Double-strand breaks on YACs during yeast meiosis may reflect meiotic recombination in the human genome.

Meiotic recombination in the yeast Saccharomyces cerevisiae is initiated at double-strand breaks (DSBs), which occur preferentially at specific locations. Genetically mapped regions of elevated meiotic recombination ('hotspots') coincide with meiotic DSB sites, which can be identified on chromosome blots of meiotic DNA (refs 4,5; S.K. et al., manuscript submitted). The morphology of yeast artificial chromosomes (YACs) containing human DNA during the pachytene stage of meiosis resembles that of native yeast chromosomes. Homologous YAC pairs segregate faithfully and recombine at the high rates characteristic of S. cerevisiae (vs. approximately 0.4 cM/kb in S. cerevisiae versus approximately 10-3 cM/kb in humans). We have examined a variety of YACs carrying human DNA inserts for double-strand breakage during yeast meiosis. Each YAC has a characteristic set of meiotic DSB sites, as do yeast chromosomes (S.K. et al., manuscript submitted). We show that the positions of the DSB sites in the YACs depend on the human-derived DNA in the clones. The degree of double-strand breakage in yeast meiosis of the YACs in our study appears to reflect the degree of meiotic recombination in humans.

Increased instability of human CTG repeat tracts on yeast artificial chromosomes during gametogenesis.

Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable. The instability is 6 to 10 times more pronounced in meiosis than during mitotic division. The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present. Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats. The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions. In our experiments, expansions were found almost exclusively during gametogenesis. Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability. These unique patterns have novel implications for possible mechanisms of repeat instability.

A short chromosomal region with major roles in yeast chromosome III meiotic disjunction, recombination and double strand breaks.

A multicopy plasmid was isolated from a yeast genomic library, whose presence resulted in a twofold increase in meiotic nondisjunction of chromosome III. The plasmid contains a 7.5-kb insert from the middle of the right arm of chromosome III, including the gene THR4. Using chromosomal fragments derived from chromosome III, we determined that the cloned region caused a significant, specific, cis-acting increase in chromosome III nondisjunction in the first meiotic division. The plasmid containing this segment exhibited high spontaneous meiotic integration into chromosome III (in 2.4% of the normal meiotic divisions) and a sixfold increase (15.5%) in integration in nondisjunctant meioses. Genetic analysis of the cloned region revealed that it contains a "hot spot" for meiotic recombination. In DNA of rad50S mutant cells, a strong meiosis-induced double strand break (DSB) signal was detected in this region. We discuss the possible relationships between meiosis-induced DSBs, recombination and chromosome disjunction, and propose that recombinational hot spots may be "pairing sites" for homologous chromosomes in meiosis.

Externally added DNA molecules support initiation of transcription in isolated nuclei from petunia.

Nuclei isolated from protoplasts transfected with the pUC8CaMVCAT and pDO432 plasmids were able to support, in run off experiments, the synthesis of specific transcripts as was evident from analysis by dot blot hybridization. Also the addition of the above plasmids to nuclei, prepared from non-transfected protoplasts, supported the synthesis of specific transcripts. Dot blot analysis showed that most of the transcripts obtained were complementary to the relevant gene sequences. alpha-Amanitin, at concentrations which are known to block the activity of RNA polymerase II, significantly inhibited the synthesis of specific transcripts by the isolated nuclei. The transcription activity was found to be predominantly associated with the nuclear fraction while the transcription products (RNA molecules) appeared in the supernatant obtained following sedimentation of the nuclei.

Application of SNPs for assessing biodiversity and phylogeny among yeast strains.

We examined the efficacy of single-nucleotide polymorphism (SNP) markers for the assessment of the phylogeny and biodiversity of Saccharomyces strains. Each of 32 Saccharomyces cerevisiae strains was genotyped at 30 SNP loci discovered by sequence alignment of the S. cerevisiae laboratory strain SK1 to the database sequence of strain S288c. In total, 10 SNPs were selected from each of the following three categories: promoter regions, nonsynonymous and synonymous sites (in open reading frames). The strains in this study included 11 haploid laboratory strains used for genetic studies and 21 diploids. Three non-cerevisiae species of Saccharomyces (sensu stricto) were used as an out-group. A Bayesian clustering-algorithm, Structure, effectively identified four different strain groups: laboratory, wine, other diploids and the non-cerevisiae species. Analysing haploid and diploid strains together caused problems for phylogeny reconstruction, but not for the clustering produced by Structure. The ascertainment bias introduced by the SNP discovery method caused difficulty in the phylogenetic analysis; alternative options are proposed. A smaller data set, comprising only the nine most polymorphic loci, was sufficient to obtain most features of the results.

Uptake and efflux of inorganic carbon in Dunaliella salina.

