ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide–sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

Taxonomic status of Triturus vittatus (Amphibia: Salamandridae) in western Turkey: trunk vertebrae count, genome size and allozyme data

The banded newt, Triturus vittatus is suggested to consist of two species (T. ophryticus and T. vittatus). The northern taxon, T. ophryticus, is subdivided into two geographic fragments: the "western group" of populations from western Anatolian Turkey, and the "eastern group" distributed in the remaining part of the Pontic Turkey and western Caucasus. The western samples are characterized by a peculiar combination of various features. In this group the modal number of trunk vertebrae is equal to 12, like in T. v. vittatus and T. v. cilicensis, whereas the eastern group of T. ophryticus has 13 vertebrae. The amount of DNA per diploid nucleus, determined by flow cytometry, is higher in the western group (66.8-68.8 pg vs. 62.5-63.7 pg in the eastern group). Significant differences between both groups of T. ophryticus were revealed in allozymes (DNei'72 = 0.383 in average) as well. We allocate the western group of T. ophryticus to a separate subspecies, Triturus ophryticus nesterovi, subsp. nov. A paleogeographic scenario for T. vittatus and other newts is proposed. The taxonomic structure of the paraphyletic genus Triturus is discussed. We proposed to split the genus in four monophyletic genera Triturus sensu stricto, Lophinus, Mesotriton and Ommatotriton.

Sexual Diploids of Aspergillus nidulans Do Not Form by Random Fusion of Nuclei in the Heterokaryon

The sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads can be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined “relative heterothallism” of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available.

Determination of cellular DNA content of Iberian salamanders by flow cytometry

The DNA content per diploid nucleus (2 C-value) was determined for the eight species of Iberian salamanders by flow cytometry. All species showed high values, which are characteristic of Caudata. The species with the lowest value was Pleurodeles waltl (48.25 pg DNA/cell) and Salamandra salamandra had the highest values (70.55 pg DNA/cell). There were no statistical differences between sexes in any of the eight species and DNA content was not directly related with phylogeny. The values obtained are often higher than previously published estimates obtained with older and potentially less accurate methodologies. We discuss the possible relationship between cellular DNA content and reproductive biology. Se determinó el contenido en ADN por núcleo diploide (valor 2-C) por citometría de flujo de ocho especies de salamandras ibéricas. Todas las especies mostraron altos valores, lo que es característico de Caudata. La especie con el menor valor fue Pleurodeles waltl (48,25 pg ADN/célula) mientras Salamandra salamandra tuvo las valores más altos (70,55 pg ADN/célula). No se encontraron diferencias estadísticas entre sexos en ninguna de las 8 especies y el contenido en ADN no se relacionó directamente con la filogenia. Los valores obtenidos son, a menudo, más altos que los publicados previamente con técnicas más antigüas, y potencialmente menos precisas. Discutimos las posibles relaciones entre el contenido en ADN celular y la biología reproductiva de cada especie.

Kar4p, a karyogamy-specific component of the yeast pheromone response pathway.

Karyogamy is the process whereby two haploid nuclei fuse to form a diploid nucleus during mating in Saccharomyces cerevisiae. Here, we describe the characterization of the KAR4 gene, previously identified in a screen for new nuclear fusion-defective mutants. During mating, kar4 mutants were defective for the microtubule-dependent movement of nuclei, a phenotype identical to that of mutations in KAR3 and CIK1. Consistent with its mutant phenotype, we found that the kar4 mutation resulted in failure to induce KAR3 and CIK1 mRNA during mating. Expression of KAR3 and CIK1 under independent regulatory control suppressed the kar4 defect, indicating that KAR4 is required primarily for the induction of KAR3 and CIK1. KAR4 was also required for meiosis, during which it may regulate KAR3; however, mitotic expression of KAR3 and CIK1 during S/G2 phase was independent of KAR4. A 30-bp region upstream of KAR3 conferred both KAR4- and STE12-dependent induction by mating pheromone. This region contained one moderate and two weak matches to the consensus pheromone response element to which the Ste12p transcriptional activator binds and five repeats of the sequence CAAA(A). Overproduction of Ste12p suppressed the kar4 defect in KAR3 induction and nuclear fusion. In contrast, Ste12p-independent expression of Kar4p did not alleviate the requirement for Ste12p during KAR3 induction. We propose that Kar4p assists Ste12p in the pheromone-dependent expression of KAR3 and CIK1. KAR4 defines a novel level of regulation for the pheromone response pathway, acting at a subset of Stel2p-inducible genes required for karyogamy.

