The same configuration of Ty elements promotes different types and frequencies of rearrangements in different yeast strains

Summary We examined Ty-mediated genomic rearrangements in three related mitotically dividing haploid yeast strains having the same configuration of Ty elements in the CYC1-sup4 interval of chromosome X. Surprisingly, quite different types and frequencies of rearrangements were found in the three strains. In one strain we found only Ty-mediated deletions, which occurred with a frequency of about 1×10^-6. Another strain yielded similar deletions, but approximately one-third of these were accompanied by adjacent Ty-mediated inversions. A third strain was found to have an extremely high rate of inversion/reinversion between two of the three Ty elements. This rate was conservatively estimated to be 1.4±0.2×10^-2 per cell per generation, which is at least 2 orders of magnitude higher than previously reported values for Ty-mediated rearrangements. These data provide evidence that local regions of the genome can, in some cases, be much more fluid than had been previously believed.     

A gel electrophoresis assay for the simultaneous determination of topoisomerase I inhibition and DNA intercalation

The DNA maintenance enzyme, topoisomerase I, is thought to play crucial roles in all living cells and for this reason inhibitors of this enzyme have been much studied. In this paper we describe a gel electrophoresis method capable of characterizing and quantifying inhibition of topoisomerase I by selected compounds. Inhibitors of topoisomerase I are often associated with intercalative binding to DNA and the method can simultaneously determine intercalative binding (as DNA unwinding) except in the cases where inhibition is prohibitively strong. The method uses closed circular (plasmid) DNA and can separate single-strand nicked, linearized (double-strand nicked), fully relaxed, partially relaxed (topoisomers), and supercoiled forms of the plasmid so that topoisomerase-dependent DNA cleavage (poisoning) can also be determined. By quantifying poisoning, inhibition, and intercalation simultaneously and separately in relation to reference compounds it is possible to make quantitative determinations of these phenomena for comparative purposes. Data for the topoisomerase I inhibitor, luteolin, are presented.     

Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots

The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains [promoter (P), ORF (O) and terminator (T)] derived from the common genetic markers HIS3, LEU2 , TRP1 and URA3. Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5 and 3 flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs. Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions. Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions. Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains (PO or OT) of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF. Therefore, we suggest that coding sequences could be considered to be cold spots for plasmid integration in yeast.Communicated by A. Aguilera     

Antitumor drugs possessing topoisomerase I inhibition: applicable separation methods

Separation methods for antitumor drugs capable of topoisomerase I inhibition were reviewed in this study. Camptothecin (CPT) its related analogues seemed to be promising anticancer drugs that exhibit topoisomerase I inhibition. This group of compounds contain a closed α-hydroxy-δ-lactone ring (lactone form) that can undergo reversible hydrolysis to form the open-ring form (carboxylate form). In vitro pharmacological study showed that the antitumor activity of the lactone form was higher than that of the carboxylate form. Thus a quantitative method to separate these two forms is important to evaluate the pharmacokinetics and pharmacodynamics of these compounds. Nevertheless, current separation methods are complicated by the pH-dependent instability of the lactone moiety. High-performance liquid chromatography (HPLC) coupled with fluorometric detection has been widely used for the quantitation of the drug as the intact lactone form or as the total lactone carboxylate forms in biological matrices. In this report we reviewed current applicable chromatographic techniques for further bioanalytical studies of CPT derivatives including sample preparations, HPLC columns, mobile phases and additives.     

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele

Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264Glu) and recA665 (Tyr264His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.     

The identification of a functional interaction between PKC and topoisomerase II

Topoisomerase II plays an essential role in the segregation of chromosomes during cell division. It is also a major component of the nuclear matrix. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between the catalytic domain of the yeast protein kinase 1 enzyme (Pkc1) and the N-terminal domain of the S. cerevisiae topoisomerase II. The S. cerevisiae Pkc1 is the homologue of the mammalian calcium dependent PKC.     

Effect of donor copy number on the rate of gene conversion in the yeast Saccharomyces cerevisiae

Summary Nonreciprocal recombination (gene conversion) between homologous sequences at nonhomologous locations in the genome occurs readily in the yeast Saccharomyces cerevisiae. In order to test whether the rate of gene conversion is dependent on the number of homologous copies available in the cell to act as donors of information, the level of conversion of a defined allele was measured in strains carrying plasmids containing homologous sequences. The level of recombination was elevated in a strain carrying multiple copies of the plasmid, compared with the same strain carrying a single copy of the homologous sequences either on a plasmid or integrated in the genome. Thus, the level of conversion is proportional to the number of copies of donor sequences present in the cell. We discuss these results within the framework of currently favoured models of recombination.     

