Activation of Mrc1, a mediator of the replication checkpoint, by telomere erosion

Background information. In budding yeast, the loss of either telomere sequences (in telomerase‐negative cells) or telomere capping (in mutants of two telomere end‐protection proteins, Cdc13 and Yku) lead, by distinct pathways, to telomeric senescence. After DNA damage, activation of Rad53, which together with Chk1 represents a protein kinase central to all checkpoint pathways, normally requires Rad9, a checkpoint adaptor. Results. We report that in telomerase‐negative (tlc1Δ) cells, activation of Rad53, although diminished, could still take place in the absence of Rad9. In contrast, Rad9 was essential for Rad53 activation in cells that entered senescence in the presence of functional telomerase, namely in senescent cells bearing mutations in telomere end‐protection proteins (cdc13‐1 yku70Δ). In telomerase‐negative cells deleted for RAD9, Mrc1, another checkpoint adaptor previously implicated in the DNA replication checkpoint, mediated Rad53 activation. Rad9 and Rad53, as well as other DNA damage checkpoint proteins (Mec1, Mec3, Chk1 and Dun1), were required for complete DNA‐damage‐induced cell‐cycle arrest after loss of telomerase function. However, unexpectedly, given the formation of an active Rad53–Mrc1 complex in tlc1Δ rad9Δ cells, Mrc1 did not mediate the cell‐cycle arrest elicited by telomerase loss. Finally, we report that Rad9, Mrc1, Dun1 and Chk1 are activated by phosphorylation after telomerase inactivation. Conclusions. These results indicate that loss of telomere capping and loss of telomere sequences, both of which provoke telomeric senescence, are perceived as two distinct types of damages. In contrast with the Rad53–Rad9‐mediated cell‐cycle arrest that functions in a similar way in both types of telomeric senescence, activation of Rad53–Mrc1 might represent a specific response to telomerase inactivation and/or telomere shortening, the functional significance of which has yet to be uncovered.     

Molecular analysis of the MHC class II region inDR4, DR7, andDR9 haplotypes

Within the class 11 region of the major histocompatibility complex (MHC) the amount of DNA in theDR-DQ interval has been shown to be haplotype dependent, with those carrying the DR4, DR7, and DR9 specificities having been reported to contain 110–160 kilobases (kb) more DNA than haplotypes carrying the DR3 specificity. Certain subtypes of haplotypes carrying particular DR specificities are more closely associated with autoimmune diseases than others. With the prospect of the DNA perhaps containing a disease susceptibility locus, we have mapped eight DR4 and two DR7 homozygous cell lines and a DR7/9 heterozygous cell line together with a control DR3 cell line using pulsed field gel electrophoresis (PFGE) with the enzymesBss H II,Pvu I, andNot I/Nru I. Our results, however, show that the presence and amount of the extra DNA is constant irrespective of the subtype. We have also tried to narrow down the position of insertion of the extra DNA using eight further rare-cutting enzymes but, due to the polymorphic nature of sites and/or differences in methylation in this region, it was not possible to refine it further than between DRA and DQA1/B1. This polymorphic nature of theDR-DQ region is unusual, considering the uniformity of rare cutter sites that has been observed within the rest of the class II, and class III, regions. The presence of this, and other, haplotype dependent variations in the DNA content of theDR subregion may be important with respect to recombination and will be particularly interesting if the additional DNA is found to contain novel genes.     

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.     

Functional compartmentalization of Rad9 and Hus1 reveals diverse assembly of the 9‐1‐1 complex components during the DNA damage response in Leishmania

The Rad9‐Rad1‐Hus1 (9‐1‐1) complex is a key component in the coordination of DNA damage sensing, cell cycle progression and DNA repair pathways in eukaryotic cells. This PCNA‐related trimer is loaded onto RPA‐coated single stranded DNA and interacts with ATR kinase to mediate effective checkpoint signaling to halt the cell cycle and to promote DNA repair. Beyond these core activities, mounting evidence suggests that a broader range of functions can be provided by 9‐1‐1 structural diversification. The protozoan parasite Leishmania is an early‐branching eukaryote with a remarkably plastic genome, which hints at peculiar genome maintenance mechanisms. Here, we investigated the existence of homologs of the 9‐1‐1 complex subunits in L. major and found that LmRad9 and LmRad1 associate with chromatin in response to replication stress and form a complex in vivo with LmHus1. Similar to LmHus1, LmRad9 participates in telomere homeostasis and in the response to both replication stress and double strand breaks. However, LmRad9 and LmHus1‐deficient cells present markedly opposite phenotypes, which suggest their functional compartmentalization. We show that some of the cellular pool of LmRad9 forms an alternative complex and that some of LmHus1 exists as a monomer. We propose that the diverse assembly of the Leishmania 9‐1‐1 subunits mediates functional compartmentalization, which has a direct impact on the response to genotoxic stress.     

