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V E Russo

Publications and source records attributed to V E Russo.

At least 19 recordsLinked to original sources
PubMed2001

Cytokinin overproducing ove mutants of Physcomitrella patens show increased riboside to base conversion.

Ove mutants in the moss Physcomitrella patens can arise from different recessive mutations. These mutants display a much larger number of buds than the wild type (wt) due to a dramatic overproduction of cytokinins (Cks), which are released into the culture medium (T.L. Wang, R. Horgan, D.J. Cove [1981] Plant Physiol 68: 735-738). The amounts of isopentenyladenine (iP) and isopentenyladenosine ([9R]iP) produced by chloronema of different ove mutants were measured. Levels of the major Ck iP in the culture medium of the mutants oveA78, oveA201, oveC200, and oveB300 (cultured at 21 degrees C) were 4-fold (oveA78) to 22-fold (oveB300) higher than for the wt. A new temperature-sensitive ove strain oveST25, which exhibits a strong ove phenotype at 25 degrees C, was also studied. It produced about 260 times more iP than the thiamine auxotrophic wt from which it was derived. To contribute to the physiological understanding of Ck overproduction, in vivo labeling experiments with (3)H-[9R]iP were performed. In all ove mutants analyzed, the rate of (3)H-[9R]iP conversion to (3)H-iP was higher as compared with the wt. In oveST25, the 3-fold increased riboside to base conversion was temperature inducible and correlated with the iP production. Analysis of Ck catabolism revealed no major differences between ove mutants and wt, thus indicating that ove mutants are unlikely to be degradation mutants. The data suggest that in ove mutants the increased riboside to base conversion is part of a generally up-regulated Ck biosynthetic pathway and may play an important role for the enhanced release of iP into the medium.

Bryopsida↗
PubMed1999

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

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.

Amino Acid Sequence↗
PubMed1998

Photoregulation of cot-1, a kinase-encoding gene involved in hyphal growth in Neurospora crassa.

Blue light plays a key role as an environmental signal in the regulation of growth and development of fungi and plants. Here we demonstrate that in Neurospora crassa hyphae branch more frequently in cultures grown in light. Previous studies had identified cot-1 as a gene that controls apical hyphal cell elongation. In the cot-1 mutant, cessation of elongation is accompanied by hyperbranching. Here we demonstrate that the cot-1 gene encodes two transcript species of about 2100 nt (cot-1 (s)) and about 2400 nt (cot-1 (l)) in length and that the ratio of both transcript species abundance is photoregulated. The origin of the difference between cot-1 (l) and cot-1 (s) was localized to the 5' end of the cot-1 transcripts, suggesting that two COT1 isoforms with different activities may be formed. Both light effects, on branching and on cot-1 expression, were dependent on functional wc-1 and wc-2 gene products. In addition to light, L-sorbose comprises another environmental cue that controls hyphal branching in N. crassa. In the presence of L-sorbose, photoregulation of cot-1 was blocked, suggesting the involvement of alternative and potentially interdependent signaling pathways for the regulation of hyphal elongation/branching.

Base Sequence↗
PubMed1997

Novel pattern of DNA methylation in Neurospora crassa transgenic for the foreign gene hph.

It has previously been reported that multiple copies of the hph gene integrated into the genome of Neurospora crassa are methylated at Hpa II sites (CCGG) during the vegetative life cycle of the fungus, while hph genes integrated as single copies are not methylated. Furthermore, methylation is correlated with silencing of the gene. We report here the methylation state of cytosine residues of the major part of the promoter region of the hph gene integrated into the genome of the multiple copy strain HTA5.7 during the vegetative stage of the life cycle. Cytosine methylation is sequence dependent, but the sequence specificity is complex and is different from the sequence specificity known for mammals and plants (CpG and CpNpG). The pattern of DNA methylation reported here is very different from that measured after meiosis in Neurospora or in Ascobulus . After the sexual cycle in those two fungi all the cytosines of multiple stretches of DNA are heavily methylated. This indicates that the still unknown methyltransferase in Neurospora has a different specificity in the sexual and the vegetative stages of the life cycle or that there are different methyltransferases. The pattern of methylation reported here is also different from the pattern of cytosine methylation of transgenes of Petunia , the only pattern published until now in plants that has DNA methylation at cytosines which are not in the canonical sequences CpG and CpNpG.

Animals↗
PubMed1996

bli-4, a gene that is rapidly induced by blue light, encodes a novel mitochondrial, short-chain alcohol dehydrogenase-like protein in Neurospora crassa.

