42. Aspergillus nidulans maintains short telomeres throughout development.
Anamitra Bhattacharyya and Elizabeth H. Blackburn Department of Microbiology and ImmunologyUniversity of California, San Francisco, San Francisco, CA 94143-0414.
We have identified and cloned the telomeres of the filamentous fungus, Aspergillus nidulans. We have demonstrated that the telomeric repeat sequence is TTAGGG, identical to that found in vertebrates, including humans, and some lower eukaryotes. Three classes of cloned chromosomal ends were identified based on the telomere-associated sequences (TASs). One category of telomere clones was found to contain internal, variant TAAGGG repeats. The A. nidulans telomeric tract length is strikingly short, based on analysis of cloned ends (4 to 22 repeats) and from Bal3l digestion of native telomeres. We have also found that telomere length is remarkably stable in different cell-types and at altered growth temperatures, suggesting a highly regulated mechanism for length control.
43. Cloning, sequencing and structural analysis of nsdD gene that controls sexual development of Aspergillus nidulans.
Kap-Hoon Han, Kyu-Yong Han, Suhn-Kee Chae1 and Dong Min Han. Department of Molecular Biology, Wonkwang University, Iksan, South Korea and 1Department of Biochemistry, Paichai University, Tae-Jeon, South Korea.
Several mutants which never undergo sexual development (nsd) of Aspergillus nidulans
have been isolated and characterized. Among them, a genomic DNA fragment (pNSD19) which
complemented the nsdD19 mutation was isolated from nsdD19+ transformants by in vitro
packaging after transforming with a genomic DNA library. When nsdD- hosts were retransformed
with pNSD19, four different kinds of phenotypes relating to the production of sexual organs were
observed. Southern blot hybridization revealed that phenotypic variation was due to the
differences in the copy number of the transformed nsdD gene. The nsdD gene has an ORF of
1,383bp, interrupted by three introns, encoding a protein of 461 amino acids. A putative 'zinc
finger' DNA binding motif was observed in NsdD protein which was also found in GATA factor
and AreA. The results suggested that the nsdD gene may play important roles in regulation of
sexual development of A. nidulans.
44. Recurrent selection for fertility in Nectria haematococca MPI.
Brian T. Hawthorne, HortResearch, Auckland, New Zealand.
Selection for increased numbers of fertile perithecia in Nectria haematococca MPI was carried out for six generations of intercrossing among 18 to 24 parents (9-12 of each of the two mating types, MAT1-1 and MAT1-2) in each generation. Perithecia producing spore horns four weeks after spermatisation were scored as fertile. The parents were selected from progeny of the three most fertile crosses from the preceding generation. After six generations the mean numbers of fertile perithecia for the crosses within a generation had increased nearly threefold. Within a generation the difference between the least and most fertile crosses, as a fraction of the most fertile cross, reduced from 500% in generation 0 to 60% in generation 6. Variation in fertility of the reciprocal crosses, MAT1-1 x MAT1-2 and MAT1-2 x MAT1-1, which varied several-fold in early generations was reduced to virtually nil in generation six. The proportion of additive genetic variance decreased three- to four-fold between generation 0 and generation 2 and remained relatively constant in the subsequent generations. Confirmation of the increased fertility was obtained by comparing the most fertile cross from each of the generations under common conditions.
The evidence from this study confirmed that there are genes additional to sex and mating type
which influence both sexual compatibility and fertility in Nectria haematococca MPI.
45. Asexual sporulation in dikaryons and a dikaryon-like AmutBmut homokaryon of Coprinus cinereus.
Eline Polak1, Ursula Kues1, M. Hollenstein1, Rene Hermann1,2 and Markus Aebi1. Institute of Microbiology1 and
Laboratory for Electron Microscopy I2, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland.
Monokaryons of Coprinus cinereus constitutively produce abundant vegetative spores (oidia) on specialised aerial structures, so called oidiophores. Although oidia formation in dikaryons is strongly repressed, a low amount of oidia is still produced, often upon light induction. Most oidia produced on dikaryons are haploid and uninucleate, and the ratio of the two possible oidia types is dependent upon the genotype of the dikaryon. Formation of the monokaryotic oidiophore can occur on a dikaryotic hypha suggesting a light-induced, regulated discrimination of the two nuclei in the initial steps of oidiophore development.
This light-induced oidiophore development was studied in the homokaryon AmutBmut.
Unlike monokaryons and similar to dikaryons, oidia production in such AmutBmut homokaryons
is repressed in the dark. However, upon light induction an AmutBmut homokaryon produces oidia
in amounts comparable to monokaryons. Taking advantage of this phenomenon, oidia and
oidiophore formation in an AmutBmut homokaryon is described morphologically and cytologically
by light and electron microscopy. A model defining the different steps in this process will be
presented.
46. Mating-type associated incompatibility in Neurospora crassa.
Patrick Ka Tai Shiu, University of British Columbia, Vancouver, BC, Canada.
In Neurospora crassa, mating is controlled by a single locus with two mating types, A and a. This mating type locus has an additional unique function during vegetative growth known as heterokaryon incompatibility: if hyphae of opposite mating types fuse during vegetative stage, the resulting heterokaryotic cells are inhibited in their growth. An unlinked mutation tol suppresses the heterokaryon incompatibility but does not affect sexual function.
Sequencing of the two mating type alleles, A and a, showed that the two alleles are totally dissimilar sequence of 5301 and 3235 base pairs respectively. The term "idiomorph" is coined to describe these dissimilar genes occupying homologous positions on a chromosome. A single open reading frame (ORF) from each of mating-type idiomorph, mt A-1 and mt a-1, confers both mating function and heterokaryon incompatibility. Both MT A-1 and MT a-1 contain a conserved DNA-binding motif, indicating that they could be transcriptional activators.
In this study, it was found that a putative LRR (leucine-rich repeat) in mt A-1 is essential to
the incompatibility function. tol was cloned by chromosome walking. It is the first suppressor of
many incompatibility loci found in different fungi to be cloned. It encodes a 922-aa protein and
contains a putative LRR and an amphipathic -helix. Further experiments will be done to
characterize tol (including expression pattern, localization, and functional domains) and to study
the mechanism of interactions among tol and the mating-type genes (using two hybrid system).
47. Analysis of A. nidulans genes involved in meiosis: uvsC, a homologue of yeast RAD51.
Diana van Heemst, Klaas Swart, Christa Heyting, Henk W.J. van den Broek and Theo Goosen
Dept. of Genetics, Agricultural University Wageningen, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
We cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted UVSC protein shows 67.4% sequence identity to S. cerevisiae Rad51 and 27.4% sequence identity to E. coli RecA proteins. Transcription of uvsC is induced by MMS, like transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequences of the uvsC114 allele have a deletion of six base pairs, which results in a deletion of two amino acids and a replacement of one amino acid in the translation product. In order to gain more insight in the biological function of the uvsC gene, a uvsC null mutant was constructed by replacing the complete uvsC coding sequences for a selectable marker gene. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. Meiotically, the uvsC114 mutant arrested in prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans protein homologous to Rad51 of yeast has a role both in the specialised processes preceding meiosis and in meiotic prophase-I.
In order to isolate more genes involved in meiosis by complementation, we started a mutant screen, which so far has resulted in a few dozen specific meiosis mutants. Part of those have now been characterised by complementation and cytology. Of one mutant, blocked in karyogamy (karA), the corresponding gene has been isolated.