Nuclear congression and membrane fusion: two distinct events in the yeast karyogamy pathway.

Karyogamy is the process where haploid nuclei fuse to form a diploid nucleus during yeast mating. We devised a novel genetic screen that identified five new karyogamy (KAR) genes and three new cell fusion (FUS) genes. The kar mutants fell into two classes that represent distinct events in the yeast karyogamy pathway. Class I mutations blocked congression of the nuclei due to cytoplasmic microtubule defects. In Class II mutants, nuclear congression proceeded and the membranes of apposed nuclei were closely aligned but unfused. In vitro, Class II mutant membranes were defective in a homotypic ER/nuclear membrane fusion assay. We propose that Class II mutants define components of a novel membrane fusion complex which functions during vegetative growth and is recruited for karyogamy.

The DDK inbred strain as a model for the study of interactions between parental genomes and egg cytoplasm in mouse preimplantation development

The DDK strain of mice has unusual genetic properties. When females of this strain are crossed to males of other strains, they generally exhibit a very low fertility, whereas reciprocal crosses are fully fertile as are the intrastrain crosses. The observed low fertility results from early embryonic lethality, the F1 embryos dying around the late morula–early blastocyst stage. Nuclear transplantation experiments between hybrid eggs of BALB/c and DDK strains has shown that failure of F1(DDK ♀× BALB/c♂) embryos to develop is not due to the combination per se of maternal (DDK) and paternal (BALB/c) genomes but rather to an incompatibility between paternal (BALB/c) genomic contribution and DDK cytoplasm. This incompatibility does not occur between a female BALB/c pronucleus and the DDK cytoplasm, suggesting the involvement of a differential imprinting of parental genomes. Introduction of cytoplasts isolated from DDK 1- to 8-cell embryos into BALB/c♀×BALB/c♂ or BALB/c♀×DDK♂ embryos of the corresponding developmental stage demonstrate that the cytoplasm of DDK embryos prevents the formation of normal blastocysts through a specific interaction with the paternal component of the BALB/c diploid nucleus. Genetic and molecular studies are underway to try and isolate the gene(s) responsible for the failure of (DDK♀×BALB/c♂)F1 embryos. These experiments should help in our understanding of nucleocytoplasmic interactions and the respective roles of parental genomes in early embryonic development.

Nuclear volume and number in long-term in vitro cultures of Puccinia graminis

Eight cultures of Puccinia graminis f. sp. tritici which had been serially subcultured in the Cereal Rust Laboratory for 2–13 years were examined for nuclear volume, number of nuclei per cell, and dimensions of hyphae. The cultures were of two types: compact with short, wide hyphal cells (ca. 10 × 22 μm); and fluffy with long, narrow cells (ca. 5 × 80 μm). The compact cultures tended to sporulate; the fluffy ones did not. By using volume of hyphal nuclei to estimate ploidy, the cultures were judged to be of three types: compact with two haploid nuclei per cell; compact with one diploid nucleus per cell; and fluffy with two diploid nuclei per cell. The compact cultures originated from American uredial isolates of races 17, 23, 38, and 56. The fluffy cultures originated only from University of Sydney uredial isolate 334, race 126Anz6,7, or as variants of compact cultures from American race 56. A uninucleate fluffy culture which had been initiated from uredial isolate 334 by Australian workers had a nuclear volume four times those of binucleate compact cultures, suggesting that the nucleus of each cell was tetraploid. A culture of P. graminis f. sp. secalis initiated at the Cereal Rust Laboratory was compact and had mostly four nuclei per cell, each presumed to be haploid. Generally, compact cultures had more characteristics in common with the parasitic form of P. graminis than fluffy cultures did.