Freshwater ascomycetes from southern Australia: Melanascomaceae fam. nov., Melanascoma panesporagen. et. sp. nov., and Pleurotheciumbruniussp. nov.

During a survey of freshwater fungi in temperate southern Australia, two new taxa were found, Melanascoma panespora and Pleurothecium brunius. Morphological and molecular data place Melanascoma panespora in the Diaporthomycetidae representing a new genus. Melanascoma, along with Proliferophorum and Paraproliferophorum, form a new lineage and the family Melanascomaceae is introduced. Phylogenetic analyses using ITS, 28S, and 18S nrRNA gene sequences,, along with morphological examination revealed Pleurothecium brunius to be a new species of Pleurothecium, sister to P. aquaticum. Citation: Fryar SC, Catcheside DEA (2023). Freshwater ascomycetes from southern Australia: Melanascomaceae fam. nov., Melanascoma panespora gen. et. sp. nov., and Pleurothecium brunius sp. nov. Fungal Systematics and Evolution 11: 85-93. doi: 10.3114/fuse.2023.11.07.

Delineation of the IGF-II C domain elements involved in binding and activation of the IR-A, IR-B and IGF-IR.

OBJECTIVE: Human insulin-like growth factor-I and -II (IGF-I and -II) ligands share a high degree of sequence and structural homology. Despite their similarities, IGF-I and IGF-II exhibit differential receptor binding and activation characteristics. The C domains of IGF-I and IGF-II are the primary determinants of binding specificity to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11- (IR-A) and exon 11+ (IR-B) isoforms. DESIGN: Three IGF-II analogues were generated in order to delineate the C domain residues that confer the differential receptor binding affinity and activation properties of the IGFs. Chimeric IGF-II analogues IGF-IICI(N) and IGF-IICI(C) contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively. RESULTS: The IGF-IICI(N) analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with a relatively lower affinity than IGF-II, suggesting a negative interaction between the exon-11 encoded peptide in the IR-B and the C-domain. The ability of IGF-IICI(N) to activate receptors and elicit cell viability responses was generally proportional to its relative receptor binding affinity but appeared to act as a partial agonist equivalent to IGF-I when binding and activating the IGF-IR. In contrast, IGF-IICI(C) bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICI(S) contained a truncated IGF-I C domain (GSSSRRAT) and generally displayed a relatively poor ability to bind, activate and elicit viability responses via each receptor. CONCLUSIONS: Together, the IGF analogues demonstrate that both flanks of the IGF-II C domain play important roles in the greater ability of IGF-II to bind and activate IR receptors than IGF-I.

Residual recombination in Neurospora crassa spo11 deletion homozygotes occurs during meiosis.

Spo11 is considered responsible for initiation of meiotic recombination in higher organisms, but previous analysis using spo11 (RIP) mutants suggests that the his-3 region of Neurospora crassa experiences spo11-independent recombination. However, despite possessing several stop codons, it is conceivable that the mutants are not completely null. Also, since lack of spo11 interferes with chromosomal pairing and proper segregation at Meiosis I, spores can be partially diploid for a period after meiosis. Thus, it is possible that the recombination observed could be an abnormal event, occurring during the period of aneuploidy rather than during meiosis. To test the former hypothesis, we generated spo11 deletion homozygotes. Using crosses heteroallelic for his-3 mutations, we showed that His(+) progeny are generated in spo11 deletion homozygotes at a frequency at least as high as in wild type and, as in the spo11 (RIP) mutants, local crossing over is not reduced. To test the latter hypothesis, we utilised mutations in either end of a histone H1-GFP fusion gene, inserted between the recombination hotspot cog and his-3, in which GFP(+) spores arise as a result of recombination in a cross between the two GFP alleles. In a control cross homozygous for spo11 (+), the frequency at which GFP(+) spores arise is comparable to the frequency of His(+) spores and glowing nuclei first appear during prophase, prior to metaphase I, as expected for a product of meiotic recombination. Similarly in spo11 deletion homozygotes, GFP(+) spores arise at high frequency and glowing nuclei are first seen before metaphase, indicating that allelic recombination occurs during meiosis in the absence of spo11. We have therefore shown that spo11 is not essential for either his-3 allelic recombination or crossing over in the vicinity of his-3, and that spo11-independent allelic recombination is meiotic, indicating that there is a spo11-independent mechanism for initiation of recombination in Neurospora.

A crossover hotspot near his-3 in Neurospora crassa is a preferential recombination termination site.

During analysis of 148 unselected Neurospora crassa octads, an above average rate of crossing over was detected within a 360-base region near the 3' end of his-3, suggesting a hotspot for crossing over about 1.8 kb away from the recombination initiation site within cog. Homozygous deletion of the 360-base region increases exchanges in his-3 and on the far side of his-3 from cog, with the heterozygote showing an intermediate increase. We conclude that recombination events initiated at cog terminate within the 360-base sequence more often than in other sections of the cog-his-3 interval and, since some of these terminations will be resolved as crossovers, a cluster of crossovers at this location is the outcome. Removal of this termination site increases the chance that an event will reach his-3, resulting in recombination within the gene, or extend past it to yield a crossover on the other side of his-3. The deleted sequence has substantial predicted secondary structure, including a complex predicted stem-loop, suggesting that DNA secondary structure may be responsible for the termination.

