The Ade6-M26 Mutation of Schizosaccharomyces Pombe Increases the Frequency of Crossing over

The ade6-M26 mutation of Schizosaccharomyces pombe increases conversion frequency in comparison with the nearby mutation ade6-M375. In order to investigate the effect of ade6-M26 on crossover frequency, heteroallelic ade6 duplications were constructed by integration of plasmids carrying the marker gene ura4. One ade6 gene carries either of the mutations M26 or M375 while the other ade6 copy carries the L469 mutation in both duplications. The duplication with ade6-M26 yields Ade(+) recombinants at significantly higher frequencies in meiosis, but not in mitosis. Tetrad analysis and physical characterization of spore clones from recombination tetrads demonstrate that conversions, unequal crossovers and intrachromatid exchanges occur at higher frequencies but with unaltered proportions among them. The conversion events show a pronounced bias when M26 is involved: they take place preferentially at the M26 allele. Thus the ade6-M26 mutation not only enhances conversion frequency as demonstrated before, but also crossover frequency. It displays the properties expected for a preferred site of initiation of general meiotic recombination. The duplications also yielded new information on ectopic recombination in S. pombe: ectopic crossovers occur in the duplications at much higher frequency than among naturally dispersed homologous sequences.     

Inactivation of nonsense suppressor transfer RNA genes in Schizosaccharomyces pombe. Intergenic conversion and hot spots of mutation

Intergenic conversion is a mechanism for the concerted evolution of repeated DNA sequences. A new approach for the isolation of intergenic convertants of serine tRNA genes in the yeast Schizosaccharomyces pombe is described. Contrary to a previous scheme, the intergenic conversion events studied in this case need not result in functional tRNA genes. The procedure utilizes crosses of strains that are homozygous for an active UGA suppressor tRNA gene, and the resulting progeny spores are screened for loss of suppressor activity. In this way, intergenic convertants of a tRNA gene are identified that inherit varying stretches of DNA sequence from either of two other tRNA genes. The information transferred between genes includes anticodon and intron sequences. Two of the three tRNA genes involved in these information transfers are located on different chromosomes. The results indicate that intergenic conversion is a conservative process. No infidelity is observed in the nucleotide sequence transfers. This provides further evidence for the hypothesis that intergenic conversion and allelic conversion are the result of the same molecular mechanism. The screening procedure for intergenic revertants also yields spontaneous mutations that inactivate the suppressor tRNA gene. Point mutations and insertions of A occur at various sites at low frequency. In contrast, A insertions at one specific site occur with high frequency in each of the three tRNA genes. This new type of mutation hot spot is found also in vegetative cells.     

Mitotic recombination between dispersed but related rRNA genes of Schizosaccharomyces pombe generates a reciprocal translocation

Summary Recombination between dispersed yet related serine tRNA genes of Schizosaccharomyces pombe does occur during mitosis but it is approximately three orders of magnitude less frequent than in meiosis. Two mitotic events have been studied in detail. In the first, a sequence of at least 18 nucleotides has been transferred from the donor sup3 gene on the right arm of chromosome I to the related acceptor gene sup12 on the left arm of the same chromosome, thereby leading to the simultaneous change of 8 bp in the acceptor gene. This event must be explained in terms of recombination rather than mutation. It is assumed that it represents mitotic gene conversion, although it was not possible to demonstrate that the donor gene had emerged unchanged from the event. The second case reflects an interaction between sup9 on chromosome III and sup3 on chromosome I. Genetic and physical analysis allows this event to be described as mitotic gene conversion associated with crossingover. The result of this event is a reciprocal translocation. No further chromosomal aberrations were found among an additional 700 potential intergenic convertants tested. Thus intergenic conversion is much less frequently associated with crossingover than allelic conversion. However, the rare intergenic conversion events associated with crossingover provide a molecular mechanism for chromosomal rearrangements.     

Purification and characterization of metallothionein from liver of cadmium exposed Rhesus monkeys (Macaca mulatta)

Metallothionein (MT) a low molecular weight, Cd-binding, cysteine rich, cytosolic protein has been isolated, purified and characterized from cadmium exposed Rhesus monkeys maintained on protein calorie malnourished (PCM) diet. Metallothionein was resolved into three isoforms i.e. MT_a, MT_b and MTV_c. The ratio of Cd, Zn and Cu varied in these isometallothioneins. MT_c was the major isometallothionein. UV Spectra of MT_c revealed the presence of mercaptide bonds and absence of aromatic amino acids. These observations were further confirmed by amino acid analysis of MT_c which demonstrated high cysteine content (22.6) followed by serine, glycine and lysine. The molecular weight of MT_c as determined by gel filtration and amino acid analysis was 13000 and 6398 daltons respectively. This demonstrates that MT_c is a non-globular ellipsoid polypeptide. MT_c showed a unique property of binding selenium. Monkey liver metallothionein was immunologically identical with human metallothionein. All the characteristics of MT_c obtained in the present study reveal a similarity between monkey and human metallothionein probably due to closer phylogenetic relationship between the two species.     

