Gene Regulation
17. Hexokinase activity in S. cerevisiae. 1. The role of mitochondrial deficiency, glucose and ethanol concentrations.
Kamel A. Ahmed, Mohamed H. Hamoda, Ahmed N. Sharaf and Hassan A. Mohamed. Faculty Agriculture, University of Cairo, and National Res. Centre, Egypt.
The genetic regulation of hexokinase enzyme in Saccharomyces cerevsiae was
investigated through the effect of the deficiency in mitochondrial function and regulatory effect of
glucose and ethanol concentration as well as aeration. Petite mutants were induced by nicotine
mutagen and identified in two genetically different haploid yeast strains. Petite mutants showed
no chromosomal reverted genes, different patterns of sugar utilization, when tested on defferent
carbon sources , and a variable increase in enzyme activity than that in its parental strains .
Different types of diploids (normal mitochondrial function, partially or totally nonmitochondral
function diploids) showed differences in their sporulation time where the petite diploid "non-functional mitochondrial diploid" never sporulated. Diploids exerted variable hybrid vigour in
their enzyme production . Tetrad analysis of different diploids resulted in variable segregation
patterns in the enzyme activity . Parental, petite mutants, diploids and tetrad segregants strains
tolerated the increase in ethanol concentration up to 10% , glucose concentration up to 40% and
aerobic or anaerobic conditions to different degrees which was reflected on their enzyme activity
as a function of their chromosomal and cytoplasmic genes.
18. Characterization of the fluffy gene of Neurospora crassa.
Lori A. Bailey and Daniel J. Ebbole. Texas A&M University.
The fluffy (fl) mutant of Neurospora crassa blocks the development of macroconidia.
Genetic mapping places fl between mus-23 and trp-3 on the right arm of chromosome III. A
cosmid was identified that complemented both mus-23 and fl. Fl was subcloned to a 4.5 kbp
fragment and sequence analysis of this fragment revealed an open reading frame (ORF) containing
a zinc finger DNA binding domain and two putative introns. The ORF has limited sequence
similarity to Nit-4 and NirA, regulators of nitrogen assimilation in N. crassa and Aspergillus
nidulans respectively. We have sequenced four alleles of fl, and these data demonstrate that the
predicted ORF is required for proper gene function. RIP inactivation of the 4.5 kbp fragment
produced progeny with the fluffy phenotype. The sequence of the RIP mutant contained 74
mutations due to base pair changes including one changing the translational start codon and one
destroying the zinc finger domain. Expression of fl appears to peak six hours after induction of
development, and it is induced by nitrogen starvation.
19. A prion-like protein in the filamentous fungus Podospora anserina.
Joel Begueret, Carole Deleu, Virginie Coustou, Beatrice Turcq and Corinne Clave, Institut CNRS de Biochimie et Genetique Cellulaires, Bordeaux, France.
The het-s locus of the fungus Podospora anserina is involved in vegetative
incompatibility. Coexpression of the two alternate alleles het-s and het-S is lethal leading to
incompatibility between strains. Strains containing the het-s allele can exhibit two different
phenotypes: either they are incompatible with strains containing the antagonistic het-S allele ([s]
phenotype) or they are neutral in incompatibility ([s*] phenotype). This later phenotype can be
propagated vegetatively and is maternally inherited through meiosis. However a [s*][s] switch
can occur either spontaneously at a very low rate or at a high frequency after a contact with a
strain which exhibits the [s] phenotype. Once induced, the [s*][s] transition spreads as an
infectious process within the mycelium. We found that the HET-s protein is expressed at the
same level in strains that display these alternate phenotypes leading to the conclusion that a post-translational modification of the protein is responsible for the difference between [s] and [s*]
strains. These different results suggested that the HET-s protein should behave as the prion
protein PrP: the protein present in [s*] and [s] strains should display different conformations and
the [s*] molecule could be converted to the [s] form by a physical interaction between the
molecules. Different results confirmed that the HET-s protein display properties common with
the prion protein. i) the HET-s protein strongly aggregates in vitro and can form homodimers in
vivo as demonstrated using the yeast two hybrid system. ii) the proteins present in [s*] and [s]
strains display different sensitivity to proteolytic enzymes. iii) overexpression of the HET-s
protein strongly enhances the frequency of the [s*][s] switch.
