Y Ogura, C. Aoyagi, N. Yabe and K. Hasunuma, Yokohama City University, Kihara Institute for Biological Research, Totsuka-ku, Yokohama, 244 Japan.
We have established that 15 kDa proteins (psl5) showed increase in the
phosphorylation in response to blue light (420 nm)(1). The psl5 protein was
partially purified from the crude extract of mycelia to 200-fold. The partially
purified fraction contained nucleoside diphosphate kinase (NDP kinase) activity
forming CTP, GTP and UTP from CDP, GDP and UDP in the presence of [gamma-
32P-]ATP. The partially purified fraction was further separated by a Tricine SDS-
PAGE and the psl5 showed autophosphorylation activity. From the protein band
ps 1 5 was extracted and hydrolyzed by chymotripsin. The polypeptides were further
fractionated by a reverse phase HPLC. Amino acid sequence of one of the
polypeptide was completely coinsided with that deduced from the cDNA for NDP
kinase; NCNDKI (No.D88148) isolated by our group.
The null type mutant of the phosphorylation of NDP kinase, designated to be
delta-ps15-1 was isolated. From the mutant mRNA was isolated and by use of RT-
PCR, the cDNA was cloned. The delta-ps15-1 included aminoacid replacement from
Pro-72 to His-72. The amino terminal part of Pro-72 formed Beta-sheet, and carboxy
terminal part formed alpha-helix. The amino acid replacement from Pro to His may
cause aberrant folding of the protein leading complete loss of the phosphorylation
activity of it. The results of RT-PCR identified two species of mRNA, both of
which was cloned and resulted to detect alternative splicing in the mRNA. Southern
blot analysis showed single copy of NDP kinase in Neurospora crassa genome, and
the genomic DNA was isolated and sequenced. Two introns and one alternative
splicing between the two introns could be detected. The consensus sequence for
ATP-binding and the mutation site of Pro-72 were spliced out.
(1) Oda, K. and Hasunuma, K. (1994) FEBS Lett. 3459 162-166.
Molecular analysis of light signal transduction regulating NTP- binding, ADP-ribosylation and phosphorylation of proteins in Neurospora crassa.
K. Hasunuma, Y Ogura, K. Oda and N. Yabe, Yokohama City University, Kihara Institute for Biological Research, Totsukaku, Yokohama, 244 Japan.
We have established an in vitro system to analyze molecular mechanism of light signal
transduction by use of the crude membrane and the soluble fractions of mycelia of Neurospora
crassa. The irradiation of the crude membrane fraction containing 10-7M[alpha-32P]ATP or
[alpha-32P]GTP by UV-A (370 nm) stimulated the increase in the binding of ATP and /or GTP to
proteins with molecular masses of 53 kDa, 77 kDa, 83 kDa and 129 kDa(l). Blue light (420 nm)
irradiation of the soluble fractions in the presence of 10-7M[32P]NAD stimulated the
ADP-ribosylation of 38 kDa and 56 kDa proteins. A blind mutant, bd lis-3 showed reduced light
response in the ADP-ribosylation of the 56 kDa protein(2). Blue light (420 nm) irradiation of
the membrane fraction in the presence of 10- 8M[gamma-32P]ATP stimulated the increase in the
phosphorylation of 15 kDa proteins (psl5)(3). We have isolated a null type mutant with no
phosphorylation of psl5, designated to be delta-ps15-1, which was mapped on LGVR, 22% distal
to al-3 and 24 % proximal to his-6. The delta-ps15-1 showed abnormal light response to the
morphogenesis of perithecia. In darkness wild type formed perithecial beak at random places on
the perithecia. However, under the light illumination parallel to the solid medium wild type
formed the beak at the top of the perithecia. Such a light induced ordering of the positioning of
perithecial beak was designated as "polarity of perithecia". The delta-ps15-1 completely lacked
such a light induced polarity of perithecia (submitted).
(1) Oda, K. and Hasunuma, K. (1 993) Cytologia 5 8, 231-240.
(2) Oda, K. and Hasunuma, K. (1994) FEBS Lett. 345, 162-166.
(3) Mizoguchi, K. et al. (1 993) J. Photochem. Photobiol., in press.
24. Is there a link between photolyases and blue light reception in fungi?
Gloria Berrocal-Tito, Liat Sametz-Baron*, Benjamin Horwitz* and Alfredo Herrera-Estrella. Centro de Investigacion y Estudios Avanzados del I.P.N, Irapuato, Gto. Mexico.*Technology Institute of Israel ,Technion, Haifa , Israel.