Blue light plays an important role in developmental control throughout nature. The bli-4 gene of Neurospora crassa, together with bli-3, al-1 and al-2, is rapidly inducible by blue light. Induction leads to a ninety-fold increase in transcription rate over the dark control level, and the gene therefore appears to be of prime importance in the blue-light induction pathway of N. crassa. We describe the sequencing and analysis of bli-4 and the 38 kDa protein it encodes. We show that the protein is very rapidly imported into the mitochondria and exhibits high homology with the family of short-chain alcohol dehydrogenases.

Alcohol Dehydrogenase↗
PubMed1994

Characterization of al-2, the phytoene synthase gene of Neurospora crassa. Cloning, sequence analysis, and photoregulation.

We have cloned the al-2 gene of Neurospora crassa and have analyzed its structure and regulation. The gene encodes a 603-residue polypeptide with a segment homologous to prokaryotic and other eukaryotic phytoene synthases. RNA measurements showed that the level of al-2 mRNA increased over 30-fold in photoinduced mycelia compared with dark-grown mycelia. This observation is consistent with the fact that carotenoid biosynthesis is induced by blue light during growth of N. crassa mycelia. The photoinduced increase in al-2 mRNA levels was not observed in two Neurospora mutants, wc-1 and wc-2, that are defective in all physiological photoresponses.

Alkyl and Aryl Transferases↗
PubMed1994

Neurospora crassa blue light-inducible gene bli-3.

Blue light induces various physiological, morphological and biochemical reactions in the filamentous fungus Neurospora crassa. This light response is accompanied by a global change in gene expression, and several light-inducible transcripts (bli-genes) have been cloned. We isolated the genomic clone of the gene bli-3, whose mRNA we have previously shown to be induced 2 minutes after the beginning of illumination. Its DNA sequence predicted a transcriptional unit of 1050 bp encoding a novel, hydrophilic protein of 209 aminoacids. Comparison to other N. crassa genes revealed a group of inducible genes which share this promoter structure: a well conserved TATA-box, similar transcription start box (TCATCANC) and repeats of pyrimidines (CT) between these boxes. Based upon this group, we propose a consensus sequence for one possible type of inducible promoter in N. crassa.

Amino Acid Sequence↗
PubMed1993

Promoter analysis of the bli-7/eas gene.

Expression of the Neurospora crassa gene bli-7, (identical with eas, and ccg-2), is induced by blue light, as well as glucose- or nitrogen-starvation. A promoter analysis was performed by an assay that does not involve promoter-reporter constructs but rather the insertion of foreign DNA into the transcribed sequence. To detect regulatory elements a series of deletions in the upstream region was generated. The inducibility of the gene, in response to the three inducing conditions mentioned, is lost by eliminating the region between -1498 bp and -1017 bp upstream of the transcription start point. A segment with an apparently negative effect was found between -595 bp and -429 bp, as well as a stretch of DNA from -429 bp to -380 bp which may exert a positive influence after light induction.

Cloning, Molecular↗
PubMed1992

The Ncypt1 gene from Neurospora crassa is located on chromosome 2: molecular cloning and structural analysis.

Small GTP-binding proteins are encoded by ras-like genes and play a central role in cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the Ascomycete fungus Neurospora crassa with Zmypt genes from Zea mays we have isolated a member of the ypt gene family, Ncypt1. The gene resides on a 4 kb fragment of genomic DNA and contains four introns, which interrupt the coding sequence of a protein of 203 amino acid residues. The Ncytp1 gene was assigned to a single-copy gene encoding a transcript of 1.5 kb and a protein of 26,000 daltons. The gene maps on linkage group IIR between DB0001 and ccg-2 close to the Fsr-3 locus. Analysis of the nucleotide sequence and the deduced protein sequence revealed a striking homology to yeast, mouse and human genes encoding small GTP-binding proteins that are related to the ras supergene family. Homology was most significant to ypt1 from Schizosaccharomyces pombe, Mus musculus and Homo sapiens sharing 84.8%, 82.3%, and 82.3% identity, respectively. Common domains present in other small GTP-binding proteins were identified in the predicted sequence of the NCYPT1 protein, and the arrangement of peptide motifs sharing similarity with well characterized, small GTP-binding proteins suggests that the NCYPT1 protein is a GTPase. The C-terminal region extending from amino acid residues 175 to 199 shares only weak amino acid sequence similarity with other eukaryotic GTPases. Like other RAS proteins the NCYPT1 protein contains two conserved C-terminal cysteine residues, suggesting post-translational modification(s) by fatty acylation required for membrane anchoring. The high degree of homology between the NCYPT1 protein and eukaryotic YPT proteins suggests that NCYPT1 could be involved in the control of secretory processes.

Amino Acid Sequence↗
PubMed1992

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

A 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 without 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.

Azacitidine↗
PubMed1992

Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora.

