Meiotic chromosome synapsis in a haploid yeast

An extensive synaptonemal complex (SC) is found at pachytene in whole mount spread preparations of a haploid yeast, Saccharomyces cerevisiae, strain. Whereas unsynapsed axial elements are present only in a few nuclei, in others non-homologous synapsis involves virtually the whole chromosome set. This suggests that homology is not an indispensable precondition for SC formation in yeast but that chromosomes engage in non-homologous synapsis if no homologous partner is available. Recent evidence that in the sporulation deficient yeast mutants rad50 and mer1 axial elements do form but remain unsynapsed in the majority of nuclei is discussed in the light of the above findings.by D. Schweizer     

Denaturation mapping of R factor deoxyribonucleic acid.

The R factor NR1 consists of two components: a resistance transfer factor which harbors the tetracycline resistance genes (RTF-TC) and the r-determinants component which harbors the other drug resistance genes. Using partial denaturation mapping it is possible to distinguish the RTF-TC region from the r-determinants region of the composite R factor NR1 DNA which has a contour length of 37 mum and a density of 1.712 g/ml. The r-determinants region was a relatively undenatured 8.5-mum segment of the molecule when the deoxyribonucleic acid was partially denatured at pH 10.7. An RTF-TC genetic segregant of NR1 which had lost the r-determinants component had a contour length of 28.7 mum and a density of 1.710 g/ml. Characterization of an RTF-TC using partial denaturation mapping at pH 10.7 confirmed that the relatively undenatured 8.5-mum r-determinants segment of the composite R factor had been deleted. Circular, transitioned NR1 DNA molecules (1.716 to 1.718 g/ml), whose contour lengths were consistent with an RTF-TC plus an integral number of tandem copies of r-determinants, were also characterized by denaturation mapping. The relatively undenatured region in these molecules had a length equal to an integral number of copies of r-determinants and was located at the same site in the partially denatured RTF-TC as the single copy of r-determinants in the 37-mum composite NR1. This indicates that there is a unique integration site for r-determinants in the RTF-TC component. The R factor UCR122, a TC deletion mutant of NR1, was also characterized by denaturation mapping. The translocation of the TC resistance gene(s) on the denaturation map permitted the alignment of the denaturation map with the heteroduplex map of Sharp et al. (u073). Linear and circular monomeric and presumed multimeric r-determinants DNA molecules (p = 1.718 g/ml) were partially denatured at a higher pH (11.10). The r-determinants multimers showed a repeating 8.3-mum (monomeric) partial denaturation pattern indicating a head-to-tail arrangement of monomers in these poly-r-determinant molecules.     

Participation of deoxyribonucleic acid polymerase alpha in amplification of ribosomal deoxyribonucleic acid in Xenopus laevis.

Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase alpha, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase alpha, but not DNA polymerase gamma, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase beta was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase alpha plays a major role in ribosomal ribonucleic acid gene amplification.     

Meiotic recombination within the centromere of a yeast chromosome

In order to examine the frequency of nonreciprocal recombination (gene conversion) within the centromere of the yeast chromosome, we constructed strains that contained heterozygous restriction sites in the conserved centromere sequences of chromosome III in addition to heterozygous markers flanking the centromere. One of these markers was the selectable URA3 gene, which was inserted less than one kb from the centromere. We found that meiotic conversion of the URA3 gene occurred at normal frequency (about 2% of unselected tetrads) and that more than one-third of these convertants coconverted the markers within the centromere. In addition, we observed tetrads in which conversion events extended through the centromere to include a marker on the opposite side from URA3. We conclude that meiotic conversion events occur within the centromere at rates similar to other genomic sequences.     

Study of ribosomal deoxyribonucleic acid of sea urchin

The structure of ribosomal DNA (rDNA) satellite of sea urchin (Lytechinus variegatus) sperm has been re-examined after purification by two widely used methods. Saturation hybridization experiments indicate that about 28–32% of the rDNA contain sequences complementary to rRNA. Results of hyperchromic spectral analysis reveal that the rDNA melts as two distinct but unequal components. The early transition presumably corresponds to the melting of most of the transcribed part and the late transition is suggestive of the melting of a G+C-rich segment of the rDNA, which may be a non-transcribed spacer.Abbreviations F_AT fraction of all A+T pairs denatured - F_GC fraction of all G+C pairs denatured - SSC standard saline citrate - rDNA template for ribosomal RNA This work is a part of the author's Ph.D. thesis (U.N.C., Chapel Hill).     

Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria

1. Nucleic acids were released from Escherichia coli by lysing with tri-iso-propylnaphthalene sulphonate and 4-aminosalicylate and then extracting with a phenol-cresol mixture. 2. Nucleic acids were similarly released from Bacillus subtilis after initial treatment with lysozyme. 3. DNA was sedimented after careful precipitation with m-cresol or 2-butoxyethanol (0.1-0.12vol.) in the presence of 20% sodium benzoate. 4. Contaminating ribosomal RNA was removed by precipitation in the presence of 4m-sodium chloride or by extracting DNA with an acetate-butyrate mixture, in which RNA is insoluble. 5. The DNA from B. subtilis has a transforming ability of 0.3-0.6% for the tryptophan marker. 6. Ribosomal RNA was then precipitated with rapidly labelled RNA by the addition of an equal volume of 2-butoxyethanol. 7. There was good separation of the nucleic acids from protein and polysaccharides.     

Linear mitochondrial deoxyribonucleic acid from the yeast Hansenula mrakii.

The mitochondrial deoxyribonucleic acid (mtDNA) from a petite-negative yeast, Hansenula mrakii, was studied. A linear restriction map was constructed with 11 restriction enzymes. The linearity of the genome was confirmed by direct end labeling of the molecule, followed by restriction analysis. The molecular weight of the DNA was found to be 55,000 base pairs. This is the first linear mtDNA found in yeast species. Using specific gene probes obtained from Saccharomyces cerevisiae mtDNA, we have constructed a gene map of H. mrakii mtDNA. The arrangement of genes in this linear genome was very different from the circular mtDNA of other known yeasts.     

Binding of rat ribosomal proteins to yeast 5.8S ribosomal ribonucleic acid.

5.8 S RNA-protein complexes were prepared using purified yeast 5.8 S RNA and proteins from the large ribosomal subunit of rat liver. Formation of such hybrid complexes, as measured by Millipore filtration, was dependent on protein concentration. Binding of proteins to the RNA could approach saturation. Such complexes were isolated from sucrose density gradient centrifugation and shown to contain proteins L6, L8, L19, L35 and L35a. These proteins were identified by their molecular weights on polyacrylamide gels containing dodecylsulfate and their mobilities on two dimensional polyacrylamide gels.     

Meiotic cohesins modulate chromosome compaction during meiotic prophase in fission yeast

The meiotic cohesin Rec8 is required for the stepwise segregation of chromosomes during the two rounds of meiotic division. By directly measuring chromosome compaction in living cells of the fission yeast Schizosaccharomyces pombe, we found an additional role for the meiotic cohesin in the compaction of chromosomes during meiotic prophase. In the absence of Rec8, chromosomes were decompacted relative to those of wild-type cells. Conversely, loss of the cohesin-associated protein Pds5 resulted in hypercompaction. Although this hypercompaction requires Rec8, binding of Rec8 to chromatin was reduced in the absence of Pds5, indicating that Pds5 promotes chromosome association of Rec8. To explain these observations, we propose that meiotic prophase chromosomes are organized as chromatin loops emanating from a Rec8-containing axis: the absence of Rec8 disrupts the axis, resulting in disorganized chromosomes, whereas reduced Rec8 loading results in a longitudinally compacted axis with fewer attachment points and longer chromatin loops.     

Enhancement of ribosomal ribonucleic acid synthesis by deoxyribonucleic acid gyrase activity in Escherichia coli.

The effect of the deoxyribonucleic acid (DNA) gyrase inhibitors coumermycin A1, novobiocin, and oxolinic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was studied in vivo and in vitro. Preferential inhibition of ribosomal RNA (rRNA) synthesis was observed. No effect of oxolinic acid and coumermycin on rRNA synthesis was seen in mutants having a DNA gyrase which is resistant to these inhibitors. In a temperature-sensitive DNA gyrase mutant rRNA synthesis was decreased at nonpermissive temperatures. Thus, a functional DNA gyrase is required for rRNA synthesis. Purified DNA gyrase had no effect on rRNA synthesis in a purified system. However, DNA gyrase does show preferential stimulation of rRNA synthesis in a system supplemented with other proteins. Apparently, DNA gyrase stimulation of rRNA synthesis requires another protein.     

Denaturation mapping studies on the circular chloroplast deoxyribonucleic acid from pea leaves.

The structure of circular pea chloroplast DNA (ctDNA) has been analyzed by denaturation mapping. All of the pea ctDNA molecules that were examined had identical gross base sequences. Denaturation maps were constructed at denaturation levels of 2.5%, 22%, and 44%. These denaturation maps showed that the circular pea ctDNA contained six small AT-rich regions on one-half of the DNA molecule, and two small GC-rich regions on the other half of the DNA molecule. The structure of pea ctDNA circular dimers was also examined. The results showed that the pea ctDNA circular dimers consisted of two monomer length units integrated in tandem repeat.