20. Cdc42-signaling during cytokinesis in Ustilago maydis .
Michael Bo1kerl and Gerhard Weinzierl, Institut of Genetics, University of Munich, Maria-Ward-Str. la, D-80638 Munchen, Germany; lpresent address: Dept. of Biology, University of Marburg, D-35032 Marburg, Germany
The phytopathogenic fungus Ustilago maydis exhibits a dimorphic life style. Haplold
sporidia grow yeast-like by budding and are non-pathogenic. The dikaryon grows filamentous
and is able to induce tumors in maize plants. To identify genes that are involved in this
morphogenetic switch we have isolated a number of mutants with aberrant morphology. Among
these we could identify two mutants that are affected in cytokinesis. Both the donl and don3
mutants show normal nuclear division but the mutant cells fail to separate after bud formation.
Cells remain connected through a septum that can be stained by calcofluor. The corresponding
genes have been isolated by complementation. Molecular analysis of these genes revealed that the
final step of cytokinesis seems to be regulated by a member of the Rho/Rac/Cdc42 family of small
GTP binding proteins. The Donl protein shows high similarity to guanine exchange factors
specific for these small Ras-like proteins, the don3 gene codes for a kinase that is homologous to
the yeast STE20 and the mammalian pak/p65 serine/threonine kinases. Using the yeast two-hybrid-system we could demonstrate that the U.maydis Cdc42 homologue interacts with both
Donl and the Don3. We propose that cell separation is triggered by a signal that is transmitted via
Cdc42.
21. Mutations, pumps, antibodies, and polar growth.
Emma Jean Bowman, Forest J. O'Neill, and Barry J. Bowman, University of California, Santa Cruz, CA. 95064.
Concanamycin A (CCA) is a potent specific inhibitor of vacuolar ATPases. Mutants were selected for growth on medium containing 1.0 uM CCA. Suprisingly, 65 of 66 mutations mapped in the region of the pma1 locus, which encodes the plasma membrane H+-ATPase. Plasma membrane H+-ATPase activity in isolated plasma membranes from the mutants was 17-84% of the level seen in the wild type. The most interesting change in the plasma membrane H+ATPase was in kinetic behavior. The wild-type enzyme showed sigmoid dependence on MgATP concentration with a Hill number of 2.0, while 7 of the 8 mutants tested exhibited hyperbolic kinetics with a Hill number of 1.0. One interpretation of these data was that the enzyme had changed from a functional dimer to a functional monomer.
Mutation of the plasma membrane H+-ATPase did not confer resistance by preventing
uptake of CCA. In the presence of CCA both wild type and mutant strains were unable to
accumulate arginine, failed to concentrate chloroquine in acidic vesicles, and exhibited gross
alterations in hyphal morphology, indicating that the CCA had entered the cells and inactivated
the vacuolar ATPase. Instead, we hypothesize that the mutations conferred resistance because
the altered plasma membrane H+-ATPase could more efficiently rid the cell of toxic levels of Ca++
or protons or other ions accumulated in the cytoplasm following inactivation of the vacuolar
ATPase by CCA.
22. Overproduction of aflatoxin precursors is associated with altered sclerotial development in Aspergillus parasiticus.
Perng-Kuang Chang1, Peter J. Cotty2, Deepak Bhatnagar2, Joan W. Bennett1 and Thomas E. Cleveland1.
1Tulane University, New Orleans, LA; 2Southern Regional Research Center, ARS/USDA, New Orleans, LA.
A genetic relationship between aflatoxin biosynthesis and sclerotial development was
tested in A. parasiticus SRRC 2043, an O-methylsterigmatocystin accumulating strain, by
transforming it with the aflatoxin pathway genes, aflR and/or aflJ. Elevated production of
aflatoxin precursors, norsolorinic acid, averantin, versicolorin A and O-methylsterigmatocystin,
resulted from introduction of extra copies of either aflR or aflR plus aflJ in transformants, but not
by introduction of aflJ alone. Similarly, sclerotial production on PDA increased only in the
transformants receiving either aflR or aflR plus aflJ and not in the transformant which received
aflJ alone. However, the sclerotial production of the aflR plus aflJ transformant was substantially
decreased on CZ plates. Increased production of aflatoxin precursors was associated with
changes in sclerotial morphology; sclerotial shape became more elongated in transformants.