The apparent photosynthetic Km (CO2) of air-grown Dunaliella salina is 2 µM as measured both by the filtering centrifugation technique and by O2 electrode. These cells are capable of accumulating inorganic carbon (Cinorg) up to 20 times its concentration in the medium. It is suggested that air-grown Dunaliella cells are able to concentrate CO2 within the cell. Analysis of the efflux of Cinorg from cells previously loaded with H(14)CO 3 (-) demonstrated the existence of an internal pool which has an half-time of depletion of 2.5-7 min depending on the conditions of the experiment. This finding indicates that the internal Cinorg pool is not readily exchangeable with the external medium. Furthermore, the influence of the presence or absence of unlabelled Cinorg in the medium during the efflux experiment on the half-time observed indicate that efflux of Cinorg is not a simple diffusion process but is rather carrier-mediated.

Meiotic double-strand breaks in Schizosaccharomyces pombe.

Meiotic DNA double-strand breaks (DSBs) are associated with recombination hot spots in the yeast Saccharomyces cerevisiae and are believed to initiate the process of recombination. Until now, meiosis-induced breaks have not been shown to occur regularly in other organisms. Here we show, by pulsed-field gel electrophoresis of DNA, that meiotic DSBs occur transiently in all three chromosomes of the fission yeast Schizosaccharomyces pombe. In a repair defective mutant, carrying a mutation in the RecA homolog gene rhp51, meiotic DSBs accumulate. In contrast to expectation from the genetic map of S. pombe, however, many chromosomal DNA molecules remain unbroken during meiosis.

Photosynthesis and Inorganic Carbon Accumulation in the Acidophilic Alga Cyanidioschyzon merolae.

The intracellular pH and membrane potential were determined in the acidophilic algae Cyanidoschyzon merolae as a function of extracellular pH. The alga appear to be capable of maintaining the intracellular pH at the range of 6.35 to 7.1 over the extracellular pH range of 1.5 to 7.5. The membrane potential increase from -12 millivolts (negative inside) to -71 millivolts and thus DeltamuH(+) decreased from -300 to -47 millivolts over the same range of extracellular pH. It is suggested that the DeltamuH(+) may set the upper and lower limits of pH for growth. Photosynthetic performance was also determined as a function of pH. The cells appeared to utilize CO(2) from the medium as the apparent K(m(co(2))) was 2 to 3 micromolar CO(2) over the pH range of 1.5 to 7.5 C. merolae appear to possess a ;CO(2) concentrating' mechanism.

Sister chromatid-based DNA repair is mediated by RAD54, not by DMC1 or TID1.

In the mitotic cell cycle of the yeast Saccharomyces cerevisiae, the sister chromatid is preferred over the homologous chromosome (non-sister chromatid) as a substrate for DNA double-strand break repair. However, no genes have yet been shown to be preferentially involved in sister chromatid-mediated repair. We developed a novel method to identify genes that are required for repair by the sister chromatid, using a haploid strain that can embark on meiosis. We show that the recombinational repair gene RAD54 is required primarily for sister chromatid-based repair, whereas TID1, a yeast RAD54 homologue, and the meiotic gene DMC1, are dispensable for this type of repair. Our observations suggest that the sister chromatid repair pathway, which involves RAD54, and the homologous chromosome repair pathway, which involves DMC1, can substitute for one another under some circumstances. Deletion of RAD54 in S.cerevisiae results in a phenotype similar to that found in mammalian cells, namely impaired DNA repair and reduced recombination during mitotic growth, with no apparent effect on meiosis. The principal role of RAD54 in sister chromatid-based repair may also be shared by mammalian and yeast cells.

Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis.

The nature of the inorganic carbon (C(i)) species actively taken up by cyanobacteria CO(2) or HCO(3) (-) has been investigated. The kinetics of CO(2) uptake, as well as that of HCO(3) (-) uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO(2) was higher than that for HCO(3) (-). Though the calculated V(max) was the same in both cases, the maximum rate of uptake actually observed was higher when HCO(3) (-) was supplied. C(i) uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO(2) was the species supplied. Observations of photosynthetic rate as a function of intracellular C(i) level (following supply of CO(2) or HCO(3) (-) for 5 seconds) led to the inference that HCO(3) (-) is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C(i) transport apparatus binds CO(2) and releases HCO(3) (-) at or near a membrane porter. The latter transports HCO(3) (-) ions to the cell interior.

Is HCO(3) Transport in Anabaena a Na Symport?

Na(+) strongly promoted HCO(3) (-) transport in Anabaena variabilis. The effect was highly specific to this cation. Kinetic analysis indicated a progressive decrease in the K(m) (HCO(3) (-)) of the transport system with increasing Na(+) concentration. V(max) was also affected. We raise the possibility that the transport is a Na(+)-HCO(3) (-) symport; alternatively, that a Na(+)-H(+) antiport (or Na(+)-OH(+) symport) system mediates the efflux of the OH(-) ions derived from the entering HCO(3) (-) ions, and that this antiport can rate-limit HCO(3) (-) influx.

