In vitro reconstruction of the Aspergillus (= Emericella ) nidulans  genome

Abstract

A physical map of the 31-megabase Aspergillus nidulans genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA͞DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the A. nidulans genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.The ascomycetous fungus Aspergillus nidulans is one of the simplest multicellular eukaryotes. Its estimated 31-megabase genome (1) with low repetitive DNA content (2), distributed among eight chromosomes, is sufficiently complex to direct multicellular development (3, 4). With its sophisticated forward and reverse genetics (5, 6), A. nidulans has been used to address fundamental questions in cell and molecular biology (7-10) and has become a model system for the study of pathogenic and biotechnologically useful relatives (11, 12). One of the major questions in genomics is the organization of repeated DNA sequences in a genome because this organization has implications for functional organization of genomes, development, chromosome structure and function, recombination, and genome evolution. Small eukaryotic genomes provide excellent models for the development of novel mapping technologies for larger genomes. For example, the A. nidulans genome is entirely covered (tiled) by 775 nonoverlapping cosmids (averaging Ϸ40-kb inserts) and the human genome, with 3,000 yeast artificial chromosomes [averaging Ϸ1,001-kb inserts, (13)]. Here we present a physical mapping strategy that produces a portrait of how repeated DNA sequences are distributed in small eukaryotic genomes. MATERIALS AND METHODSLibraries. Two independent libraries were constructed in the Lambda-based cosmid vector pLORIST2 and the pBRbased cosmid vector pWE15 (1).Physical Mapping Strategy. For the physical mapping experiment, pulsed field gel electrophoresis isolated chromosomes were radiolabeled and used, one by one, to probe the two genomic libraries (5,134 clones stamped onto seven highdensity gridded 5 ϫ 3.5-inch nylon membranes containing 864 clones each) to show the chromosomal affiliations of each clone (1). With this information, clones were sorted into subsets (S for specific to one chromosome, R for repeated hybridization to several, but not all, chromosomes, and O for octochromosomal). Octochromosomal (O) clones hybridized to all eight A. nidulans chromosomes. The numbers of S, R, and O clones in the genomic library were 3,307 (64.4%), 1,769 (34.4%), and 58 (1.1%), respectively. Then, only S clones were radiolabeled and used to probe the genomic library of 5,134 clones (on seven identical membranes)

Terbinafine Resistance Mediated by Salicylate 1-Monooxygenase inAspergillus nidulans

“…The physical location of the salA DNA fragment was verified by three means: (i) by DNA-DNA hybridization to pulsed-field-gel-electrophoresis-separated chromosomes, (ii) hybridization to a single chromosome VI cosmid of the A. nidulans physical map (28), and (iii) the determination that salA is on chromosome VI of the fully sequenced genome A. nidulans database at the Whitehead Institute (Cambridge, Mass.). Thus, we hypothesize that an apparent multicopy effect of the salA gene observed by Southern analysis of the pD02-salA transformant strains (Fig.…”

Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans The GenBank accession number for the sequence reported in this paper is AF043230.

“…Using chromosome-specific libraries (Brody et al, 1991), three positive cosmids, L09H05, L14G06 and L24E04, were identified. These cosmids have been assigned to the same region of A. nidulans chromosome V (Prade et al, 1997), suggesting that the tpsA gene is located on this chromosome. Bell et al, 1992), K. lactis (klggs1 ; Luyten et al, 1993), Schiz.…”

Genome sequencing and analysis of Aspergillus oryzae

“…The mosaic structure of the genome, considered to be evidence for horizontal gene transfer 17 , was found by synteny analysis of the A. oryzae genome and was further characterized by the localization of the EST expression (see above) of non-metabolic genes (P ¼ 1.78 £ 10 295 and 1.32 £ 10 251 for information and storage (J to B) and cellular function and signalling (D to O), respectively) and the genes of high codon adaptation index (top 5% genes, P ¼ 9.8 £ 10 228 ). The phylogenetic distance between the genes in the orthologous cluster and the A. oryzae-specific ones was similar to that between the genes of Aspergillus and the other genera belonging to Sordariomycetes.…”