Nuclear Behaviour In Heterokaryons : Genetic And Molecular Analysis Of (his-3+ his-3+) Heterokaryons Of Neurospora Crassa

Abstract

In contrast to plant and animal cells, the fungal cells are multinucleate. A consequence of their multinucleate condition is heterokaryosis — the occurrence of genetically different nuclei in a common cytoplasm. In nature this condition occurs because of spontaneous mutations in the haploid nuclei in the coenocytic mycelium. Inspite of heterokaryosis being a fundamental aspect of fungal biology, the behaviour and dynamics of nuclei in fungal mycelium are little understood. This study was prompted by the following questions: (1) Why does a fungus need so many nuclei? (2) Are they all active simultaneously? (3) Does the proportion of the different nuclear types in fungal mycelium alter in response to change in conditions of growth? (4) Is the activity of an enzyme related to the dose of nuclei containing the encoding gene?

Experimental approach. The approach taken was to generate heterokaryons in which one of the nuclear types carries a mutant allele for a specific enzyme while the other nuclear type carries the functional allele, introduced by transformation. Because in filamentous fungi, the transforming DNA commonly integrates randomly into the chromosomal DNA, the transformants would be genetic 'variants' in which the ratios of transformed to non-transformed nuclei might be controlled differently. The transformants could thus be useful in investigating the relationship between the frequency of transformed nuclei and the activity of encoded enzyme. In addition the transformants might be useful for studying nuclear behaviour. The availability of developmental information, genetic and molecular methodology, and biochemical mutant in Neurospora crassa made this fungus a material of choice for this investigation.

Strain construction. A histidinol dehydrogenase (his-3) mutant strain was used into which an albino colour marker and a biochemical marker, inositoL were introduced by crossing. The latter two markers served as check against possible laboratory contamination. In addition, a gene mem, was introduced into the strain. In the mem genetic background, the strain has a wild-type morphology on agar medium but when grown in liquid shake culture it produces uninucleate microconidia that are useful in estimating nuclear ratio. Protoplasts of a constructed strain (his-3 al-1; mem; inl) were transformed with a plasmid containing the wild-type his-3 allele, thereby converting the original strain into a heterokaryotic strain having a mixture of transformed (his-3+t) and untransformed (his-3) nuclei. [The superscript +/ is used here to denote an his~3+ allele ectopically introduced by transformation]. Integration of plasmid DNA sequence in three selected transformants, 2T5, 3T3 and 4T12, was confirmed by genomic Southern analysis using the vector DNA as probe. The exponential growth rate of all three transformants was similar (~0.08mgh"1).

Nuclear ratio. Assuming a uniform distribution of nuclei in mycelium, and a correspondence between nuclear ratio in mycelium and conidia, the ratio his-3* {: his-3 was estimated by plating microconidia. In transformant 3T3, the nuclear ratio was 7:1. In 2T5, all nuclei were his-3n. Transformant 4T12 did not produce microconidia. The nuclear ratio in this transformant was therefore estimated by macroconidial plating and found to be 1:5, in favour of his-3 nuclei. Behaviour of transformants in vegetative and sexual phase. Although the transformants had originally been selected for the expression of his-3+T gene, a majority of macroconidia produced in cultures of 3T3 and 2T5 required histidine to trigger their germination. This condition, referred to as cphenotypic lag', led to a gross underestimation of the proportion of prototrophic macroconidia by the direct plating method and biased the estimation of nuclear ratios. Therefore nuclear ratio was estimated by first germinating macroconidia on histidine supplemented medium before testing colonies in histidine dropout slants and comparing the numbers of auxotrophic and prototrophic mycelia. Phenotypic lag was not observed in 4T12. The variation in the degree of expression of phenotypic lag among the transformants was ascribed to transgene position effect. The transformants differed also in meiotic instability of the transforming DNA — the transforming DNA in 3T3 was passed through unchanged but it was deleted or modified in4T12and2T5.

Experimental alteration of nuclear ratio. The transformants differed with respect to the self-adjusted ratio of transformed to non-transformed nuclei and also to the degree to which their nuclear ratio could be altered by nutritional manipulation of the growth medium, i.e., by growing the transformants in the presence or absence of histidine in the medium. In 3T3, the proportion of his-3+t nuclei progressively decreased by 3.5-fold in the sixth subculture on histidine medium. The change in 4T12 was even more striking: in the sixth serial subculture, the proportion of his-3+t nuclei decreased from 17-20% to -0.05%.However, when it was propagated again in medium that lacked histidine, the frequency of his-3+t nuclei was immediately restored to original level (-17%). That drastic alterations in nuclear ratio occurred upon nutritional manipulation was verified by Southern analysis. The intensity of signal specific for transformed DNA (nuclei) in cultures grown without histidine supplement was strong, but barely detectable in cultures grown with histidine. The signal reappeared when 4T12 was propagated in medium lacking histidine. Histidine induced change in nuclear ratio in 4T12 was further confirmed by three tests: (i) inoculum test using conidia, (ii) hyphal tip analysis, and (iii) genetic test using colour markers. Nuclear ratio and enzyme activity. Because in 4T12 changes in nuclear ratio could be manipulated, this transformant was used to investigate whether the proportion of his-3+t nuclei is correlated with the levels of encoded enzyme, histidinol dehydrogenase. Surprisingly, the specific activity of histidinol dehydrogenase was the same regardless of the percentage of his-3+t nuclei. This observation suggested that the physiological demand of a metabolite may be satisfied with only a few nuclei carrying the relevant gene. Or in other words the majority of nuclei in the coenocytic mycelium may, perhaps, not be active simultaneously.

Silencing of transforming DNA in nuclei. Two experiments were done to test the possibility that in a majority of nuclei, the transforming DNA is selectively silenced by methylation of cytosine: (1) Southern analysis of chromosomal DNA digested with isoschizomers, and (2) Reactivation by growth of transformants in presence of 5-azacytidine, an inhibitor of methylation. The results suggested that a majority of transformed nuclei may, perhaps, be inactive. The results of Northern analysis suggested that the amount of his-3+t transcript was correlated (but 5-azacytidine experiment indicated that only few his-3+t nuclei may be active) with the proportion of his-3+t nuclei, but not histidinol dehydrogenase activity. The above results suggested that expression of his-3+t gene was controlled both at the levels of transcription and posttranscription.

Nuclear selection. To study competition between nuclei containing mutant (his-3) nuclei and prototrophic nuclei containing his-3+ gene at its normal chromosomal location or at the ectopic location, heterokaryons were synthesized using strains in which the nuclear types had been marked by non-allelic genetic colour markers, al-1 and al-2. The results suggested that in heteronuclear mixture, the replication rate of the transformed nuclei is affected as compared to the nuclei having the gene in normal chromosomal location.

Major contributions. This study generated (his-3 + his-3+) heterokaryons by transformation. The behaviour of transformants differed in some respects both in the vegetative and sexual phases. It was demonstrated that nuclear ratio could be experimentally altered. However, there was no correlation between nuclear ratio and enzyme activity. The observations imply asynchronous division rate among nuclei and raise the possibility that not all nuclei in the coenocytic mycelium are active simultaneously.