Genetic screens and selections for cell and nuclear fusion mutants.
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Mutants of Saccharomyces cerevisiae defective for nuclear fusion have been isolated. Their mutations have been characterized by meiotic analysis, dominance-recessivity and complementation. Twelve of the mutations are allelic to the previously described kar 1-1; five affect a second gene designated KAR 2 and three affect a third gene designated KAR 3. There is evidence suggesting that other two mutants are affected in a gene different from the three mentioned. Mutations in KAR 1 and KAR 2 genes are recessive and do not cause obvious effects other than the failure of the karyogamy. Mutations in KAR 3 are semidominant and do cause pleiotropic effects affecting both a mitosis and meiosis.
We propose a new mechanism for the production of high-energy ( E>3 keV) deuterons, suitable to induce dd nuclear fusion, based on multielectron ionization and Coulomb explosion of heteronuclear deuterium containing molecular clusters, e.g., (D2O)n, in intense ( 10(16)-2x10(18) W/cm2) laser fields. Cluster size equations for E, in conjunction with molecular dynamics simulations, reveal important advantages of Coulomb explosion of (D2O)n heteronuclear clusters, as compared with (D)n clusters. These involve the considerably increased D+ kinetic energy and a narrow, high-energy distribution of deuterons.
Genetic exchange may occur between two particular Trypanosoma brucei clones simultaneously transmitted by the same tsetse fly. We report here that this exchange takes place in the fly, through nuclear fusion. The resulting hybrids appear to be sub-tetraploid, some particular DNA sequences from one of the parental stocks being lost before enough cloned hybrid trypanosomes could be harvested for DNA analysis. A further reduction of the DNA content of these hybrids occurs gradually upon growth and yields near diploid value in a major part of the population. This mode of hybrid generation is different from the fusion of haploid gametes, which is thought to occur normally upon inoculation of metacyclic trypanosomes in their mammalian host. In this respect, the sub-tetraploid hybrids appear to undergo meiosis in the fly, generating sub-diploid metacyclic forms, then fusion in the mammalian blood.
A mutant unable to fuse nuclei during mating has been isolated from standard wild-type Saccharomyces cerevisiae. Tetrad analysis of the mutation responsible for this defect (kar1-1) shows that it segregates as a single Mendelian factor. The defect kn kaf1-1 appears to be nuclear limited. Cytological and genetic evidence shows that in this mutant the events associated with zygote formation are normal until the point of nuclear fusion. The consequence of this defect is the formation of a multinucleate zygote which in subsequent divisions can segregate heterokaryons and haploid heterplasmons.
Recent experiments on the interaction of intense, ultrafast laser pulses with large van der Waals bonded clusters have shown that these clusters can explode with substantial kinetic energy. By driving explosions in deuterium clusters with a 35 fs laser pulse, we have accelerated ions to sufficient kinetic energy to produce DD nuclear fusion. By diagnosing the fusion yield through measurements of 2.45 MeV fusion neutrons, we have found that the fusion yield from these exploding clusters varies strongly with the cluster size, consistent with acceleration of deuterons via Coulomb explosion forces.
The effect of 0.2% caffeine combined with 5-phenyl-2-imino-4 oxazolidone (pemoline, Tradon), in different ways, on mitosis in lateral roots of Vicia faba L. has been studied by means of aerated hydrocultivation. The results show that prolonged treatment with 10(-6) g/ml or 3 x 10(-4) g/ml (=saturated solution) pemoline to a certain degree compensates the negative influences of caffeine on mitotic index during and after the caffeine treatment. Depending on the duration of treatment 10(-6) g/ml pemoline are able to increase rates of nuclear fusions, bimitoses and 4n-mitoses, while 3 x 10(-4) g/ml have the opposite effects. The number of irregular cytokineses is increased by 10(-6) g/ml, whereas no abnormal cell walls are formed during treatment with 3 x 10(-4) g/ml pemoline. The conclusion is that pemoline has the same sites of action as caffeine, partly antagonistically, partly synergistically.