Scanning electron micrographs showed that the sclerotia of the aflR plus aflJ transformant from
PDA was not as compact as that observed for the wild-type strain. These results suggest a
regulatory association between sclerotial morphogenesis and aflatoxin biosynthesis.
23. Regulation of the acetate utilization pathway in the basidiomycete Coprinus cinereus.
Pushpalata T. Chaure, Lorna A. Casselton and Ian F. Connerton, Univ of Oxford and Inst of Food Research, Reading, UK.
Growth on acetate as sole carbon source requires the induction of enzymes of the
glyoxylate cycle and the acetate mobilising enzyme, acetyl CoA synthetase. Induction is
dependent on the function of a regulatory gene, acu-1. acu-1 was isolated by functional
complementation in C. cinereus. DNA sequence analysis identifies a Zinc finger DNA-binding
domain in its protein that has homology to the finger in corresponding acetate regulatory proteins
of Aspergillus nidulans (facB) and Saccharomyces cerevisiae (cat8). Surprisingly, there is little
other sequence conservation in the three proteins despite the obvious functional homology. We
will present our sequence analysis of this gene and its predicted protein and describe experiments
that confirm its regulatory role with respect to two of the key enzymes induced by acetate, acetyl
CoA synthetase and isocitrate lyase. Elements within the promoters of the acs-1 and acu-7 genes
have been identified as possible acu-1 binding sites and these are also present within the promoter
of acu-1 itself indicating autogenous regulation. Other interesting aspects of acu-1 regulatory
function will be presented.
24. Protein-protein interactions between arginine biosynthetic enzymes of Neurospora.
Jessica Y. Chung, Suhn-Kee Chae* and Richard L. Weiss. Department of Chemistry and Biochemistry, University of California, Los Angeles, * Present address Pai Chai University, Korea.
N-acetyl glutamate synthase (AGS) and N-acetyl glutamate kinase (AGK) are the first two
enzymes in the arginine biosynthetic pathway in Neurospora crassa. AGS and AGK are encoded
by two independent genes, arg-14 and arg-6, respectively. Early biochemical and genetic
evidence indicated that mutations in AGK affected both AGK and AGS activities, and that a wild-type AGK gene could restore AGS activity in arg-6 mutants. In order to investigate the nature of
this activation, we tested the possibility of protein-protein interaction between AGS and AGK
proteins using the yeast Two-Hybrid System. We report the first molecular evidence for direct
AGS and AGK interaction. AGS and AGK interaction is strong and visualization through a -galactosidase assay is rapid: blue colonies were observed in less than 30 minutes. The interaction
domains of AGS and AGK have been identified using various deletion constructs. The interaction
domain of AGS resides at the N-terminus and the interaction domain of AGK is at the C-terminus.
25. Arginine feedback resistant mutation in Neurospora.
Jessica Y. Chung, Suhn-Kee Chae* and Richard L. Weiss. Department of Chemistry and Biochemistry, University of California, Los Angeles, *present address, Pai Chai University, Korea
Mutations linked to the complex arg-6 locus (su(pro-3) ) are feedback resistant and
suppress proline auxotrophic mutations (pro-3). N-acetyl glutamate kinase (AGK) is one of the
proteins encoded by the arg-6 gene and is inhibited by arginine. We postulated that the su(pro-3)
mutation could be located at the arginine feedback site of AGK. In order to identify the feedback
resistant site in AGK and the nature of su(pro-3) mutations, we cloned AGK from several su(pro-3) mutant stains. Genomic DNA from several su(pro-3) strains was isolated and PCR was
performed. The PCR products were introduced into a N. crassa double mutant (arg-6, pro-3)
and transformants were identified by arginine prototrophy. Transformants that contained the
su(pro-3) mutant gene suppressed the proline requirement and were able to grow in the absence
of proline. The su(pro-3) mutations were mapped to the N-terminus of AGK, and all su(pro-3)
mutant genes have a single amino acid change from phenylalanine (F) to leucine (L) at amino acid
81. To confirm that the F to L mutation alone caused the su(pro-3) phenotype, wild-type AGK
was mutated using overlap extension PCR mutagenesis. The mutated AGK was introduced into
the N. crassa double mutant (arg-6, pro-3) and transformants were identified by arginine
prototrophy. A single F to L change in the wild-type AGK gene resulted in the su(pro-3)
phenotype: transformants grew in the absence of proline. Southern blot analysis verified the
presence of the mutant AGK gene in transformants.
