TRANSPLANTATION OF NUCLEI IN NEUROSPORA CRASSA

Wilson , James F. (Hartnell Coll., Salinas, Calif.) Transplantation of nuclei in Neurospora crassa. Amer. Jour. Bot. 50(8): 780–786. Illus. 1963.—Heterocaryons of N. crassa were produced artificially by cell-to-cell transfer of protoplasm of genetically marked strains. The injected hyphal sections were excised and cultured separately. The criteria of prototrophic growth and recovery of conidia of the donor genotype were used to prove that functional nuclei had been transplanted. Reciprocal injections were made with 2 pairs of macroconidial and 3 pairs of microconidial strains. Growth curves and conidial ratios of these artificial heterocaryons were compared to the corresponding natural heterocaryons. In each pair of heterocaryons produced by reciprocal injection, the growth curve of only one was equivalent to the naturally formed heterocaryon of that pair. The growth curve of the other artificial heterocaryon differed either in rate or in the lag period before growth occurred. There was no apparent relation between conidial ratios of the natural heterocaryons and the artificial heterocaryons with comparable growth curves. Injection of normal nuclei into a morphological temperature-sensitive mutant produced an apparent heterocaryon which grew with normal morphology at 34 C. The apparent stability of Neurospora nuclei may make in vitro studies possible.     

Cytoplasmic activation of human nuclear genes in stable heterocaryons

We have induced the stable expression of muscle-specific genes in human nonmuscle cells. Normal diploid human amniocytes were fused with differentiated mouse muscle cells by using polyethylene glycol. The fusion product, a stable heterocaryon in which the parental cell nuclei remained distinct, did not undergo division and retained a full complement of chromosomes. This is in contrast with typical interspecific hybrids (syncaryons), in which the parental nuclei are combined and chromosomes are progressively lost during cell division. The human muscle proteins, myosin light chains 1 and 2, MB and MM creatine kinase and a functional mouse-human hybrid MM enzyme molecule were detected in the heterocaryons. Synthesis of these proteins was evident 24 hr after fusion and increased in a time-dependent manner thereafter. Our results indicate that differentiated mouse muscle nuclei can activate human muscle genes in the nuclei of a cell type in which they are not normally expressed, and that this activation occurs via the cytoplasm. The activators are still present in cells which have already initiated differentiation, are recognized by nuclei of another species, and do not diffuse between unfused cells. The reprogrammed amniocyte nuclei of stable heterocaryons provide a unique system in which to study the mechanisms regulating gene expression during cell specialization.     

Nuclear division cycle in Neurospora crassa hyphae under different growth conditions.

Treatment with picolinic acid blocked Neurospora crassa nuclei in G1, and recovery from the treatment allowed a synchronous wave of deoxyribonucleic acid synthesis to occur. Nuclei, which appeared as compact globular bodies during the period of blockage, assumed a ring shape during the following S phase, which was also maintained in the G2 phase. The proportion of compact globular nuclei was much higher in hyphae growing at lower rates, whereas that of ring nuclei increased when the hyphae were growing at higher rates. Horseshoe nuclei (probably mitotic nuclei) and double ring nuclei were also observed in growing hyphae, but their frequencies were low and fairly independent of the rate of growth. The length of the S phase of the Neurospora nuclear division cycle was determined to be about 30 min. From the frequencies of the phase-specific nuclear shapes, the durations of the G1 phase and the combined S plus G2 phases were calculated. The results showed that variations in the growth rates of the mycelia were mainly coupled with variations in the G1 phase of the nuclear division cycle. For mycelia growing in minimal sucrose, the lengths of all of the phases of the nuclear division cycle were estimated.     

Cycloheximide inhibits light-induced phase shifting of the circadian clock in Neurospora

Cycloheximide inhibits light-induced phase shifting of the circadian clock and protein synthesis in Neurospora. Light resetting is not inhibited in mutants whose protein synthesis is resistant to cycloheximide. When light and cycloheximide are presented together at various circadian phases, the final phase shift is always determined by cycloheximide. This dual-treatment phase response curve approach may be useful for other studies using pharmacological treatments to analyze clock pathways. Taken together, the results suggest that synthesis of a protein (or proteins) is involved in the phototransduction pathway of the circadian clock in Neurospora.     