Involvement of a Primary Electrogenic Pump in the Mechanism for HCO(3) Uptake by the Cyanobacterium Anabaena variabilis.

The response of the membrane potential to HCO(3) (-) supply has been studied in the cyanobacterium Anabaena variabilis strain M-3 under various conditions. Changes in potential were followed with the aid of the lipophilic cation tetraphenyl phosphonium bromide.Addition of HCO(3) (-) to CO(2)-depleted cells resulted in rapid hyperpolarization. The rate and extent of hyperpolarization were greater in low-CO(2)-adapted than in high-CO(2)-adapted cells. Addition of the electron acceptor p-nitrosodimethylaniline which resulted in O(2) evolution in CO(2)-depleted cells did not cause hyperpolarization. The hyperpolarization was not attributable to a change in pH or in ionic strength of the medium. Pretreatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented the hyperpolarization. KCN depolarized hyperpolarized cells. Addition of HCO(3) (-) also brought about immediate K(+) influx which was succeeded after about 2 minutes by K(+) efflux.TWO OF THE MODELS CONSIDERED WOULD BE CAPABLE OF EXPLAINING THESE AND PREVIOUS FINDINGS: (a) a primary electrogenic pump for transporting HCO(3) (-) ions; (b) proton-HCO(3) (-) contransport, the driving force for which is generated by a proton pump which is sensitive to the HCO(3) (-) concentration.

Induction of HCO(3) Transporting Capability and High Photosynthetic Affinity to Inorganic Carbon by Low Concentration of CO(2) in Anabaena variabilis.

The apparent affinity of photosynthesis for inorganic carbon in Anabaena variabilis strain M-3 increased during the course of adaptation from high to low CO(2) concentration (5% and 0.03% v/v CO(2) in air, respectively). This was attributed to an increased ability of the cells to accumulate inorganic carbon during the course of adaptation to low CO(2) conditions. The release of phycobiliproteins was used to evaluate the sensitivity of the cells to lysozyme treatment followed by osmotic shock. High CO(2)-grown cells were more sensitive to this treatment than were low CO(2) ones. The efflux of inorganic carbon from cells preloaded with radioactive bicarbonate is faster in high than it is in low CO(2)-adapted cells. It is postulated that the cell wall or membrane components undergo changes during the course of adaptation to low CO(2) conditions. This is supported by electron micrographs showing differences in the cell wall appearance between high and low CO(2)-grown cells. The increasing ability to accumulate HCO(3) (-) and the lessened sensitivity to lysozyme during adaptation to low CO(2) conditions depends on protein synthesis. The increase in affinity for inorganic carbon during the adaptation to low CO(2) conditions is severely inhibited by the presence of spectinomycin. Incubation in the light significantly lessens the time required for the adaptation to low CO(2) conditions.

A versatile method for efficient YAC transfer between any two strains.

The ability to transfer yeast artificial chromosome (YAC) clones among yeast hosts greatly enhances their utility as cloned DNAs by increasing the range of methods available for experimental manipulation. An effective method for the transfer of YACs between strains in Kar1- matings is described in the accompanying paper (F. Spencer et al., 1994, Genomics 22, 118-126). To evaluate the general nature of the new methodology, we compare YAC transfer in matings in which the YAC donor, the recipient, or both partners carry the kar1 mutation. A set of four universal kar1 intermediary strains that allow YAC transfer from any source to any target strain of the same or of opposite mating type is described. The procedure requires elementary microbial manipulations, including yeast culture and replica plating, and pulsed-field gel electrophoresis for verification of the YAC transfer and integrity. Transfer of YACs by Kar1- mating provides an efficient, reliable, and highly flexible technique that will greatly facilitate YAC manipulation required for a wide variety of applications.

Energization and activation of inorganic carbon uptake by light in cyanobacteria.

The requirement of the inorganic carbon (C(i)) transport system for light in cyanobacteria was investigated in Anabaena variabilis by the filtering centrifugation technique and in a mutant (E(1)) isolated from Anacystis nidulans using a gas exchange system. C(i) transport capability increased with time of preillumination and decreased following darkening. Full activity could not be obtained by operating either photosystem II (PSII) or photosystem I alone. 3(3,4 Dichlorophenyl)-1,1 dimethylurea strongly inhibited C(i) uptake. Very low activity of PSII was sufficient to activate C(i) uptake. However, in the presence of dithiothreitol PSII activity was not required. We conclude that light may be required to activate as well as to energize C(i) uptake in cyanobacteria.