26. Transgene induced gene silencing "quelling" in Neurospora crassa.
Carlo Cogoni and Giuseppe Macino. Universita' "La Sapienza" Roma Italy.
Transgene-induced gene silencing of several genes used in transformation experiments in Neurospora crassa has been termed "quelling". In studies using the carotenoid biosynthetic gene albino- 1 as a visual reporter for quelling, silencing was found to be reversible, and reversion was accompanied by loss of exogenous copies. Gene silencing acts at a post-transcriptional level leading to a strong reduction of steady-state mRNA level of the duplicated gene. Silencing was shown to be a dominant trait, operative in heterokaryotic strains, indicating the involvement of diffusable, trans-acting molecules. The production of an unintended transgene derived sense RNA was found to correlate with the gene silencing.These findings are compatible with a model in which RNA-RNA interaction is involved in gene silencing.
The isolation of a transformant strain silenced for the albino-1 gene in which quelling is stable,
allowed us to perform UV mutagenesis to isolate, by simple visual screening, mutant strains
impaired in gene silencing. Among 120,000 survivors screened after exposure to UV, 25 strains
showing the recovery of wild type (orange) phenotype were isolated. Genetic analysis indicates
that all the strains, containing recessive mutations, fall into four different complementation
groups. The isolation of the corresponding genes will be of fundamental importance in the
elucidation of the molecular mechanism of gene silencing in Neurospora as well as in plants.
27. Clock Mutants and Light Entrainment in Neurospora.
Anne Cole, Susan Crosthwaite, Michael Collett, Jay Dunlap and Jennifer Loros. Dartmouth Medical School, Hanover, NH
A defining feature of circadian oscillators is their ability to be entrained by environmental stimuli. The most universal of these stimuli are light and temperature. In Neurospora crassa, the effects of light and temperature on the clock can be followed at the phenotypic level by monitoring the circadian conidiation rhythm, and at the molecular level by monitoring the expression of known clock components.
The clock in Neurospora is comprised of a negative feedback loop in which the frequency (frq) gene encodes the FRQ protein which, in turn, either directly or indirectly represses its own expression. With appropriate delays built in, this feedback loop yields the oscillation in the amount of frq transcript and FRQ protein which collectively comprises the Neurospora clock. Light delivered at any point within the circadian cycle acts rapidly to increase the level of frq transcript and thus resets the clock.
In two "photo-blind" strains, white collar-1 (wc-1) and white collar-2 (wc-2), we have found that both light and temperature treatments that normally synchronize clock wild-type cultures do not result in a rhythmic phenotype. Furthermore, light induced accumulation of frq transcript is blocked in wc-1, and the sustained, light-driven increase in frq mRNA that is seen in wild type strains is blocked in wc-2. Both frq transcript and FRQ protein levels are low and arrhythmic in both of these mutants. These results together suggest that in addition to their role in light signal transduction, WC-1 and WC2 may be required for operation of a functional circadian clock.
In addition, we are currently using insertional mutagenesis to identify other genes involved
in circadian rhythmicity. By screening for mutants with altered clock output phenotype, we hope
to identify mutations which disrupt clock input, clock output and the central oscillator, as well as
developmental mutants involved in conidiation. To date, several phenotypically arrhythmic strains
have been isolated.
28. Studies on pectinolytic enzymes produced in submerged culture by mutant exo-1 of Neurospora crassa.
Luciana B. Crotti, J.A. Jorge, H.F. Terenzi and Maria de Lourdes T.M. Polizeli. Depto. de Biologia - FFCLRP-USP; - Brasil.
The pectinolytic complex is composed by enzymes which degrade pectic substances and produce oligogalacturonates (hidrolases) or insatured products (lyases). These enzymes play an important role in processing and manufacturing industries as the agents of maceration, fruit juice clarification, and others. The exo-1 synthesized and secreted five to six times more pectic enzymes than the wild type. They are produced by many microorganisms, higher plants and some insects. the best condition to produce these enzymes was obtained in two-stage cultures: (1) pre-cultivation at 30 C for 24 h in Vogel's medium with 2% glucose; (2) transfer of the mycelial mass to fresh media with different carbon sources, and incubation for 72 h. The exo-1 in this condition produced four extracellular pectinases (pectin-lyase, pectate-lyase and two polygalacturonase) those were purified in two peaks using DEAE- and CM-cellulose column and one intracellular polygalacturonase activity purified by DEAE-cellulose column. The optimum of temperature and pH for lyases activity were respectively 50 C and above 9.0, and for polygalacturonases activities, these values were about 40-45 C and 5.0-5.5, respectively. The amino acid composition of intracellular polygalacturonase was analysed and compared with a computer program (PROSEARCH) using as a data the molecular weight (31.565 Da) and pI (7.4) and this composition analysis resembled with a polygalacturonase of Prunus persica (peach).