Effects of Membrane ATPase Inhibitors on Light-Induced Phase Shifting of the Circadian Clock in Neurospora crassa

Effects of several membrane ATPase inhibitors on light-induced phase shifting of the circadian conidiation rhythm in Neurospora crassa were examined using mycelial discs in liquid culture. Suppression of phase shifting by the inhibitors was strongly dependent on the pH of the liquid medium in which the discs were cultured during the time from light-dark transition (beginning of free-run) to light irradiation. When discs were cultured in pH 6.7 medium, azide, the inhibitors of plasma membrane ATPase (diethylstilbestrol and N, N′-dicyclohexylcarbodiimide), and ethanol completely suppressed the effect of light on the clock. In contrast, mycelial discs cultured in pH 5.7 medium were fully phase-shifted by light in the presence of the same and even higher concentrations of the chemicals. However, sensitivity to light of the discs cultured in relatively acidic medium was eight times higher than that of the discs cultured at neutral pH. Oligomycin and venturicidin, inhibitors of mitochondrial ATPase, did not suppress phase shifting by light at either pH.     

Spatial Distribution of Circadian Clock Phase in Aging Cultures of Neurospora crassa

Neurospora crassa has been utilized extensively in the study of circadian clocks. Previously, the clock in this organism has been monitored by observing the morphological and biochemical changes occurring at the growing front of cultures grown on solid medium. A method has been developed for assaying the clock in regions of the culture behind the growing front, where no apparent morphological changes occur during the circadian cycle. Using this assay with Petri dish cultures that were 2 to 7 days old, the presence of a functional circadian clock not only at the growing front but in all other regions of the culture as well was demonstrated. Furthermore, the entire culture is not in the same phase, but shows a gradient of phases which is a function of the length of time the clock in a given part of the culture has been free-running. This gradient may be the result of a somewhat longer period of the oscillator behind the growing front compared to that at the growing front. The phase differences within a single culture of interconnected mycelium demonstrate the absence of total internal synchronization between adjacent regions of the hyphae under these conditions.     

Selection of fitter type nuclei in heterocaryons ofAspergillus oryzae

Summary To examine the consequences of spontaneous mutation in multinucleateAspergillus oryzae, the shift of nuclear ratio in heterocaryons produced artificially between a fast grower and a slow grower on casein-containing medium was studied throughout the course of growth. It was found that the cultivation of the heterocaryon on casein-containing medium always favored the selection of the faster grower. The faster grower had a better selective advantage when cultivated in a special tube, designed to give larger amounts of oxygen to the growing cells.With 4 Figures in the Text     

A re-examination of the role of the nucleus in generating the circadian rhythm in Acetabularia.

The role of the nucleus in the generation of the circadian rhythm in Acetabularia has been nuclear. Early experiments showed that the plant could exhibit a circadian rhythm in the absence of a nucleus. However, other experiments appeared to show that the nucleus could impart phase information to the rhythm, and so therefore must be a part of the system that generates the rhythm. We have conducted experiments similar to these--in particular, one in which the nuclear end of the plant was entrained on a light-dark cycle that was opposite that of the rest of the plant. The phase of the free-running rhythm of this type of plant is not consistent with the conclusion that the nucleus is part of the circadian oscillator. We have also tried entraining opposite ends of plants with no nuclei on opposite light-dark cycles. The ultimate phases of these plants appear to be nearly random. A possible interpretation of these experiments is discussed.     

Genes that act before conjugation to prepare the Saccharomyces cerevisiae nucleus for caryogamy

Mutations in four nuclear genes, kar1 cdc4, 28, and 37, block or impair nuclear fusion during conjugation of Saccharomyces cerevisiae. Mutations in all four genes are recessive for the caryogamy defect; in matings between diploid cells both of which are heterozygous for any one of the four mutations (-/+ X -/+), caryogamy occurs with normal proficiency. However, mutations in all four genes are "nuclear dominant"; that is, both parent nuclei must contribute one wild-type allele of each gene for successful caryogamy. In order to discriminate between two possible models to explain nuclear dominance, we have examined the caryogamy proficiency of mutant nuclei after they had passed through a heterocaryotic cytoplasm. The kar1, cdc28, and cdc37 caryogamy defects are all phenotypically suppressed in this experiment (cdc4 could not be tested). We conclude from our results that the KAR1, CDC28, and CDC37 gene products can diffuse between nuclei in a heterocaryon and that they probably perform their function for caryogamy prior to cell fusion. One simple model consistent with the roles of CDC28 and CDC37 in mitosis as well as in caryogamy is that these gene products are structural components of the nucleus that must be built into it during one cell cycle in order to permit successful caryogamy at the next G1.     