The surface of many fungal spores is covered by a hydrophobic sheath termed the rodlet layer. We have determined that the rodlet protein of Neurospora crassa is encoded by a cloned gene designated bli-7, and that bli-7 is identical to the known gene eas (easily wettable). Using eas DNA as a probe we show that eas mRNA is abundant in illuminated mycelia and conidiophores but is not detectable or is barely detectable in dark-grown mycelia, mature macroconidia, microconidia, and ascospores. Mutations in the genes acon-2, acon-3, and fl block early conidiophore development; of these, only fl prevents normal eas transcription. The EAS protein is homologous to the rodlet protein (RodA) of Aspergillus nidulans, and the hydrophobins of Schizophyllum commune. eas is the first cloned conidiation (con) gene of N. crassa that is associated with a phenotypic alteration.

Amino Acid Sequence↗
PubMed1992

Neurospora crassa blue-light-inducible gene bli-7 encodes a short hydrophobic protein.

Blue light induces a number of physiological reactions in Neurospora crassa. We have cloned and sequenced the gene bli-7, which is inducible by blue light, and both glucose and nitrogen starvation. This gene is strongly expressed at the RNA-level and contributes up to 0.2% of N. crassa total RNA when fully induced. The deduced amino acid sequence reveals a short (108 amino acids), hydrophobic protein which has homology to the protein SC-3, encoded by a gene of Schizophyllum commune which was isolated as a gene abundantly expressed at the time of fruiting. The amino-terminus of BLI-7 protein shows resemblance to a number of transit peptides. The comparison of the nucleotide sequence of the promoter region with various N. crassa promoters reveals a striking similarity to the promoter of grg-1, a glucose repressible gene.

Amino Acid Sequence↗
PubMed1991

Blue light induction of conidiation-specific genes in Neurospora crassa.

The con genes of Neurospora crassa are preferentially expressed during a developmental process known as conidiation. We present evidence indicating that transcription of con-5 and con-10 is also stimulated by blue light. Transcription of these genes was not photoinducible in wc-1 and wc-2 mutant strains. The response of con-5 and con-10 to blue light was similar to that of al-1 and al-2, genes involved in carotenoid biosynthesis, and bli-3 and bli-4, blue light inducible genes.

Blotting, Northern↗
PubMed1990

Light-induced dephosphorylation of a 33 kDa protein in the wild-type strain of Neurospora crassa: the regulatory mutants wc-1 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.

Darkness↗
PubMed1990

Fast induction of translatable mRNA by blue light in Neurospora crassa wt: the wc-1 and wc-2 mutants are blind.

After blue-light irradiation of Neurospora crassa (wt) mycelia we observed an increase of about 13 translatable mRNA species within a period of 30 min. The induction of translatable mRNA species followed a specific temporal pattern which permitted the identification of four distinct classes. One of the translatable mRNAs was induced in less than 2 min, while the others showed lag periods of 5, 10 or 20 min from the beginning of illumination. The white collar mutants, wc-1 and wc-2, which do not display any of the blue-light-induced physiological effects tested until now were found to be defective for the photoinduction of translatable mRNAs.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1990

Cloning, sequence, and photoregulation of al-1, a carotenoid biosynthetic gene of Neurospora crassa.

Carotenoid biosynthesis is regulated by blue light during growth of Neurospora crassa mycelia. We have cloned the al-1 gene of N. crassa encoding the carotenoid-biosynthetic enzyme phytoene dehydrogenase and present an analysis of its structure and regulation. The gene encodes a 595-residue polypeptide that shows homology to two procaryotic carotenoid dehydrogenases. RNA measurements showed that the level of al-1 mRNA increased over 70-fold in photoinduced mycelia. Transcription run-on studies indicated that the al-1 gene was regulated at the level of initiation of transcription in response to photoinduction. The photoinduced increase of al-1 mRNA levels was not observed in two Neurospora mutants defective in all physiological photoresponses. Analysis of cosmid containing al-1 and of a translocation strain with a breakpoint within al-1 indicated that al-1 transcription proceeds towards the centromere of linkage group I of N. crassa.

Amino Acid Sequence↗
PubMed1989

Fast light-regulated genes of Neurospora crassa.

Several physiological reactions including the sexual differentiation of the ascomycete Neurospora crassa are triggered by blue light. Mutants in the white-collar genes wc-1 and wc-2 are blind for all the blue light effects tested so far. We have previously shown that blue light induces some translatable mRNAs at different times after beginning the illumination. Here we report the cDNA cloning of four genes that are induced by blue light. Induction of these transcripts is temporally ordered (lag times from 2 to 45 min). Analysis of run-on transcripts show that the increases in mRNA levels are due to de novo transcription. None of these transcripts is inducible in white-collar mutants.

Blotting, Northern↗