Meiotic Behavior and Linkage Relationships in the Secondarily Homothallic Fungus Agaricus Bisporus

This study followed the transmission of 64 segregating genetic markers to 52 haploid offspring, obtained from both homokaryotic and heterokaryotic meiospores, of a cross (AG 93b) of Agaricus bisporus, the commonly cultivated ``button mushroom.' The electrophoretic karyotypes of the AG 93b component nuclei were determined concurrently (n = 13). Eleven distinct linkage groups were identified by two-point analysis. DNA-DNA hybridization showed that nine of these corresponded to unique chromosome-sized DNAs. Two other chromosomal DNAs were marked with nonsegregating markers, including the rDNA repeat. Two remaining chromosomes remained unmarked but hybridized to repeated-sequence probes. Cross 93b had an essentially conventional meiosis in which both independent assortment and joint segregation of markers occurred, but in which crossing over was infrequent over much of the mapped genome. The 48 homokaryotic spore-offspring had overall crossover frequencies that were similar to, but possibly slightly less than, those of three homokaryon constituents of heterokaryotic spore-offspring. These data provide support for our earlier cytogenetic model of sporogenesis in A. bisporus, that explains why heterokaryotic spore-offspring usually appear to exhibit no recombination. No evidence favoring an alternative, mitotic model of sporogenesis was found. The resulting genetic map appears to survey the genome extensively and for the first time permits localization of loci determining economically important traits in this fungal crop species. Large differences in the vigor of homokaryotic offspring were correlated with the inheritance of certain chromosome segments and were also often associated with significant departures from Mendelian segregation ratios.     

Hot Spots of Recombination in Fission Yeast: Inactivation of the M26 Hot Spot by Deletion of the Ade6 Promoter and the Novel Hotspot Ura4-Aim

The M26 mutation in the ade6 gene of Schizosaccharomyces pombe creates a hot spot of meiotic recombination. A single base substitution, the M26 mutation is situated within the open reading frame, near the 5' end. It has previously been shown that the heptanucleotide sequence 5' ATGACGT 3', which includes the M26 mutation, is required for hot spot activity. The 510-bp ade6-delXB deletion encompasses the promoter and the first 23 bp of the open reading frame, ending 112 bp upstream of M26. Deletion of the promoter in cis to M26 abolishes hot spot activity, while deletion in trans to M26 has no effect. Homozygous deletion of the promoter also eliminates M26 hot spot activity, indicating that the heterology created through deletion of the promoter per se is not responsible for the loss of hot spot activity. Thus, DNA sequences other than the heptanucleotide 5' ATGACGT 3', which must be located at the 5' end of the ade6 gene, appear to be required for hot spot activity. While the M26 hotspot stimulates crossovers associated with M26 conversion, it does not affect the crossover frequency in the intervals adjacent to ade6. The flanking marker ura4-aim, a heterology created by insertion of the ura4(+) gene upstream of ade6, turned out to be a hot spot itself. It shows disparity of conversion with preferential loss of the insertion. The frequency of conversion at ura4-aim is reduced when the M26 hot spot is active 15 kb away, indicating competition for recombination factors by hot spots in close proximity.     

Electron microscopic study of endogenous peroxidase activity in human liver macrophages

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.     

Acceleration of the degradation of tyrosine aminotransferase in rat hepatome (HTC) cells by inhibitors of cyclic nucleotide phosphodiesterase.

The following inhibitors of cyclic nucleotide phosphodiesterase reduce the specific activity of tyrosine aminotransferase when added to cultures of rat hepatoma (HTC) cells: theophylline, 1-methyl-3-isobutylxanthine, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, and papaverine. Immunochemical measurements show that each inhibitor reduces the rate of tyrosine aminotransferase synthesis and increases the rate of degradation of the enzyme. The effect on synthesis also occurs with general proteins while that on degradation appears specific for tyrosine aminotransferase.