Circadian rhythms of the sex steroids of female Papio hamadryas in long-term hypokinesia

Radioimmunoassay was used to study the effect of restricted motor activity on circadian rhythms of blood plasma sexual steroids and cortisol in female Papio hamadryas during different phases of the menstrual cycle. Instact animals manifested clear-cut circadian rhythms of the testosterone and cortisol content during both the phases and those of estradiol and progesterone during the follicular phase of the cycle. Two-week immobilization did not produce any changes in the general pattern of circadian rhythms of cortisol and testosterone but led to a decrease in their mean daily concentrations, with that decrease being more pronounced for testosterone of females immobilized during the luteal phase. The authors established unbalance of sexual steroids with a dramatic fall of the mean daily concentrations of estradiol and progesterone and desynchronism of their circadian rhythms regardless of the cycle phase at the moment of immobilization. The mechanisms responsible for alterations in circadian rhythms of sexual steroids in hypokinetic females are discussed.     

STUDIES ON A CYTOPLASMIC CHARACTER IN NEUROSPORA CRASSA

Two morphologically distinctive slow growing strains of Neurospora crassa have been isolated and studied. These, abn-1 and abn-2, differ from wild type in that their growth rates are greatly reduced and often irregular, aerial hyphae are absent, conidia are extremely rare, and no protoperithecia are formed. Growth was not improved by addition of any nutrients tested, oxygen consumption was similar to that of wild type, and cytochrome c appeared abnormally high, and b low or absent. Both abn strains gave rise only to normal progeny in crosses with normal strains. The abn characteristics appear in heterocaryons, and have been transmitted to other genetic strains by means of heterocaryosis followed by plating of conidia. Conidia formed by such heterocaryons typically showed low viability, and gave rise to cultures with great variability in growth rate, morphology, and survival. Even apparently normal derived cultures often later became abnormal or died. It is concluded that the abnormal characteristics are determined primarily by cytoplasmic factors. This conclusion was strengthened by the transmission of the typical characteristics to normal strains by microinjection of cytoplasm from abn cultures, even without demonstrable transfer of nuclei. This constitutes the first time microinjection techniques have been successfully applied to the analysis of a cytoplasmic character in Neurospora.     

Calcium Inhibits Phase Shifting of the Circadian Conidiation Rhythm of Neurospora crassa by the Calcium Ionophore A23187

Effects of the calcium ionophore, A23187, and antimycin A on the circadian conidiation rhythm of Neurospora crassa were examined. A23187 at a concentration of 1 mum in medium not containing divalent cations delayed the phase by 10 hours at CT 10 and advanced it by 5 hours at CT 14 (CT 12 corresponds to the time that discs are transferred from light to dark). This phase shifting was completely inhibited by addition of 0.1 millimolar CaCl(2) but not by MgCl(2) at any concentrations examined.Antimycin A inhibited respiration by 90% at a concentration of 0.2 micrograms per milliliter and lowered the ATP content by 85%. Antimycin A alone caused small phase advances but in combination with A23187 resulted in a large phase delay at CT 10. This phase shifting was not reversed by addition of CaCl(2) lower than 10 millimolar.     

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.     

Linkage analysis for mapping genes of sex-influenced traits

Abstract Statistical methods are developed to estimate gender-specific and gender-average recombination frequencies between a biallelic or multiallelic marker and a sex-influenced gene. Iterative solutions are developed for intercross (or F-2 design). For biallelic markers, two iterative solutions are required, one for coupling and repulsion parental linkage phases and one for mixed parental linkage phases. For multiallelic markers, one set of iterative equations applies to all possible parental linkage phases. The resulting formulae for estimating recombination frequency use the full data set and yield estimates that are exactly the same as the true parameters if the observed and expected phenotypic distributions are equal. When one parent is homozygous for the sex-influenced gene as is expected with the backcross design, simple solutions exist for estimating recombination frequencies. However, offspring of one gender (male or female) do not have linkage information depending on whether the homozygous parent has two male-dominant or male-recessive alleles. Consequently, an F-2 design is more efficient than a backcross design for mapping a sex-influenced gene. Knowing each parental linkage phase is important to apply the methods developed in this article. It is shown that an individual's linkage phase of the sex-influenced locus can be determined based on allele transmission from the parents for all crosses except under the mating between an expressed male and an unexpressed female.     