Trichoderma harzianum is a common filamentous fungus which lives in the soil as a
mycoparasite or saprophyte. As a mycoparasite, Trichoderma has potential to control soil-borne
fungal diseases. Furthermore, a brief pulse of light induces sporulation, making the organism a
convenient model for the study of morphogenesis. There is evidence that some blue light
receptors in higher plants share homology with DNA photolyases, an enzyme that uses visible
light to repair one of the major forms of UV-damage to DNA. We used action spectroscopy to
study whether photoreactivation of UV-damaged spores, and the induction of sporulation, could
share the same photoreceptor. The action spectra of sporulation and photoreactivation turned out
to be very different and provided evidence that the Trichoderma enzyme belongs to class I
photolyases. In parallel with the photobiological studies, we isolated and characterized the
complete DNA photolyase gene of T. harzianum . The sequence of the gene indicates that it
belongs to class I photolyases, confirming the photobiological approach. The expression of the
corresponding mRNA during light induced sporulation and germination was analyzed The
photolyase mRNA is detectable in inmature spores and it is abundant in mature spores. The
message is also detectable in germinating spores until germ-tube emergence. These data suggest a
role of the enzyme in repairing DNA-UV damage in spores and germlings.
25. Circadian clock mutants of Neurospora crassa.
Michael Collett, Julia Doster, Sue Crosswaite, Jay Dunlap and Jennifer Loros. Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755.
The frequency (frq) gene has been shown to be a central component of the Neurospora circadian clock. frq mRNA and FRQ protein are components of a negative feedback loop which comprises the circadian clock in Neurospora (Loros, Seminars in Neuroscience 7: 3-13, 1995). frq expression cycles with a period identical to that of the Neurospora circadian cycle. In addition frq expression is rapidly induced by light. Induction of frq by light resets the clock in a time of day dependent manner, either advancing or delaying the clock towards circadian dawn or dusk (Crosthwaite et al., Cell 81: 1003-12, 1995). Continuous light suppresses clock function, due to induction of frq and FRQ to high levels suppressing frq cycling.
The lis (light insensitive) mutants express the circadian rhythm under continuous dim light, a condition when the function of the clock is normally repressed in Neurospora. frq mRNA in the lis mutants is indeed induced to high levels by light, as in wild type, but still cycles with the appropriate period under these conditions. Thus it may be that the circadian oscillator is altered in some manner in these mutants such that high levels of frq no longer suppresses clock function, although the clock is still reset when first exposed to light, alternatively, perception of light levels may be altered.
In addition we are using insertional mutagenesis to identify other genes involved in circadian
rhythmicity. By screening for mutants with an altered conidiation rhvthm or altered responses to
environmental stimuli we hope to identify mutations which alter clock input, output, or the central
oscillator itself, and thus further our molecular dissection of the circadian clock in Neurospora.
26. Photomorphogenesis mutants of Phycomyces.
Rafael Flore Luis M. Corrochano, and Enrique Cerda-Olmedo Departmento de Genetica, Universidad de Sevilla, Sevilla, Spain.
The sporangiophores of the zygomycete Phycomyces blakesleeanus grow out into the air lifting a sporangium filled with spores. Phycomyces develops two types of sporangiophores: Macrophores are giant sporangiophores that grow several cm long guided by many stimuli. The microphores are dwarf sporangiophores, about 1 mm long. At a certain stage in its development, the mycelium becomes photosensitive: blue light governs the production of the yellow pigment, -carotene (photocarotenogenesis), and the development of the sporangiophores (photomorphogenesis). Illumination increases the production of macrophores and decreases the production of microphores.
The mad mutants were isolated because of the defective phototropism of their macrophores. There are ten unlinked mad genes. Mutations in genes madA and madB are defective not only in phototropism, but in the mycelial responses, photocarotenogenesis and photomorphogenesis.
The car mutants suffer various structural and regulatory alterations in the biosynthesis of -carotene. The mutants have revealed that -carotene is not required for photocarotenogenesis or phototropism, but is essential for photomorphogenesis.
We have designed an experimental procedure to isolate mutants affected in
photomorphogenesis. We have isolated three such mutants and characterized their
photomorphogenesis, photocarotenogenesis, and phototropism. The threshold for
photomorphogenesis is 10 to 1000 times higher in the mutants, but their phototropic response is
unaffected. In addition, one of the mutants is also affected in photocarotenogenesis. Thus, the
genes that we have identified by mutation are specific for mycelial photoresponses and presumably
encode new proteins involved in the phototransduction chain. None of the mutants resemble the
phenotype of previously isolated mad mutants which suggests that our search is far from
complete.
27. Mutants in light regulation of fruitbody development in C. cinereus.
J. Granado, Y. Liu, M. Aebi, U. Kues. Institut fur Mikrobiologie, ETH Zurich, Switzerland.
During the life cycle of Coprinus cinereus, two compatible monokaryons with different mating types fuse to form a dikaryon in order to accomplish fruitbody development and sexual reproduction. Compatibility is controlled by the two mating type loci, A and B. Specific mutations in A and B can transform a monokaryon into a so called AmutBmut homokaryon that produces fruitbodies like dikaryons. Fruitbody development in C. cinereus is controlled by temperature, nutrition and, at two different stages (initiation and fruitbody maturation), by light signals.
