summary Hybrid plasmids containing the bacterial resistance-transfer factor pBR322 and the yeast leu2 ⁺gene have been used to isolate DNA fragments of Physarum that are capable of initiating DNA replication in a yeast host. Five of forty hybrid plasmids containing Physarum sequences transform leu2 ^-yeast to Leu⁺ at high frequency. The resulting Leu⁺ transformants are characterized by phenotypic instability. Supercoiled plasmid molecules containing pBR322 sequences can be detected in the transformed yeast, indicating that the transforming DNA replicates autonomously. Plasmid DNA isolated from Leu⁺ yeast can transform leuB bacteria. The hybrid plasmid recovered from the Leu⁺ bacterial transformants is identical to the original plasmid, indicating structural integrity is maintained during passage through the yeast host. These hybrid plasmids containing Physarum sequences have the same characteristics as those containing autonomously replicating yeast chromosomal sequences. As the temporal sequence of DNA replication is particularly accessible to study in Physarum plasmodia, the functional significance of these segments should be amenable to study.     

Variable copy number of macronuclear DNA molecules in Tetrahymena

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley-Liss, Inc.     

Characterization of AP lyase activities of Saccharomyces cerevisiae Ntg1p and Ntg2p: implications for biological function

Saccharomyces cerevisiae possesses two Escherichia coli endonuclease III homologs, NTG1 and NTG2, whose gene products function in the base excision repair pathway and initiate removal of a variety of oxidized pyrimidines from DNA. Although the glycosylase activity of these proteins has been well studied, the in vivo importance of the AP lyase activity has not been determined. Previous genetic studies have suggested that the AP lyase activities of Ntg1p and Ntg2p may be major contributors in the initial processing of abasic sites. We conducted a biochemical characterization of the AP lyase activities of Ntg1p and Ntg2p via a series of kinetic experiments. Such studies were designed to determine if Ntg1p and Ntg2p prefer specific bases located opposite abasic sites and whether these lesions are processed with a catalytic efficiency similar to Apn1p, the major hydrolytic AP endonuclease of yeast. Our results indicate that Ntg1p and Ntg2p are equally effective in processing four types of abasic site-containing substrates. Certain abasic site substrates were processed with greater catalytic efficiency than others, a situation similar to Apn1p processing of such substrates. These biochemical studies strongly support an important biological role for Ntg1p and Ntg2p in the initial processing of abasic sites and maintenance of genomic stability.     

The transforming activity of sonicated Haemophilus influenzae DNA

Summary The inactivation of transforming Haemophilus influenzae DNA by sonication in aqueous solution was investigated. The molecular weight decrease of the molecules is the major factor in DNA inactivation. It impairs strongly the uptake of the DNA by the recipient bacteria, especially when the molecular weight is lower than 3x10⁶ daltons. The uptake of sonicated DNA by the bacterial cells seems not to be further reduced when molecules of about 0.5x10⁶ daltons are submitted to further depolymerisation. However the transforming activity of these molecules is still sensitive to further sonication. The transforming activity of the sonicated DNA is related in the last resort to the intact length of the DNA molecules, at the level of their single-strand structure, available for recombination. Rupture by ultrasound was found to be twice as efficient in reducing transforming activity as a nick induced by pancreatic DNAse.     

Intermediates in genetic recombination of bacteriophage T7 DNA. Biological activity and the roles of gene 3 and gene 5

When Escherichia coli cells were infected with ³²P- and 5-bromodeoxyuridine-labeled T7 bacteriophage defective in genes 1.3, 2.3, 4 and 5, doubly branched T7 DNA molecules with “H” or “X”-like configurations were found in the half-heavy density fractions. Physical study showed that they are dimeric molecules composed of two parental DNA molecules (Tsujimoto & Ogawa, 1977a). The transfection assay of these molecules revealed that they were infective. Genetic analysis of progeny in infective centers obtained by transfection of dimeric molecules formed by infection of genetically marked T7 phage showed that these dimeric molecules were genetically biparental.To elucidate the roles of the products of gene 3 (endonuclease I) and gene 5 (DNA polymerase) of phage T7 in the recombination process, the ³²P/BrdUrd hybrid DNA molecules which were formed in the infected cells in the presence of these gene products were isolated, and their structures were analyzed. The presence of T7 DNA polymerase seems to stimulate and/or stabilize the interaction of parental DNAs. At an early stage of infection few dimeric molecules were formed in the absence of T7 DNA polymerase, whereas a significant number of doubly branched molecules were formed in its presence. With increasing incubation time, the multiply branched DNA molecules with a high sedimentation velocity accumulated.In contrast to the accumulation of multiply branched molecules in phage with mutations in genes 2, 3 and 4, almost all of the ³²P/BrdUrd hybrid DNA formed in phage with mutations in genes 2 and 4 were monomeric linear molecules. Shear fragmentation of monomeric linear ³²P/BrdUrd-labeled DNA shifted the density of [³²P]DNA to almost fully light density. It was also found that approximately 50% of [³²P]DNA was linked covalently to BrdUrd-labeled DNA. These linear monomer DNA molecules had infectivity and some of those formed by infection of genetically marked parents yielded recombinant phages. Therefore the gene 3 product seems to process the branched intermediates to linear recombinant molecules by trimming the branches.     

Mapping and characterization of the DQ subregion of the ovine MHC

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.     

Molecular Cloning of the Fujinami Sarcoma Virus Genome and Its Comparison with Sequences of Other Related Transforming Viruses

Full-length proviral DNA of Fujinami sarcoma virus (FSV) of chickens was molecularly cloned and characterized. An analysis of FSV DNA integrated in mammalian cells showed that restriction endonuclease SacI has a single cleavage site on FSV DNA. Unintegrated closed circular FSV DNA obtained from newly infected cells was linearized by digestion with SacI and cloned into λgtWES·λB. The following three different molecules were isolated: FSV-1 (4.4 kilobases [kb]) and FSV-2 (4.7 kb), which appeared to be full-length FSV DNA molecules containing either one or two copies of the long terminal repeat structure, and FSV-3 (6 kb), which consisted of part FSV DNA and part DNA of unknown origin. An analysis of the structure of cloned FSV-1 and FSV-2 DNA molecules by restriction endonuclease mapping and hybridization with appropriate probes showed that about 2.6 kb of the FSV-unique sequence called FSV-fps is located in the middle of the FSV genome and is flanked by helper virus-derived sequences of about 1.3 kb at the 5′ end and 0.5 kb at the 3′ end. The long terminal repeats of FSV were found to have no cleavage site for either EcoRI or PvuI. Upon transfection, both FSV-1 DNA and FSV-2 DNA were able to transform mammalian fibroblasts. Four ³²P-labeled DNA fragments derived from different portions of the FSV-fps sequence were used for hybridization to viral RNAs. We found that sequences within the 3′ half of the FSV-fps gene are homologous to RNAs of PRCII avian sarcoma virus and the Snyder-Theilen strain of feline sarcoma virus, both of which were previously shown to contain transforming genes related to FSV-fps. These results suggest that the 3′ portion of the FSV-fps sequence may be crucial for the transforming activity of fps-related oncogenic sequences.