Interaction of the antimalarial drug fluoroquine with DNA,tRNA, and poly(A): 19F-NMR chemical-shift and relaxation,optical absorption,and fluorescence studies

The interaction of the fluorinated antimalarial drug fluoroquine [7-fluoro-4-(diethyl-amino-1-methylbutylamino)quinoline] with DNA, tRNA, and poly(A) has been investigated by optical absorption, fluorescence, and ¹⁹F-nmr chemical-shift and relaxation methods. Optical absorption and fluorescence experiments indicate that fluoroquine binds to nucleic acids in a similar manner to that of its well-known analog chloroquine. At low drug-to-base pair ratios, binding of both drugs appears to be random. Fluoroquine and chloroquine also elevate the melting temperature (T_m) of DNA to a comparable extent. Binding of fluoroquine to DNA, tRNA, or poly(A) results in a downfield shift of about 1.5 ppm for the ¹⁹F-nmr resonance. The chemical shift of free fluoroquine depends on the isotopic composition of the solvent (D₂O vs H₂O). The solvent isotope shift is virtually eliminated by fluoroquine binding to any one of the nucleic acids. ¹⁹F-nmr relaxation experiments were carried out to measure the spin-lattice relaxation time (T₁), ¹⁹F{¹H} nuclear Overhauser effect (NOE), off-resonance intensity ratio (R), off-resonance rotating-frame spin-lattice relaxation time (T), and linewidth for fluoroquine in the nucleic acid complexes. By accounting for intramolecular proton-fluorine dipolar and chemical-shift anisotropy contributions to the fluorine relaxation, all of the relaxation parameters for the fluoroquine–DNA complex can be well described by a motional model incorporating long-range DNA bending on the order of a microsecond and an internal motion of the drug on the order of a nanosecond. Selective NOE experiments indicate that the fluorine in the drug is near the ribose protons in the RNA complexes, but not in the DNA complex. Details of the binding evidently differ for the two types of nucleic acids. This study provides the foundation for an investigation of fluoroquine in intact cells.     

Increased tRNA modification and gene-specific codon usage regulate cell cycle progression during the DNA damage response

S-phase and DNA damage promote increased ribonucleotide reductase (RNR) activity. Translation of RNR1 has been linked to the wobble uridine modifying enzyme tRNA methyltransferase 9 (Trm9). We predicted that changes in tRNA modification would translationally regulate RNR1 after DNA damage to promote cell cycle progression. In support, we demonstrate that the Trm9-dependent tRNA modification 5-methoxycarbonylmethyluridine (mcm?U) is increased in hydroxyurea (HU)-induced S-phase cells, relative to G? and G?, and that mcm?U is one of 16 tRNA modifications whose levels oscillate during the cell cycle. Codon-reporter data matches the mcm?U increase to Trm9 and the efficient translation of AGA codons and RNR1. Further, we show that in trm9Δ cells reduced Rnr1 protein levels cause delayed transition into S-phase after damage. Codon re-engineering of RNR1 increased the number of trm9Δ cells that have transitioned into S-phase 1 h after DNA damage and that have increased Rnr1 protein levels, similar to that of wild-type cells expressing native RNR1. Our data supports a model in which codon usage and tRNA modification are regulatory components of the DNA damage response, with both playing vital roles in cell cycle progression.     