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)⁺ RNA. This clone contains an insert which is 2775 by in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (K_m = 7.5 ± 0.6 M for SO ₄ ^2– ) was observed. A genomic mutant of S. cerevisiae in which 1096 by were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.     

Examination of the Intron in the meiosis-specific recombination gene REC114 in Saccharomyces

REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3′ end of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3′ end. Furthermore, the 3′ splice site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of yeast contain an intron at the same location in their REC114 genes. Received: 16 October 1996 / Accepted: 10 February 1997     

Mutations at the human minisatellite MS32 integrated in yeast occur with high frequency in meiosis and involve complex recombination events

Minisatellites are composed of tandem repetitive DNA sequences and are present at many positions in the human genome. They frequently mutate to new length alleles in the germline, by complex and incompletely understood recombination mechanisms which may operate during meiosis. In several minisatellites the mutation events are restricted to one end of the repeat array, indicating a possible association with elements that act in cis. Mutant alleles do not show exchange of flanking regions. To construct a model system suitable for further investigations of the mutation process, we have integrated the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot upstream of the LEU2 locus in the yeast Saccharomyces cerevisiae. Here we provide direct evidence for a meiotic origin of MS32 mutations. Mutation events were polarised towards both ends of the minisatellite and varied from simple duplications and deletions to complex intra- and interallelic events. Interallelic events were frequently accompanied by exchange of regions flanking the minisatellite. The results also support the notion that cis-acting elements are involved in the mutational process. The fact that MS32 mutant structures are similar in yeast and human shows that meiotic recombination plays a crucial role in both organisms and emphasises the usefulness of yeast strains harbouring minisatellites as a model system for the study of minisatellite mutation. Received: 1 March 1997 / Accepted: 16 May 1997     

E-ring-modified 7-oxyiminomethyl camptothecins: Synthesis and preliminary in vitro and in vivo biological evaluation

In contrast to five-membered E-ring analogues, 7-oxyiminomethyl derivatives of homocamptothecins showed ability to form stable ternary complexes with DNA and topoisomerase I. The 7-oxyiminomethyl derivatives of homocamptothecins were evaluated as a racemic mixture. Following the isolation of the two enantiomers, the 20 (R)-hydroxy isomer confirms the best activity. By using a panel of human tumor cells, all tested homocamptothecins showed a potent antiproliferative activity, correlating to the persistence of the cleavable complex. No significant difference was observed between the natural scaffold and the corresponding homocamptothecin homologue. A selected compound of this series exhibited an excellent antitumor activity against human gastrointestinal tumor xenografts.     

Quadruplex-Duplex Motifs as New Topoisomerase I Inhibitors

In this article, 13 short chains that can form G-quadruplex and quadruplex-duplex motif have been designed. Fourteen oligonucleotides, including 13 short chains as well as a reference short chain all have certain level of inhibition to topoisomerase I, whether or not they form G-quadruplex and quadruplex-duplex motif, and the G-quadruplex and quadruplex-duplex motif show better activity than single short chain. The result confirmed that after forming G-quadruplex and quadruplex-duplex motif these 14 oligonucleotides are competitive inhibition, that is, through the priority binding with the topoisomerase I and precluding from its binding with the normal substrate pBR322 and, therefore, inhibiting the next reaction.     

Functionally important amino acids in Saccharomyces cerevisiae aspartate kinase

Saccharomyces cerevisiae aspartate kinase (AK(Sc)) phosphorylates L-Asp as the first step in the aspartate pathway responsible for the biosynthesis of L-Thr, L-Met, and L-Ile in microorganisms and plants. Using site-directed mutagenesis, we have evaluated the importance of residues in AK(Sc) that are strongly conserved among aspartate kinases or in other small molecule kinases. Steady state kinetic analysis of the purified AK(Sc) variants reveals that several of the targeted amino acids, particularly K18 and H292, have important roles in the enzymatic reaction. These results provide the first identification of amino acid residues crucial to the action of this important metabolic enzyme.     

Purification and properties of the RuvA and RuvB proteins of Escherichia coli

Summary The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M_r ca. 100000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and -mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M_r ca. 85000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.     

Modulation of the SOS response by truncated RecA proteins

RecA protein plays several key roles in the SOS response. We have constructed truncated proteins and examined their capacity to accomplish Weigle reactivation and mutagenesis of bacteriophage lambda and recombination in Escherichia coli. Our data indicate that the 17 carboxyl terminal amino acids are not essential to RecA function. However in the presence of wild-type RecA protein, the truncated protein reduces the efficiency of recombination without affecting either mutagenesis or induction of an SOS gene or Weigle reactivation. The data presented here suggest that activation of RecA protein does not involve mixed multimers or is not affected by their presence.