DNA motif associated with meiotic double-strand break regions in Saccharomyces cerevisiae.

Meiotic recombination in yeast is initiated by DNA double-strand breaks (DSBs) that occur at preferred sites, distributed along the chromosomes. These DSB sites undergo changes in chromatin structure early in meiosis, but their common features at the level of DNA sequence have not been defined until now. Alignment of 1 kb sequences flanking six well-mapped DSBs has allowed us to define a flexible sequence motif, the CoHR profile, which predicts the great majority of meiotic DSB locations. The 50 bp profile contains a poly(A) tract in its centre and may have several gaps of unrelated sequences over a total length of up to 250 bp. The major exceptions to the correlation between CoHRs and preferred DSB sites are at telomeric regions, where DSBs do not occur. The CoHR sequence may provide the basis for understanding meiosis-induced chromatin changes that enable DSBs to occur at defined chromosomal sites.

Switching yeast from meiosis to mitosis: double-strand break repair, recombination and synaptonemal complex.

BACKGROUND: When Saccharomyces cerevisiae cells that have begun meiosis are transferred to mitotic growth conditions ('return-to-growth', RTG), they can complete recombination at high meiotic frequencies, but undergo mitotic cell division and remain diploid. It was not known how meiotic recombination intermediates are repaired following RTG. Using molecular and cytological methods, we investigated whether the usual meiotic apparatus could repair meiotically induced DSBs during RTG, or whether other mechanisms are invoked when the developmental context changes. RESULTS: Upon RTG, the rapid disappearance of meiotic features--double-strand breaks in DNA (DSBs), synaptonemal complex (SC), and SC related structures-was striking. In wild-type diploids, the repair of meiotic DSBs during RTG was quick and efficient, resulting in homologous recombination. Kinetic analysis of double-strand breakage and recombination indicated that meiotic DSB formation precedes the commitment to meiotic levels of recombination. DSBs were repaired in RTG in dmc1, but not rad51 mutants, hence repair did not occur by the usual meiotic mechanism which requires the Dmc1 gene product. In haploids, DSBs were also repaired quickly and efficiently upon RTG, showing that DSB repair did not require the presence of a homologous chromosome. In all strains examined, SC and related structures were not required for DSB repair or recombination following RTG. CONCLUSIONS: At least two pathways of DSB repair, which differ from the primary meiotic pathway(s), can occur during RTG: One involving interhomologue recombination, and another involving sister-chromatid exchange. DSB formation precedes commitment to recombination. SC elements appear to prevent sister chromatid exchange in meiosis.

Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae.

We present a scheme for locating double-strand breaks (DSBs) in meiotic chromosomes of Saccharomyces cerevisiae, based on the separation of large DNA molecules by pulsed field gel electrophoresis. Using a rad50S mutant, in which DSBs are not processed, we show that DSBs are widely induced in S. cerevisiae chromosomes during meiosis. Some of the DSBs accumulate at certain preferred sites. We present general profiles of DSBs in chromosomes III, V, VI and VII. A map of the 12 preferred sites on chromosome III is presented. At least some of these sites correlate with known 'hot spots' for meiotic recombination. The data are discussed in view of current models of meiotic recombination and chromosome segregation.

Patterns of meiotic double-strand breakage on native and artificial yeast chromosomes.

The preferred positions for meiotic double-strand breakage were mapped on Saccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts. Each chromosome had strong and weak double-strand break (DSB) sites. On average one DSB-prone region was detected by pulsed-field gel electrophoresis per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites. There were no preferred meiotic DSB sites near the telomeres. DSB-prone regions were associated with all of the known "hot spots" for meiotic recombination on chromosomes I, III and VI.

Double-strand breaks on artificial chromosomes in yeast.

Yeast artificial chromosomes composed primarily of bacteriophage gamma DNA exhibit very low levels of meiotic crossing over compared with similarly sized intervals of natural yeast DNA. When these recombinationally quiet chromosomes were augmented with a 12.5 kb insert of sequences from yeast chromosome VIII, genetic studies demonstrated that the artificial chromosomes had acquired recombination properties characteristic of this region of chromosome VIII. On authentic yeast chromosomes, most meiotic recombination events are initiated at sites where the DNA is cleaved to create a double-strand break (DSB). This report describes physical analyses that were carried out to examine the relationship between DSB sites and the recombination behavior of the artificial chromosomes. The results show that DSBs are rare on these artificial chromosomes, except for the 12.5 kb insert. Mapping of the DSB sites shows that their positions correlate with the previously determined positions of DSB sites on chromosome VIII. Deletion of two characterized chromosome VIII DSB sites from the 12.5 kb insert on the artificial chromosome resulted in the loss of the predicted DSB fragments and a reduction in crossing over between artificial chromosomes.