Meiotic chromosomes move by linkage to dynamic actin cables with transduction of force through the nuclear envelope.

Chromosome movement is prominent during meiosis. Here, using a combination of in vitro and in vivo approaches, we elucidate the basis for dynamic mid-prophase telomere-led chromosome motion in budding yeast. Diverse findings reveal a process in which, at the pachytene stage, individual telomere/nuclear envelope (NE) ensembles attach passively to, and then move in concert with, nucleus-hugging actin cables that are continuous with the global cytoskeletal actin network. Other chromosomes move in concert with lead chromosome(s). The same process, in modulated form, explains the zygotene "bouquet" configuration in which, immediately preceding pachytene, chromosome ends colocalize dynamically in a restricted region of the NE. Mechanical properties of the system and biological roles of mid-prophase movement for meiosis, including recombination, are discussed.

Single molecule detection of direct, homologous, DNA/DNA pairing.

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the "default" option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.

Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes.

Receptors for N-methyl-D-aspartate (NMDA) are involved in many plastic and pathological processes in the brain. Glycine has been reported to potentiate NMDA responses in neurons and in Xenopus oocytes injected with rat brain messenger RNA. Glycine is now shown to be absolutely required for activation of NMDA receptors in oocytes. In voltage-clamped oocytes, neither perfusion nor rapid pressure application of NMDA onto messenger RNA-injected oocytes caused a distinct ionic current without added glycine. When glycine was added, however, NMDA evoked large inward currents. The concentration of glycine required to produce a half-maximal response was 670 nanomolar, and the glycine dose-response curve extrapolated to zero in the absence of glycine. Several analogs of glycine could substitute for glycine, among which D-serine and D-alanine were the most effective. The observation that D-amino acids are effective will be important in developing drugs targeted at the glycine site.

Rat brain N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

N-methyl-D-aspartate (NMDA) activates a class of excitatory amino acid receptor involved in a variety of plastic and pathological processes in the brain. Quantitative study of the NMDA receptor has been difficult in mammalian neurons, because it usually exists with other excitatory amino acid receptors of overlapping pharmacological specificities. Xenopus oocytes injected with messenger RNA isolated from primary cultures of rat brain have now been used to study NMDA receptors. The distinguishing properties of neuronal NMDA receptors have been reproduced in this amphibian cell, including voltage-dependent block by magnesium, block by the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid, and potentiation by glycine. This preparation should facilitate the quantitative study of the regulation of NMDA receptor activation and serve as a tool for purification of the encoding messenger RNA.

Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast.

RecA protein is essential in eubacteria for homologous recombination and promotes the homologous pairing and strand exchange of DNA molecules in vitro. Recombination proteins with weak sequence similarity to bacterial RecA proteins have been identified in bacteriophage T4, yeast, and other higher organisms. Analysis of the primary sequence relationships of DMC1 from Saccharomyces cerevisiae and UvsX of T4 relative to the three-dimensional structure of RecA from Escherichia coli suggests that both proteins are structural homologs of bacterial RecA proteins. This analysis argues that proteins in this group are members of a single family that diverged from a common ancestor that existed prior to the divergence of prokaryotes and eukaryotes.

NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae.

We describe the identification of a new meiosis-specific gene of Saccharomyces cerevisiae, NDT80. The ndt80 null and point mutants arrest at the pachytene stage of meiosis, with homologs connected by full-length synaptonemal complexes and spindle pole bodies duplicated but unseparated. Meiotic recombination in an ndt80 delta mutant is relatively normal, although commitment to heteroallelic recombination is elevated two- to threefold and crossing over is decreased twofold compared with those of the wild type. ndt80 arrest is not alleviated by mutations in early recombination genes, e.g., SPO11 or RAD50, and thus cannot be attributed to an intermediate block in prophase chromosome metabolism like that observed in several other mutants. The ndt80 mutant phenotype during meiosis most closely resembles that of a cdc28 mutant, which contains a thermolabile p34, the catalytic subunit of maturation-promoting factor. Cloning and molecular analysis reveal that the NDT80 gene maps on the right arm of chromosome VIII between EPT1 and a Phe-tRNA gene, encodes a 627-amino-acid protein which exhibits no significant homology to other known proteins, and is transcribed specifically during middle meiotic prophase. The NDT80 gene product could be a component of the cell cycle regulatory machinery involved in the transition out of pachytene, a participant in an unknown aspect of meiosis sensed by a pachytene checkpoint, or a SPO11- and RAD50-independent component of meiotic chromosomes that is the target of cell cycle signaling.