Estimation of nuclear ratio and its influence on the rate of growth of Aspergillus niger heterocaryon

Summary A heterocaryon (hist/hypox) was employed to study the nuclear ratio in relation to the composition of the medium and to growth. The data showed that supplementation of the growth factors in any ratio in the medium had no influence on the shift of nuclear ratio, unless and until one of the growth factors was reduced to its limiting value. In such circumstances, if the amount of the other growth factor was increased, it did effect the increase of the appropriate nuclei in the heterocaryon, for which this growth factor was required. Both nuclear types responded in this manner.Although the nuclear ratio in the heterocaryon did not show a direct relation to its rate of growth, the rate of growth was effected by the amount of growth factors added to the medium.     

Recurrence of Repeat-Induced Point Mutation (Rip) in Neurospora Crassa

Duplicate DNA sequences in the genome of Neurospora crassa can be detected and mutated in the sexual phase of the life cycle by a process termed RIP (repeat-induced point mutation). RIP occurs in the haploid nuclei of fertilized, premeiotic cells before fusion of the parental nuclei. Both copies of duplications of gene-sized sequences are affected in the first generation at frequencies of approximately 50-100%. We investigated the extent to which sequences altered by RIP remain susceptible to this process in subsequent generations. Duplications continued to be sensitive to RIP, even after six generations. The fraction of progeny showing evidence of RIP decreased rapidly, however, apparently as a function of the extent of divergence of the duplicated sequences. Analysis of the stability of heteroduplexes of DNA altered by RIP and their native counterpart indicated that linked duplications diverged further than did unlinked duplications. DNA methylation, a common feature of sequences altered by RIP, did not seem to inhibit the process. A sequence that had become resistant to RIP was cloned and reintroduced into Neurospora in one or more copies to investigate the basis of the resistance. The altered sequence regained its methylation in vegetative cells, indicating that the methylation of sequences altered by RIP observed in vegetative cells is a consequence of the mutations. Duplication of the sequence restored its sensitivity to RIP suggesting that resistance to the process was due to loss of similarity between the duplicated sequences. Consistent with this, we found that the resistant sequence did not trigger RIP of the native homologous sequences of the host, even when no other partner was available. High frequency intrachromatid recombination, which is temporally associated with RIP, was more sensitive than RIP to alterations in the interacting sequences.     

Vasopressin Antagonist Disrupts the Circadian Rhythm of Water Intake on Suprachiasmatic Injection

The present study makes an attempt to find out the action of arginine vasopressin (AVP) and its antagonist d-(CH₂)₅Tyr (Me) AVP applied at the suprachiasmatic nuclei (SCN) on the circadian rhythm of water intake. Chronic implantation of a 22 G stainless steel cannula for injection was performed using a stereotaxic technique under Nembutal anesthesia. AVP and its antagonist were injected into the SCN of free-moving rats at the beginning of light and dark phases of the light-dark (LD) cycle. Injections of AVP during either phase did not disrupt the circadian pattern of water intake while the injections of the antagonist disrupted it. The findings are suggestive of the involvement of AVP as a mediator of the circadian rhythm of water intake at the level of the neural pacemaker, SCN.     

Estimation of circadian variations in cell cycle phase durations in murine epidermal basal cells

Circadian variations in the proliferative activity of squamous epithelia are well known. However, circadian variations in the duration of the various cell cycle phases (S, G2 and mitosis) have been disputed. The percent labelled mitoses method, which is traditionally used to obtain duration of cell cycle phases, is poorly suited for identification of circadian variations. Therefore methods combining changes in compartment size (cell cycle phase) and cellular flux through the compartments have been used. Three different methods using such data are presented. These incorporate various simplifying assumptions that cause methodological errors. Limits for use of the different methods are indicated. The use of all three methods gives comparable and pronounced circadian variations in the duration of S and G2 phase. These results are also compatible with circadian variations in the mitotic duration, but they may also represent artefacts due to sensitivity to model errors.