Supported by CAPES, CNPq and FAPESP.
29. Characterization of the FluG protein required for asexual sporulation in Aspergiflus nidulans.
Cletus A. D'Souza, Bee Na Lee and Thomas H. Adams, Texas A & M University, College Station, TX.
We propose that the A. nidulans sporulation gene product FluG directs a low constitutive
production of an extracellular diffusible factor with development initiating once a threshold level
accumulates. Although FluG levels are relatively constant throughout the life cycle, development
in submerged culture is limited by fluG expression levels because overexpression can obviate the
need for air in sporulation. Deletion analysis has revealed that the C-terminal half bearing 28%
identity with GSI-type prokaryotic glutamine synthetases is sufficient for development and its
overexpression can cause spurious development in submerged culture. However, FluG appears to
have a function distinct from glutamine biosynthesis because flug mutants are not glutamine
auxotrophs. Moreover, the induced expression of the heterologous Anabaena glutamine
synthetase in A. nidulans does not initiate sporulation. To identify other components acting
downstream of FluG and presumably functioning in the response to the factor, we have isolated a
dominant suppressor of a fluG mutation. Suppression results in precocious (conidiophores at
the end of germ tubes) and prolific conidiation as well as inhibited hyphal growth on solid medium
and in submerged culture. This indicates a bypass of FluG function as well as developmental
signals acting upstream of FluG.
30. Processing and Function of a Polyprotein Precursor of Two Mitochondrial Enzymes in Neurospora crassa.
Lilian Parra-Gessert, Jay Fajardo, Kenneth Koo and Richard L. Weiss, Department of Chemistry & Biochemistry, University of California, Los Angeles.
In Neurospora crassa, the mitochondrial proteins N-acetylglutamate kinase (AGK) and
N-acetyl--glutamyl-phosphate reductase (AGPR) are processed from a 96-kDa cytosolic
precursor encoded by the ARG6 gene. The proximal kinase and distal reductase domains are
separated by a short connector region. Processing was analyzed in vitro by import assays with
isolated mitochondria and in vivo by immunoblot and N-terminal sequencing of processed
products. Substitutions of arginines at positions -2 and -3 upstream of the N-terminus of the
AGPR domain or replacement of threonine at position +3 in the mature AGPR domain revealed a
second processing site. Substitution of arginine at position -22, in combination with changes at -2
and -3, prevented cleavage of the precursor. These results indicate a two-step proteolytic
cleavage of the connector region at the sequences Arg-Gly Tyr-Leu-Thr at the N-terminus of
the AGPR domain and at Arg-Gly-Tyr Ser-Thr located 20 residues upstream. Proteolytic
cleavage at both sites was prevented by inhibitors of the mitochondrial processing peptidase.
Enzyme assays and complementation studies have shown that misprocessed and unprocessed
precursors yield catalytically active enzyme(s). The AGPR specific activity of strains expressing
the unprocessed precursor is much higher than that of a wild type strain, suggesting that the
function of the polyprotein precursor may be to protect the AGPR domain from proteolysis
before reaching the mitochondrial matrix.
31. Coordinate induction of the genes encoding PKA subunits during sporulation in B. emersonii.
Suely L. Gomes Universidade de Sao Paulo, Sao Paulo, Brazil.