In our current project, we use an AmutBmut homokaryon to study regulation of fruitbody
development in a homogenetic background. We submitted the strain to a REMI and UV
mutagenesis. Fruiting of isolated mutants was tested on yeast extract-malt-extract-glucose
(YMG) agar supplemented with tryptophan. Mutants were grouped according to their defects in
fruitbody development (primordia initiation, primordia maturation, fruitbody maturation, spore
formation). 35 out of 179 REMI mutants and 20 out of 114 UV mutants unable to initiate
fruitbody development on YMG-trp do also not fruit on horse dung, the natural substrate of C.
cinereus. Among these, we hope to find mutants with defects in the light pathway. One special
class of mutants with an unusual light response form in the light so called etiolated stipes, extra
long stipes with undeveloped caps. This phenotype normally occurs if a wild type strain is left in
the dark after primordia induction. We describe one REMI mutant of this class in more detail.
Interestingly, the defect in light response in this mutant can be reversed by changing the polarity
of gravity (turning fully developed cultures by 180 degrees).
28. Blue light overrides mating type repression of asexual sporulation in Coprinus cinereus.
K. Kertesz-Chaloupkov, U. Kues, P. Walser, M. Aebi. Institut fur Mikrobiologie, ETH Zurich, Switzerland.
The basidiomycete Coprinus cinereus undergoes a life cycle with transitions between two types of mycelia. The monokaryotic mycelium constitutively produces abundant vegetative spores (oidia) on aerial hyphae. A dikaryotic mycelium arises after fusion of two compatible monokaryons. Oidiation is repressed in dikaryons. Upon light induction, fruitbodies are formed in which karyogamy and meiosis occur. In addition, light can induce the formation of haploid, monokaryotic spores on a dikaryon.
Developmental processes in C. cinereus are controlled by two distinct mating type loci, A
and B. To form a dikaryon two nuclei with different A and B specificities have to be present in the
same cell. Specific activating mutations in both mating type loci lead from a monokaryon to a
homokaryon that forms fruitbodies like a dikaryon but also produce oidia in amounts comparable
to monokaryons. However, in contrast to monokaryons, oidiation in AmutBmut is repressed in the
dark and is induced by light. Extensive studies on Amut homokaryons, Bmut homokaryons, "A
on" transformants of monokaryons and dikaryons show that the A mating type loci is responsible
for repression of oidiation in the dark. Light overrides this A mediated repression and the B
mating-type locus seems to modulate this light effect. As in fruiting, it is blue light that induces
oidiation.
29. Light/dark cycles and the control of meiosis in Coprinus cinereus.
B.C. Lu University of Guelph, Guelph, Canada.
In the synchronous meiotic system of Coprinus cinereus, meiosis is controlled at meiotic S and at metaphase I, and light intensity and light/dark cycles play an important role. The time of exposure to light requried to enter meiotic S is inversely proportional to the exponent of light intensities. The shift-up experiments showed that the critical light dependent period is 10h before the initiation of meiotic S-phase.
The control of entry to meiotic metaphase is also light intensity and strain dependent. The London
strain is light/dark blind while the Java and Japanese strains are dark dependent under moderate to
high light intensities; a 3 h dark period is essential for proper progression to metaphase I and its
effect is very stage specific. It is quite possible that the dark period signals the cellular processes
leading to metaphase events. Cytological studies showed that chromosomes are normal in meiotic
prophase but are unable to undergo condensation under arrest conditions. Genetic crosses and
backcrosses showed that light blindness is dominant and appeared to be a single gene inheritance.
30. Light-dependent conidiation in Aspergillus nidulans is influenced by fluG and veA gene expression.
Lawrence N. Yager, Hyung-Ok Lee, and Patricia A. Kennedy, Temple University, Department of Biology, Philadelphia, PA 19122.
Conidiation in Aspergillus nidulans is induced by exposure to red light. Using a strain that contains a blue light gain-of-function mutation, we have isolated a mutant, designated fluG701, that fails to conidiate in red light. The fluG gene has been shown by Lee and Adams (1994, Genes Dev. 8, 641-651) to be necessary for the synthesis of a small diffusible factor that is required for the initiation of asexual development. Although conidiation in a null fluG strain can be rescued by growth next to a wild-type colony, conidiation in the fluG701 strain cannot be rescued by the extracellular factor. Moreover, the loss of transcriptional negative autoregulation observed with other fluG mutant alleles is not observed in a fluG701 strain. The fluG701 mutation has been shown to be a G to A transition that replaces glutamic acid at amino acid position 467 with lysine.
The veA1 mutation allows conidiation to occur in the absence of light. The veA gene has been cloned, but the predicted ORF shows no homology to other known sequences. We have isolated veA1 suppressors, one class of which are alleles of fluG. These suppressors show varying degrees of rescue by the extracellular factor and do not show transcriptional negative autoregulation. The nucleotide identity of these suppressors has been determined and suggests, first that the amount of available fluG product influences light-dependent conidiation and second, that light-dependent functions are located in the C terminal portion of the fluG protein. These data indicate that fluG and veA have a genetic interaction. Although no evidence for a physical interaction has been demonstrated, one potential model is that veA and fluG operate together to interpret the light signal required for the production of the extracellular factor and initiation of conidiation.