Multiple IS10 rearrangements in Escherichia coli.

We have investigated the occurrence of multiple transposon-promoted chromosomal rearrangements in Escherichia coli K12 strains containing transposon Tn10. We show that a single Tn10 element, with its two closely spaced insertion sequence (IS10) elements, frequently gives rise to complex rearrangements that can be accounted for as the sum of two "classical" IS10 events. Using a strain containing differentially marked Tn10 elements at widely separated locations, we have investigated the possibility that IS10-promoted rearrangements occur in cell-wide "bursts", as expected if cells could occasionally undergo brief periods when all IS10 transposition events were activated, interspersed with longer periods of relative quiescence. We find no evidence for strong (greater than 60-fold), periodic cell-wide activation under our experimental conditions. The sensitivity of this experiment has been evaluated using an expression for the accumulation of double mutations in populations with heterogeneous, fluctuating mutation rates (see Appendix). We discuss several mechanisms by which two closely linked IS10 elements could undergo coupled double events without cell-wide activation: local activation of small chromosomal regions, periodic bursts of synthesis of cis-acting transposase protein, and/or a propensity for elements that have actually engaged in one rearrangement event to initiate a second successive event immediately thereafter. We favor the last possibility.

Insertion sequence IS10 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA.

Transposition of insertion sequence IS10 is regulated by an anti-sense RNA which inhibits transposase expression when IS10 is present in multiple copies per cell. The anti-sense RNA (RNA-OUT) consists of a stem domain topped by a flexibly paired loop; the 5' end of the target molecule, RNA-IN, is complementary to the top of the loop, and complementarity extends for 35 base-pairs down one side of RNA-OUT. We present here genetic evidence that anti-sense pairing, both in vitro and in vivo, initiates by interaction of the 5' end of RNA-IN and the loop domain of RNA-OUT; other features of the reaction are discussed. In the context of this model, we discuss features of this anti-sense system which are important for its biological effectiveness, and suggest that IS10 provides a convenient model for design of efficient artificial anti-sense RNA molecules.

A new generalizable test for detection of mutations affecting Tn10 transposition.

We describe here a new rapid screen that allows easy detection of transposon or host mutations that affect Tn10 transposition in Escherichia coli. This test involves a new Tn10 derivative called the "mini-lacZ-kanR fusion hopper" or mini-Tn10-LK for short. This element does not direct expression of beta-galactosidase when present at its original starting location on a suitably engineered plasmid or phage genome because it lacks appropriate transcription and translation start signals. However, transposition of this element into the chromosome of E. coli lacZ- bacteria leads to productive fusions in which the lacZ gene within the transposon is expressed from external chromosomal signals. Such fusions are readily detectable on MacConkey lactose indicator plates as red (Lac+) papillae inside of white (LacZ-) colonies. The length of time required to see red papillae appearing in a white colony sensitively and accurately reflects the transposition frequency of the mini-transposon within the colonies. Differences in times for color formation are sensitive enough that 10-fold differences in transposition frequency can readily be detected. This papillation assay can be used to identify mutant clones in which the frequency of Tn10 transposition is either increased or decreased. We have successfully used the assay to identify mutations in the terminal sequences of Tn10; mutations in the Tn10 transposase gene or the bacterial host can be isolated just as easily. This screen should be readily adaptable to transposable elements other than Tn10.

A new type of fusion analysis applicable to many organisms: protein fusions to the URA3 gene of yeast.

We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5'-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.

Two classes of Tn10 transposase mutants that suppress mutations in the Tn10 terminal inverted repeat.

Tn10 transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6-13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named "SEM" for suppression of ends mutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class II mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.

Mismatch repair mutations of Escherichia coli K12 enhance transposon excision.