The aquatic fungus B. emersonii presents an interesting developmental cycle, which begins
with the zoospore, a motile non growing cell, that is triggered to germinate by the presence of
nutrients or the addition of cAMP, dfferentiating into the germling cell. This cell is capable of
vegetative growth, which is characterized by intense nuclear proliferation, without cell division,
leading to the formation of a multinucleated cell: the sporangium. At any time during growth,
shortage of nutrients can induce the sporulation process, which culminates with the
intrasporangial formation of the zoospores and their release from the mother cells. A single PKA
is present in B. emersonii. Its activity is low in vegetative cells, rising sharply during sporulation,
reaching maximum levels in the zoospores. After germination PKA activity decreases back to low
basal levels. Work from our laboratory has shown that these variations in activity are due to a
coordinate transcriptional control of the genes encoding the regulatory (R) and the catalytic (C)
subunits. To investigate sequence elements common to both R and C gene promoters, which
could be involved in the coordinate regulation of these genes, their 5' flanking regions were
analysed by gel mobility shift and DNA-footprinting assays. It was determined that different
DNA-protein complexes are generated when fragments of the R and C gene promoters are
incubated with extracts from cells expressing or not expressing both subunits. Furthermore,
DNaseI-footprinting experiments have indicated that a 7-nucleotide sequence element present in
both promoters, was protected from DNase I digestion only by non-expressing cell extracts,
suggesting a negative control for the induction of R and C genes during sporulation.
32. Effects of removing the mitochondrial outer membrane protein TOM70 Neurospora crassa.
Leslie I. Grad, Roland Lill*, Walter Neupert* and Frank E. Nargang, University of Alberta, Edmonton, Alberta, Canada and *Institut fur Physiologische Chemie-Universitat Munchen, Munich, Germany.
Recognition, unfolding, insertion, and translocation of preproteins at the mitochondrial
outer membrane is achieved by the TOM complex (translocase of the outer membrane of
mitochondria). The largest component of this complex, TOM70, is an integral outer membrane
protein with a large cytosolic domain thought to serve as a preprotein receptor. To investigate
the role of TOM70 in mitochondrial import, a tom7O null mutant was generated using the
method of RIP (repeat induced point mutations). In contrast to lethal phenotype of null
mutations in TOM20 or TOM22, two other receptor components of the TOM complex, the
effects of destroying TOM70 function are relatively mild. The TOM70-deficient mutant is still
viable, has a growth rate about 60% that of wild-type, and produces few conidia. Unexpectedly
TOM70-deficient mutant was also found to contain enlarged mitochondria with outer membranes
more prone to breakage under stressful conditions than those of wild-type mitochondria. The null
mutant has been successfully rescued by transformation with a wild-type copy of tom7O
demonstrating that the effects of RIP are specific to only the tom7O gene. Preliminary assays of
in vitro mitochondrial protein import suggest a significant decrease in the import efficiency of the
ADP/ATP carrier protein (ACC).
33. sreA, a new GATA factor encoding gene of Aspergillus nidulans.
Hubertus Haas, Klaus Angermayr, Ivo Zadra and Georg Stoffler, Dept. of Microbiol., Medical School, University of Innsbruck; A-6020 Innsbruck, Austria.
GATA-binding proteins constitute a family of transcription factors that recognize the
consensus motif GATA. This group includes a range of major regulatory proteins from organisms
as different as fungi, mammals, birds, insects and plants. The different members of this protein
family are related by a high degree of amino acid sequence identity within their DNA-binding
domains. In Aspergillus nidulans at least three GATA binding activities other than the extensively
characterized nitrogen regulatory protein AREA can be distinguished using gel mobility shift
assays. As a prerequisite to defining the function and interaction of different GATA factors in A.
nidulans, we employed different PCR approaches to isolate additional genes of this family. In the
course of this study we identified a gene (sreA) encoding a new GATA factor of A. nidulans,
containing two GATA type zinc fingers. The deduced amino acid sequence reveals 62% overall
identity to SREP of Penicillium chrysogenum. Furthermore, the 200 amino acid sequence
embracing the two zinc fingers and the intervening region displays 50% identity to URBS1 from
Ustilago maydis, a repressor of siderophore biosynthesis. Northern blot and cDNA analysis
revealed two transcripts, 2.3 kb and 2.8 kb in length, due to two different major transcriptional
start sites. If A. nidulans was cultured in low iron media, transcription of sreA was found to be
repressed. In synopsis the data suggest a regulatory function of SREA in iron metabolism
analogous to URBS1 in U. maydis.
34. Isolation of two new genes encoding subunits of the V-type ATPase of Neurospora crassa.
Ian Hunt and Barry Bowman. Department of Biology, University of California, Santa Cruz.
The vacuolar H+-ATPase (or V-ATPase) is a large multimeric enzyme composed of several subunits that acidify intracellular compartments in eukaryotic cells and play an important role in a variety of cellular processes.