Comparative Genome Mapping of the Ataxia–Telangiectasia Region in Mouse, Rat, and Syrian Hamster

Chromosomal locations of theAtm(ataxia–telangiectasia (AT)-mutated) andAcat1(mitochondrial acetoacetyl-CoA thiolase) genes in mouse, rat, and Syrian hamster were determined by direct R-banding FISH. Both genes were colocalized to the C-D band of mouse chromosome 9, the proximal end of q24.1 of rat chromosome 8, and qa4–qa5 of Syrian hamster chromosome 12. The regions in the mouse and rat were homologous to human chromosome 11q. Fine genetic linkage mapping of the mouse AT region was performed using the interspecific backcross mice.Atm, Acat1,andNpat,which is a new gene isolated from the AT region, and 12 flanking microsatellite DNA markers were examined. No recombinations were found among theAtm, Npat, Acat1,andD9Mit6loci, and these loci were mapped 2.0 cM distal toD9Mit99and 1.3 cM proximal toD9Mit102.Comparison of the linkage map of mouse chromosome 9 (MMU9) and that of human chromosome 11 (HSA11) indicates that there is a chromosomal rearrangement due to an inversion betweenEts1andAtm–Npat–Acat1and that the inversion of MMU9 originated from the chromosomal breakage at the boundary betweenGria4andAtm–Npat–Acat1on HSA11. This type of inversion appeared to be conserved in the three rodent species, mouse, rat, and Syrian hamster, using additional comparative mapping data with theRckgene.     

Sequence specificity of Bacillus subtilis DNA gyrase in vivo

Linearization of pBG0 (a hydrid between Escherichia coli plasmid pBR322 and Staphylococcus aureus plasmid pUB110) was performed by lysis of the oxolinic acid treated Bacillus subtilis protoplasts with sodium dodecyl sulfate. This plasmid DNA linearization was used both for a detailed mapping of DNA gyrase cleavage sites of various strength and for the nucleotide sequence determinations at the points of gyrase-mediated scission by introducing the XhoI linker DNA. A total of 40 plasmids carrying inserted XhoI linker were sequenced by labeling 3' termini of XhoI sites; 38 of them were found to contain a duplication of four base-pairs of the plasmid sequence flanking the linker, which were characteristic of the oxolinic acid-induced DNA cleavage by E. coli DNA gyrase in vitro and in vivo. The relative strength of these sequenced sites was established by comparing their positions to the sites mapped on the appropriate plasmid genome. This allowed us to propose a consensus sequence of B. subtilis DNA gyrase in vivo cleavage site:GNAT GATCATNC% MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D% aebbfv3ySLgzGueE0jxyaibaiiYdd9qrFfea0dXdf9vqai-hEir8Ve% ea0de9qq-hbrpepeea0db9q8as0-LqLs-Jirpepeea0-as0Fb9pgea% 0lrP0xe9Fve9Fve9qapdbaqaaeGacaGaaiaabeqaamaabaabcaGcba% GaaeikaiaabsfacaqGPaGaaeiiaiaabccacaqGGaGaaeiiaiaabcca% caqGOaGaae4raiaabMcacaqGGaGaaeiiaiaabccacaqGGaGaaeiiai% aabccacaqGGaGaaeiiaiaabccacaqGGaGaaeiiaiaabccacaqGGaGa% aeiiaiaabccacaqGOaGaaeyqaiaabMcaaaa!4E92!\[{\rm{(T) (G) (A)}}\]where N is any nucleotide. The bases in parentheses were preferred secondarily. The involvement of DNA gyrase in illegitimate recombination events in Bacillus subtilis is discussed.     

Altered expression of the PTR/NRT1 homologue OsPTR9 affects nitrogen utilization efficiency,growth and grain yield in rice

The plant PTR/NRT1 (peptide transporter/nitrate transporter 1) gene family comprises di/tripeptide and low‐affinity nitrate transporters; some members also recognize other substrates such as carboxylates, phytohormones (auxin and abscisic acid), or defence compounds (glucosinolates). Little is known about the members of this gene family in rice (Oryza sativa L.). Here, we report the influence of altered OsPTR9 expression on nitrogen utilization efficiency, growth, and grain yield. OsPTR9 expression is regulated by exogenous nitrogen and by the day‐night cycle. Elevated expression of OsPTR9 in transgenic rice plants resulted in enhanced ammonium uptake, promotion of lateral root formation and increased grain yield. On the other hand, down‐regulation of OsPTR9 in a T‐DNA insertion line (osptr9) and in OsPTR9‐RNAi rice plants had the opposite effect. These results suggest that OsPTR9 might hold potential for improving nitrogen utilization efficiency and grain yield in rice breeding.     