Excision of the prokaryotic transposon Tn10 is a host-mediated process that occurs in the absence of recA function or any transposon-encoded functions. To determine which host functions might play a role in transposon excision, we have isolated 40 mutants of E. coli K12, designated tex, which increase the frequency of Tn10 precise excision. Three of these mutations (texA) have been shown to qualitatively alter RecBC function. We show that 21 additional tex mutations with a mutator phenotype map to five genes previously identified as components of a methylation-directed pathway for repair of base pair mismatches: uvrD, mutH, mutL, mutS and dam. Previously identified alleles of these genes also have a Tex phenotype.--Several other E. coli mutations affecting related functions have been analyzed for their effects on Tn10 excision. Other mutations affecting the frequency of spontaneous mutations (mutT, polA, ung), different excision repair pathways (uvrA, uvrB) or the state of DNA methylation (dcm) have no effect on Tn10 excision. Mutations ssb-113 and mutD5, however, do increase Tn10 excision.--The products of the mismatch correction genes probably function in a coordinated way during DNA repair in vivo. Thus, mutations in these genes might also enhance transposon excision by a single general mechanism. Alternatively, since mutations in each gene have qualitatively and quantitatively different effects on transposon excision, defects in different mismatch repair genes may enhance excision by different mechanisms.

A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains.

In this paper, we describe a 3.8-kb molecular construct that we have used to disrupt yeast genes. The construct consists of a functional yeast URA3 gene flanked by 1.1-kb direct repeats of a bacterial sequence. It is straightforward to insert the 3.8-kb segment into a cloned target gene of interest and then introduce the resulting disruption into the yeast genome by integrative transformation. An appropriate DNA fragment containing the disruption plus flanking homology can be obtained by restriction enzyme digestion. After introducing such fragments into yeast by transformation, stable integrants can be isolated by selection for Ura+. The important feature of this construct that makes it especially useful is that recombination between the flanking direct repeats occurs at a high frequency (10(-4)) in vegetatively grown cultures. After excision, only one copy of the repeat sequence remains behind. Thus in the resulting strain, the Ura+ selection can be used again, either to disrupt a second gene in similar fashion or for another purpose.

The yeast RAD50 gene encodes a predicted 153-kD protein containing a purine nucleotide-binding domain and two large heptad-repeat regions.

The RAD50 gene of Saccharomyces cerevisiae is required for chromosome synapsis and recombination during meiosis and for repair of DNA damage during vegetative growth. The precise role of the RAD50 gene product in these processes is not known. Most rad50 mutant phenotypes can be explained by the proposal that the RAD50 gene product is involved in the search for homology between interacting DNA molecules or chromosomes, but there is no direct evidence for this model. We present here the nucleotide sequence of the RAD50 locus and an analysis of the predicted 153-kD RAD50 protein. The amino terminal region of the predicted protein contains residues suggestive of a purine nucleotide binding domain, most likely for adenine. The remaining 1170 amino acids consist of two 250 amino acid segments of heptad repeat sequence separated by 320 amino acids, plus a short hydrophobic carboxy-terminal tail. Heptad repeats occur in proteins such as myosin and intermediate filaments that form alpha-helical coiled coils. One of the two heptad regions in RAD50 shows similarity to the S-2 domain of rabbit myosin beyond that expected for two random coiled coil proteins.

A general method for identifying recessive diploid-specific mutations in Saccharomyces cerevisiae, its application to the isolation of mutants blocked at intermediate stages of meiotic prophase and characterization of a new gene SAE2.

We describe a general new approach for identifying recessive mutations that affect diploid strains of yeast Saccharomyces cerevisiae and the application of this method to the identification of mutations that confer an intermediate block in meiotic prophase chromosome metabolism. The method uses a temperature-sensitive conjugation mutation ste7-1 in combination with homothallism. The mutations of interest confer a defect in spore formation that is dependent upon a gene required for initiation of meiotic recombination and development of meiosis-specific chromosome structure (SPO11). Identified in this screen were null mutations of the DMC1 gene, nonnull mutations of RAD50 (rad50S), and mutations in three new genes designed SAE1, SAE2 and SAE3 (Sporulation in the Absence of Spo Eleven). Molecular characterization of the SAE2 gene and characterization of meiotic and mitotic phenotypes of sae2 mutants are also presented. The phenotypes conferred by a sae2 null mutation are virtually indistinguishable from those conferred by the previously identified nonnull mutations of RAD50 (rad50S). Most notably, both mutations confer only weak sensitivity to the radiomimetic agent methyl methane sulfonate (MMS) but completely block resection and turnover of meiosis-specific double-strand breaks. These observations provide further evidence that this constellation of phenotypes identifies a specific molecular function.