Here, we report the isolation of two new genes responsible for the expression of putative V-ATPase subunits in Neurospora crassa. The first gene (vma10) encodes a protein with a molecular weight of 13,693 Daltons. The protein (subunit G) contains 113 amino acids and is highly hydrophilic. With similar subunits reported in yeast, bovine and insect it appears this subunit is ubiquitous to all V-ATPases. The second gene (vma9) appears to encode a 19,125 Dalton protein that contains 177 amino acids and although observed in other organisms on SDSPAGE gels has yet to be isolated and sequenced. This report thus represents the first characterization of this subunit, tentatively called subunit H. The role these two subunits play with regard the structure-function of the V-ATPase is unsure. However, both show sequence homology to the b-subunit of the closely related F-ATPase that is thought to be involved in coupling proton conduction and ATP synthesis. We thus propose a similar mechanism.
Serendipitously, we have also isolated a gene that encodes an amino acid transport protein with a molecular weight of 65kDa. The protein shows no sequence identity with any of the known Neurospora crassa amino acid transporters but does have strong homology with several basic amino acid permeases in yeast. We are presently pursuing characterization studies of this protein.
*Both vma genes were originally identified from EST's sequenced by the Neurospora Genome
Project, Department of Biology, University of New Mexico.
35. Molecular and functional analysis of the hymA gene in Aspergillus nidulans.
Marvin Karos and Reinhard Fischer, Universitat Marburg and MPI fur terrestrische Mikrobiologie, Marburg, Germany
The filamentous fungus Aspergillus nidulans is able to reproduce asexually with conidia and sexually with ascospores. In this study insertional mutagenesis was used to isolate mutants defective in asexual sporulation. One of these mutants was analyzed at a molecular level. In this strain hyphal growth rate was slightly decreased and branching frequency increased in comparison to wildtype. The conidiophore development was specifically blocked at the metula stage. Metulae rather resembled hyphae, because they were elongate, multinucleate and septate. Thus, the gene was named hymA (hypha-like metula).
A 2.4 kb hymA complementing genomic DNA fragment was isolated which detected a 1.8 kb
trancript in hyphae and in conidiophores. Sequence comparision of genomic DNA and
corresponding cDNAs revealed 4 Introns between 58 and 65 bp in size. The 384 amino acid
deduced HymA protein showed homology to proteins from Saccharomyces cereviseae,
Caenorhabditis elegans and Mus musculus. The mouse protein is thought to be a Ca2+-binding
protein. The region harbouring the Ca2+-binding site in the mouse protein was highly conserved in
HymA. However, a functional role of HYMA in Ca2+-dependent processes remains to be
determined.
36. Catalase is specifically oxidized by singlet oxygen during conidiation.
Fernando Lledias, Shaday Michan and Wilhelm Hansberg, Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico.
We have proposed cell differentiation as an avoidance response to a hyperoxidant state. Total protein and specific enzymes were oxidized and degraded at the start of each of the three morphogenetic transitions of the conidiation process in Neurospora crassa. In this work, we asked if catalase was also oxidized at the hyperoxidant states of cell-differentiation.
Cat-l a and Cat-lc activity increased in hyphae at the prestationary growth phase and in the first two morphogenetic transitions of the conidiation process. With formation of conidia, in addition to a Cat-la and Cat-lc increase, Cat-le and Cat-2 appeared transiently. Conidia had over 60 times more catalase activity than exponentially growing hyphae. Increments were biphasic: after an initial rise, a decrease in specific activity coincident in time with the hyperoxidant state followed by catalase induction in the differentiated cell-structures. Accumulation of Cat-1 mRNA followed this biphasic activity pattern.
Purified catalase Cat-la gave rise to Cat-lc and Cat-le. Cat-la was shown to be oxidized
through the sequential reaction of the four monomers with singlet oxygen, generating active
catalase conformers with more acidic isoelectric points. Modification was brought about
specifically by singlet oxygen, singlet oxygen scavengers prevented modification; superoxide and
hydroxyl radical had no effect on electrophoretic mobility. Modified Cat-1 monomers had a
similar molecular mass than the unmodified ones. Cat-2 from N. crassa and catalases from
different organisms were also susceptible to modification by singlet oxygen.
