Vincenzo E. A. Russo

Reversible inactivation of a foreign gene, hph, during the asexual cycle in Neurospora crassa transformants

SummaryA plasmid construct carrying the hygromycin phosphotransferase (hph) gene fused to the expression elements of the trpC gene of Aspergillus nidulans was used to obtain hygromycin B (Hyg)-resistant transformants of Neurospora crassa. The plasmid does not have any homology with the N. crassa genome. Here we demonstrate that most of the transformants arise from integration of the transforming DNA into only one of the nuclei present in the protoplasts. Furthermore, in most of the transformants the integrated transforming DNA is physically stable after growth of the transformants for about 25 nuclear divisions without Hyg selection, in spite of being present in multiple copies. In transformants carrying only a single insertion, phenotypic expression of the hph gene remains unaltered in conidial isolates obtained withoug Hyg selection. On the other hand, about 40% of transformants harbouring plasmid DNA integrated at more than one location yield conidial isolates showing reversible inactivation of the hph genes. Interestingly, the presence of methylated cytosine residues in the integrated DNA is strongly correlated with the number of plasmid copies. The hph genes are heavily methylated in transformants harbouring multiple copies but not in those harbouring only one copy of the plasmid. Phenotypic expression of the inactive hph genes can be restored by growing the transformants either under Hyg selection pressure or in the presence of 5-azacytidine. In the first case the hph genes are again inactivated when Hyg selection pressure is removed, while the activation of the hph gene by 5-azacytidine gives stable Hygr strains.

A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens

Abstract The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time.

PHOTOINITIATION OF SPORANGIOPHORES IN PHYCOMYCES MUTANTS DEFICIENT IN PHOTOTROPISM AND IN MUTANTS LACKING β‐CAROTENE

Abstract—The formation of sporangiophores from mature Phycomyces mycelium is inhibited in a closed system. Irradiation of the mycelium with blue light reverses the inhibition of spordngiophore formation. Dose response curves for this reaction are established for wild type. β‐caroteneless mutants and for mutants that are deficient in phototropism.

Blue light induces circadian rhythms in the bd mutant of Neurospora: double mutants bd, wc 1 and bd, wc-2 are blind

This paper describes a new blue light effect for Neurospora crassa, the photoinduction of circadian rhythms in the bd mutant. The wc-1 and wc-2 genes are necessary for this effect.

The organization of Physcomitrella patens RAD51 genes is unique among eukaryotic organisms

Genetic recombination pathways and genes are well studied, but relatively little is known in plants, especially in lower plants. To study the recombination apparatus of a lower land plant, a recombination gene well characterized particularly in yeast, mouse, and man, the RAD51 gene, was isolated from the moss Physcomitrella patens and characterized. Two highly homologous RAD51 genes were found to be present. Duplicated RAD51 genes have been found thus far exclusively in eukaryotes with duplicated genomes. Therefore the presence of two highly homologous genes suggests a recent genome duplication event in the ancestry of Physcomitrella. Comparison of the protein sequences to Rad51 proteins from other organisms showed that both RAD51 genes originated within the group of plant Rad51 proteins. However, the two proteins form a separate clade in a phylogenetic tree of plant Rad51 proteins. In contrast to RAD51 genes from other multicellular eukaryotes, the Physcomitrella genes are not interrupted by introns. Because introns are a common feature of Physcomitrella genes, the lack of introns in the RAD51 genes is unusual and may indicate the presence of an unusual recombination apparatus in this organism. The presence of duplicated intronless RAD51 genes is unique among eukaryotes. Studies of further members of this lineage are needed to determine whether this feature may be typical of lower plants.

Nitrogen Regulation of Blue Light-Inducible Genes in Neurospora-Crassa

Sexual and asexual differentiation of Neurospora crassa are influenced by nitrogen availability and blue light. Nitrogen limitation induces the production of protoperithecia on solid medium and conidia in liquid medium. Both developmental processes are stimulated by blue light. We have analysed changes in mRNA levels for a variety of light-inducible genes (al, bli and con) under conditions of nitrogen limitation. We show that the photoregulated genes al-1, al-2, bli-4, bli-7, con-5 and con-10 are also regulated by nitrogen limitation. These genes exhibited nitrogen regulation in the nonphotoresponsive mutant strains wc-1 and wc-2. Therefore, the wc-1 and wc-2 gene products, although necessary for photoregulation of these al, bli and con genes, are not required for nitrogen regulation.

Sensory physiology of phycomyces Blakesleeanus

C. Desc r ip t ion of the Var ious Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 1. Lightand Da rk -Growth Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 2. P h o t o t r o p i s m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 3. G e o t r o p i s m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 4. A u t o c h e m o t r o p i s m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 5. A n e m o t r o p i s m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 6. S l r e l ch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 7. G r o w t h Response lo Var ious Gases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 8. P h o t o d i f f e r e n t i a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Ethylene is involved in the autochemotropism of Phycomyces.

The sporangiophore of the fungus Phycomyces blakesleeanus has the property of growing away from a barrier which is few mm from the growing zone of the sporangiophore (avoidance or autochemotropic response). A model has been published (Cohen, R.J., Jan, N.Y., Matricon, J., Delbrück, M.: J. Gen. Physiol. 66, 67–95 (1975)). To explain the avoidance response which postulates that the sporangiophore emits and readsorbs a volatile growth-promoting effector (gas X) and that the barrier modifies the effector distribution by acting as an aerodynamic obstacle, causing a higher concentration of gas X on the side of the sporangiophore closer to the barrier. From this model we deduced three properties of the gas X. Of the several gases tested (N2, CO2, CH4, C2H2, C2H4, C2H6) only ethylene (C2H4) had all these three properties, a finding which suggests that it has a role in the avoidance response (autochemotropism).

Light-induced Dephosphorylation of a 33 kDa protein in the wild-type strain of Neurospora crassa: The regulatory mutants wc-i and wc-2 are abnormal

Light induces the dephosphorylation of a 33 kdalton protein within 8 min in the wild-type strain of Neurospora crassa. The regulatory mutants, wc-1 and wc-2, have an altered pattern of phosphoproteins in darkness and also after irradiation. Because the wc genes have previously been implicated in photodifferentiation (F. Degli Innocenti and V. E. A. Russo, Genetic analysis of blue light-induced responses in Neurospora crassa, in H. Senger (ed.), Blue Light Effects in Biological Systems, Springer-Verlag, Berlin, Heidelberg, 1984, pp. 213-219), we suggest that protein dephosphorylation may constitute a necessary step in the light-transduction chain of Neurospora crassa.

Genetic Analysis of Blue Light-Induced Responses in Neurospora crassa

A genetic analysis of blue light sensory transduction chains has been done until now essentially only in Phycomyces. In this organism it was found that two genes, mad A and madB, are necessary for the photoinduction of five different effects. This pleiotropism has been considered an indication of the different photoresponses having their early steps in common, including the photoreceptor [12, 17, 18]. The disadvantage of Phycomyces lies in the fact that its genetics, as well as its biochemistry are still at a very primitive stage. On the contrary,Neurospora crassa, one of the most studied eukaryotic organisms, is well known from both a genetic [15] and from a biochemical point of view [6]. Furthermore, it has been studied also from a photobiological point of view (see below). The possibility of integrating information coming from genetic, biochemical, and photobiological studies makes this organism particularly suitable to approach the problem of photoreception at the molecular level.