Mutations in Saccharomyces cerevisiae that block meiotic prophase chromosome metabolism and confer cell cycle arrest at pachytene identify two new meiosis-specific genes SAE1 and SAE3.

Two new meiosis-specific genes, SAE1 and SAE3, have been identified in a screen for mutations that confer an intermediate block in meiotic prophase. Such mutations confer a block to spore formation that is circumvented by addition of a mutation that eliminates meiotic recombination initiation and other aspects of chromosome metabolism, i.e., spo11. We show that sae1-1 and sae3-1 mutations each confer a distinct defect in meiotic recombination. sae1-1 produces recombinants but very slowly and ultimately to less than half the wild-type level; sae3-1 makes persistent hyper-resected meiotic double-strand breaks and has a severe defect in formation of recombinants. Both mutants arrest at the pachytene stage of meiotic prophase, sae1-1 temporarily and sae3-1 permanently. The phenotypes conferred by sae3-1 are similar to those conferred by mutation of the yeast RecA homologue DMC1, suggesting that SAE3 and DMC1 act at the same step(s) of chromosome metabolism. These results provide further evidence that intermediate blocks to prophase chromosome metabolism cause cell-cycle arrest. SAE1 encodes a 208-residue protein homologous to vertebrate mRNA cap-binding protein 20. SAE3 corresponds to a meiosis-specific RNA encoding an unusually short open reading frame of 50 codons.

Genetic evidence against intramolecular rejoining of the donor DNA molecule following IS10 transposition.

Tn10 and IS10 transpose by a nonreplicative mechanism in which the transposon is excised from the donor molecule and integrated into a target DNA site, leaving behind a break at the original donor site. The fate of this broken donor DNA molecule is not known. We describe here two experiments that address this issue. One experiment demonstrates that a polar IS10 element gives rise to polarity-relief revertants at less than 1% the frequency of transposition of the same element in the same culture. In a second experiment, transpositions of an IS10 element from one site in the bacterial genome to another are selected and the resulting isolates examined for alterations at the donor site; none of 1088 such isolates exhibited a detectable change at the donor locus. These results are compatible with two possible fates of the transposon donor molecule: degradation ("donor suicide"), or restoration of the original information at the donor site by a recombinational repair mechanism analogous to double-strand break repair. These results argue against the possibility that the donor molecule gap is simply resealed by intramolecular rejoining.

Terminal redundancy heterozygotes involving the first-step-transfer region of the bacteriophage T5 chromosome.

Individual progeny of two-factor crosses between A1am and A2am T5 phages give rise to bursts containing more than one type of plaque. The simplest explanation for these mixed bursts is that the A1 and A2 genes are located within the terminally repeated portion of the T5 genome and that the mixed bursts are made by "terminal redundancy heterozygotes". The observation of genetic heterozygosity means that the A1 and A2 genes are repeated intact. This implies that the terminal segments of T5 are genetically interchangeable.

Transposition of plasmid-borne Tn10 elements does not exhibit simple length-dependence.

The transposition frequencies of Tn10 elements from the bacterial chromosome to an F epitome decrease 40% for every kilobase increase in transposon length. The basis for this relationship is not known. We have now examined complemented transposition of defective Tn10 elements off small multicopy plasmids. We find that length dependence in this situation is either reduced or absent, depending on the specific class of transposition events involved. These observations can be interpreted as evidence against the model that chromosomal length dependence occurs because of decay of a transposition-associated replicative complex. This interpretation is consistent with unrelated experiments suggesting that Tn10 transposition is normally nonreplicative. Alternative explanations of length dependence phenomena are discussed.