37. Catalase is oxidized by singlet oxygen during germination.
Fernando Lledias, Pablo Rangel and Wilhelm Hansberg, Institute de Fisiologia Celular, Universidad Nacional Autonoma de Mexico.
Specific modification of Cat-1 by singlet oxygen occurred during the hyperoxidant states of the conidiation process. In this work we asked if a hyperoxidant state and Cat-1 oxidation by singlet oxygen also takes place during germination.
Neurospora crassa conidia did not germinate under an atmosphere of nitrogen or argon. However, they did germinate under N2 or Ar if a pulse of hydrogen peroxide or oxygen saturated water was given to generate a transient hyperoxidant state, measured by chemioluminescence.
Cat-l a stored in conidia was oxidized during germination giving rise to Cat-1c. This could be detected even when germinating in the dark. Under conditions of increasingly higher concentration of singlet oxygen, such as intense light, a source of singlet oxygen, or in carotenoid deficient mutant strains, a higher proportion of Cat-l a was oxidized to Cat-lc and Cat-le. Increased and precocious accumulation of Cat-1 mRNA was observed. Oxidized catalase conformers disappeared rapidly and new Cat-la appeared.
A hyperoxidant state is probably required for breakage of dormancy. Oxidation of Cat-la
indicated intracellular generation of singlet oxygen during germination under all conditions tested.
Modified Cat-1 conformers probably have a shorted half life than the unmodified enzyme.
Carotenoids appeared to protect Cat-1 from in vivo modification by singlet oxygen.
38. Regulation of a conidiation gene, con-10, of Neurospora by starvation and stresses is unmasked by mutation of a developmental regulator, rco-1.
Kwangwon Lee and Daniel J. Ebbole, Texas A&M University.
The Neurospora crassa con-10 gene encodes a small polypeptide that has homology with a
general stress-induced gene, gsiB, of Bacillus subtilis. A regulatory gene, rco-1, controls con-10
expression and sporulation in N. crassa. RCO1 appears to function as a repressor of con-10
during mycelial growth. In wild-type strains, prolonged carbon starvation results in development
and con-10 expression, however, con-10 is not highly induced by glucose starvation in the
absence of development. In the rco-1 mutant strain, con-10 expression is rapidly and highly
induced by carbon or nitrogen starvation and heat shock. Thus, in the wild-type, rco-1 functions
to repress con-10 expression in mycelia and to block rapid induction of the gene in response to
starvation and stresses.
39. Partial Purification and Biochemical Properties of Trehalose-6-Phosphate Synthase of Neurospora crassa.
Maria de Lourdes T.M. Polizeli, Luciana B. Crotti, J.A. Jorge and H.F. Terenzi. Dep. Biologia - FFCLRP-USP - Brasil.
Trehalose is synthesized in yeast from glucose-6-phosphate (G6P) and uridine-diphosphoglucose (UDPG) in a two step reaction catalyzed by the trehalose-6-phosphate synthase (Tre6P synthase)/trehalose-6-phosphate phosphatase (Tre6P phosphatase) complex. Previous report of our laboratory showed that the synthesis and breakdown of trehalose in N. crassa conidiospores submitted to temperature shifts. Tre6P synthase exhibited optimum of temperature and pH of 55 C and pH 7.0, respectively. During heatshock this activity increased about 100% simultaneously with accumulation of trehalose, independent of synthesis of protein. This metabolic event may be primarily due to temperature-dependent changes in the kinetic properties of Tre6P synthase. Different sensibility to inhibition by phosphate, F6P, ATP and T6P was verified when the enzyme was assayed either at 30, 40 or 50 C. The increase of temperature diminished inhibition by phosphate compounds. A similar difference in degree of inhibition was also observed between crude extracts of cells submitted or not to heat shock. Tre6P synthase was induced four-fold during germination in glucose, reaching in 6 h its maximum sp. act. In glycerol, the activity was 15-17 fold lower. The enzyme was partially purified by phosphocellulose chromatography and FPLC(BioGel SP5 plus). Tre6P exhibited Mwapp of about 430 kDa, suggesting its presence in a complex. Km and Vmax, were respectively 0.814 mM and 528 nmoles/min/mg prot for UDPG, and 9.5 mM and 77.7 nmoles/min/mg prot for G6P. Next we shall test whether the fundamental regulatory roles attributed to yeast Tre6P also apply to filamentous fungi. Support: FAPESP and CNPq