High density analysis of randomly selected Neurospora octads reveals conversion associated with crossovers located between cog and his-3.

We analysed 148 octads from a Neurospora cross maximised for sequence heterology in the his-3 region and detected non-Mendelian segregation at his-3, cot-1 and lys-4 loci. This was in all cases 6:2 or 2:6, with no evidence of post-meiotic segregation (PMS) in these genes. High density snp analysis was used to place crossovers between his-3 and the centromere-distal marker ad-3, and sequencing to refine the location of crossovers between his-3 and the recombination hotspot cog. Crossovers appeared to have a non-random distribution, falling close to his-3 or more than 40 kb distal, and all those in which the location was determined were flanked by sequences showing gene conversion and/or PMS amongst the polymorphisms. This octad study confirms the validity of assumptions made during random spore analyses and suggests that recombination hotspots at cot-1 and lys-4 may, unlike the relatively cold recombination initiator at the am locus, be high frequency recombinators similar to cog.

Alleles of the hotspot cog are codominant in effect on recombination in the his-3 region of Neurospora.

There are two naturally occurring functional alleles of the recombination hotspot cog, which is located 3.5 kb from the his-3 locus of Neurospora crassa. The presence of the cog+ allele in a cross significantly increases recombination in the his-3 region compared to a cross homozygous for the cog allele. Data obtained shortly after discovery of cog+ suggested that it was fully dominant to cog. However, a dominant cog+ conflicts with observations of hotspots in Saccharomyces cerevisiae and Schizosaccharomyces pombe, in which recombination is initiated independently of homolog interactions, and suggests recombination mechanisms may differ in Neurospora and yeast. We present evidence that cog alleles are codominant in effect on both allelic recombination in his-3 and crossing over between loci flanking his-3. In addition, we show that genetic background variation has at least a twofold effect on allelic recombination. We speculate that variation in genetic background, together with the complexities of recombination in crosses bearing close mutant alleles, accounts for the previous conclusion that cog+ is dominant to cog.

Guest, a transposable element belonging to the Tc1/mariner superfamily is an ancient invader of Neurospora genomes.

Guest is a transposable element of the Tc1/mariner superfamily with 30-40bp terminal inverted repeats and a TA dinucleotide target site duplication. Guest was originally discovered in the St. Lawrence 74A laboratory strain of the filamentous fungus Neurospora crassa. In this report, Guest iterations subcloned from a cosmid library of the Oakridge 74A strain were used to design PCR primers that permitted the detection of Guest in wild isolates of N. crassa. Guest is present in N. crassa as multiple copies ranging between 100bp and 2.4kb and is present in the mating type locus of several Neurospora species. Bioinformatic analysis of the entire N. crassa genome (Oakridge 74A strain) detected 48 Guest iterations. All iterations appeared to have been inactivated either by repeat-induced point mutation or sequence deletion, with the majority being remnants less than 400bp in length. The possible involvement of Guest in the evolution of the variable region that flanks the mating type idiomorphs in several Neurospora species is discussed.

Diversification of exogenous genes in vivo in Neurospora.

We have adapted the meiotic recombination hotspot cog of Neurospora crassa for shuffling exogenous DNA, providing a means of generating novel genes in situ from sequences introduced into chromosomes. Genes to be diversified are inserted between the his-3 locus and cog. Diversification crosses are heterozygous both for alleles of the exogenous DNA and for auxotrophic alleles of his-3. Progeny selected for ability to grow without histidine supplementation are enriched for exchange events within the exogenous DNA. Exchange events initiated by cog can propagate past DNA sequences mismatched for more than 370 bp and complete exchanges in patches of matched sequence as short as 24 bp, parameters that make the system suited for use in the directed evolution of genes for protein engineering. Here we demonstrate the system by shuffling human immunoglobulin kappa chain genes and also endoglucanase genes derived from different species of fungi.

Recombination at his-3 in Neurospora declines exponentially with distance from the initiator, cog.

By deletion of 1.8 kb of sequence between cog(L) and his-3 and replacement with sequences of different lengths, we have generated a set of Neurospora strains in which the distance between cog(L) and the site at which recombination is selected varies from 1.7 to nearly 6 kb. Each of the manipulated strains includes cog(L), a highly active recombination hotspot, and rec-2, thus allowing high-frequency recombination. In addition, each is a his-3 mutant, either K26 or K480. The frequency of His(+) recombinants in progeny of these crosses is inversely proportional to the distance between his-3 and cog. Specifically, there is a linear relationship between log(10) (recombination frequency) and the distance in base pairs, indicating that as distance decreases, the rate of interallelic recombination increases exponentially. An exponential relationship between distance separating markers and the chance of co-conversion has been found in both Drosophila and fission yeast, indicating that the extension of recombination events may be a stochastic process in most organisms. On the basis of these and additional data presented in this article, we conclude that recombination is initiated at cog(L) in >17% of meioses, that most conversion tracts are very short, and that few extend >14 kb.