High-level expression of the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem multimers in Escherichia coli

To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1 in molecular mass, amino acid sequence and ACE-inhibiting activity. Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998     

A meiosis-specific protein kinase, Ime2, is required for the correct timing of DNA replication and for spore formation in yeast meiosis

 In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase α-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. At late meiotic times, ime2Δ diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase α is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase α are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation. Received: 20 February 1996 / Accepted: 7 June 1996     

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.     

Generation and fluorescent in situ hybridization mapping of yeast artificial chromosomes of 1p, 17p, 17q, and 19q from a hybrid cell line by high-density screening of an amplified library

A yeast artificial chromosome (YAC) library has been constructed from a somatic cell hybrid containing a t(1p;19q) chromosome and chromosome 17. After amplification, part of this library was analyzed by high-density colony filter screening with a repetitive human DNA probe (Alu). The human YACs distinguished by the screening were further analyzed by Alu fingerprinting and Alu PCR. Fluorescent in situ hybridization (FISH) was performed to localize the YACs to subchromosomal regions of chromosome 1p, 17, or 19q. We have obtained a panel of 123 individual YACs with a mean size of 160 kb, and 77 of these were regionally localized by FISH: 33 to 1p, 10 to 17p, 25 to 17q, and 9 to 19q. The YACs cover a total of 19.7 Mb or 9% of the 220 Mb of human DNA contained in the hybrid. No overlapping YACs have yet been detected. These YACs are available upon request and should be helpful in mapping studies of disease loci, e.g., Charcot-Marie-Tooth disease, Miller-Dieker syndrome, hereditary breast tumor, myotonic dystrophy, and malignant hyperthermia.     

A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli

Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of - and -subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (K_m=0.77 M) than for 2-aminophenol (K_m=0.89 M) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the - and -subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the - and -subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.     

Segregation,viral phenotype,and proviral structure of 23 avian leukosis virus inserts in the germ line of chickens

Summary We have artificially introduced 23 avian leukosis virus (ALV) proviral inserts into the chicken germ line by injection of wild-type and recombinant subgroup A ALV near the blastoderm of fertile eggs just before incubation. Eight viremic males were identified as germline mosaics because they transmitted proviral DNA to their generation 1 (G-1) progeny at a low frequency. Eleven female and 9 male G-1 progeny carried 23 distinct proviruses that had typical major clonal proviral-host DNA junction fragments detectable after digestion of their DNA with SacI, Southern blotting and hybridization with a probe representing the complete ALV genome. These proviruses, identified by their typical proviral-host DNA junction fragments, were transmitted to approximately 50% of their G-2 progeny after mating the G-1 parents to a line of chickens lacking endogenous ALV proviral inserts. One G-1 female carried 2 proviruses and another 3. The proviruses appeared to be scattered throughout the genome. One of the 14 proviruses carried by females was on the sex (Z) chromosome. Two of the 3 proviruses carried by a single G-1 female were linked with a recombination frequency of about 0.20. Twenty-one of the proviruses coded for infectious ALV. Two proviruses coded for envelope glycoprotein, and cell cultures carrying them were relatively resistant to subgroup A sarcoma virus, but failed to produce infectious ALV. One of these proviruses coded for internal gag proteins, had a deletion in pol, but produced non-infectious virus particles. The other failed to code for gag proteins and had no detectable internal deletions nor did it produce virus particles. Thus, we have shown that replication-competent ALV can artificially infect germ-line cells and that spontaneous defects in the inherited proviruses occur at a rather low rate.     

A new Pax gene,Pax-9, maps to mouse Chromosome 12

Members of the Pax gene family have recently been shown to play important roles in mouse embryogenesis. Of eight so far characterized Pax genes, three have been associated with mouse developmental mutants. Here we report the cloning of a new Pax gene, Pax-9. Most of the DNA sequence encoding the highly conserved paired domain has been determined and compared with previously known paired domains. This comparison classifies Pax-9 as a member of the same subgroup as Pax-1/undulated. By analysis of the segregation of a Pax-9 restriction fragment length polymorphism and a large number of simple sequence length polymorphisms in an interspecific C57BL/6 x Mus musculus mollosinus backcross, Pax-9 was mapped close to the D12Nds1 locus on the proximal part of Chromosome (Chr) 12.