A cytological study of barley microspores during pretreatment of the uninucleate stage to the early culture stage was conducted utilizing six genotypes. Among the three main pretreatments investigated, microspores completed the first mitotic division during 28 d cold pretreatment of spikes, with or without leaf sheath attached, and during 0.3 M mannitol pretreatment of anthers at 25 degrees C. However, during a 4 d pretreatment in 0.3 M mannitol at 4 degrees C this first mitotic division was blocked or delayed and subsequently most often occurred during the first day on culture medium. The first mitotic division of most microspores pretreated in 0.3 M mannitol was mostly symmetrical (55-60%), whereas it was asymmetric (94%) during the 28 d cold pretreatment of spikes. Following the first mitotic division during the mannitol pretreatment at 25 degrees C, closely associated daughter nuclei often appeared to fuse via membrane coalescence, leading to a high frequency of large uninucleate microspores. Based upon nuclear size, the frequencies of fused uninucleate microspores in genotypes GBC 778, GBC 777 and Igri were estimated to be 87%, 54% and 75%, respectively, after a 4 d mannitol pretreatment at 25 degrees C. Chromosome numbers in dividing nuclei and relative densitometry measurements of nuclear DNA in microspores from cv. Igri confirmed the apparent fused nature of large nuclei in uninucleate microspores. The high frequency of fused nuclei indicates that nuclear fusion occurred between both symmetric and asymmetric nuclei. Microspores of cv. Igri cultured on filter paper following three different pretreatments provided an average of about 12 000 embryo-like structures (ELS) per plate. In samples, 85-97% of these ELS regenerated green shoots. The frequency of doubled haploids (74-83%) following all pretreatments was similar to the frequencies of fused nuclei. The pretreatment of spikes in 0.3 M mannitol at 4 degrees C for 4 d is preferred as it appears to provide genotype independent induction and suspension of nuclear division, as well as regenerating green plants in a shorter time than cold alone.
DnaJ-like proteins are functional partners for Hsp70 molecular chaperones. Complete nucleotide sequencing of yeast chromosome X has revealed that an open reading frame YJL073w encodes a novel member of the DnaJ-like protein family. The open reading frame represents a protein of 692 amino acids with a J-domain and one putative membrane-spanning segment. An epitope-tagged version of the protein was anchored in the endoplasmic reticulum (ER) membrane and its J-domain faced the ER lumen. We therefore propose to designate this gene JEM1 (DnaJ-like protein of the ER membrane) and to designate its gene product JEM1p. The JEM1 gene is not essential for cell growth, but double disruption of the JEM1 gene and the SCJ1 gene, which encodes another DnaJ-like protein in the ER lumen, causes growth arrest at elevated temperature. The Deltajem1 mutant is defective in nuclear fusion, karyogamy, during mating. A mutant JEM1p carrying a mutation in the highly conserved His-Pro-Asp sequence in the J-domain could not complement either temperature-sensitive growth of the Deltajem1 Deltascj1 double mutant or defects in karyogamy of the Deltajem1 mutant. JEM1p likely assists the functions of BiP, Hsp70 in the ER, including karyogamy.
Jem1p is a DnaJ-like protein of the yeast ER membrane, which is required for nuclear membrane fusion during mating. We have determined the position of translation initiation codon for the JEM1 gene. Translation of Jem1p starts from the second ATG codon of the previously assumed JEM1 open reading frame, leading to the synthesis of a precursor protein of 645 amino acids long. The translated Jem1p precursor contains an N-terminal hydrophobic sequence that functions as a signal sequence and is removed upon import into the ER lumen. Jem1p is peripherally associated with the ER membrane probably through protein-protein interactions.
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We have repeated the experiment of Taleyarkhan et al. [Science 295, 1868 (2002)]] in an attempt to detect the emission of neutrons from d-d fusion during bubble collapse in deuterated acetone. Using the same cavitation apparatus, a more sophisticated data acquisition system, and a larger scintillator detector, we find no evidence for 2.5-MeV neutron emission correlated with sonoluminescence form collapsing bubbles. Any neutron emission that might occur is at least 4 orders of magnitude too small to explain the tritium production reported in Taleyarkhan et al. as being due to d-d fusion. We show that proper allowance for random coincidence rates in such experiments requires the simultaneous measurement of the count rates in the individual detectors.
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