Doris M. Kupfer

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus)

The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 108 synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor, a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.

Introns and splicing elements of five diverse fungi.

ABSTRACT Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements. An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5′GU…AG3′), and that they have polypyrimidine tracts predominantly in the region between the 5′ ss and the branch point. Information content is high in the 5′ ss, branch site, and 3′ ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined. The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi. Generally, as intron length increases in the fungi, so does intron information content. Homologs of U2AF spliceosomal proteins were found in all species except for S. cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns. Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point. Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found. There appear to be sufficient differences between S. cerevisiae and S. pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571

BackgroundBiological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem.ResultsThe 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor.ConclusionThe genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

Allelic variants of ovine prion protein gene (PRNP) in Oklahoma sheep.

1,144 sheep belonging to 21 breeds and known crosses were sequence analyzed for polymorphisms in the ovine PRNP gene. Genotype and allele frequencies of polymorphisms in PRNP known to confer resistance to scrapie, a fatal neurodegenerative disease of sheep, are reported. Known polymorphisms at codons 136 (A/V), 154 (H/R) and 171 (Q/R/H/K) were identified. The frequency of the171R allele known to confer resistance to type C scrapie was 53.8% and the frequency of the 136A allele known to influence the resistance to type A scrapie was 96.01%. In addition, we report the identification of five new polymorphisms at codons 143 (H/R), 167 (R/S), 180 (H/Y), 195 (T/S) and 196 (T/S). We also report the identification of a novel allele (S/R) at codon 138.

Transcriptome analysis of the salivary glands of the female tick Amblyomma americanum (Acari: Ixodidae).

Ticks infest a wide range of hosts while bypassing their immune, inflammatory and haemostatic responses during their extended feeding, which may last for more than two weeks. Here, we present a transcriptome analysis of 3868 expressed sequence tags (ESTs) from three cDNA libraries generated from the salivary glands of adult female Ambyomma americanum ticks at different stages of feeding. We applied a normalization step for one library, significantly decreasing the abundance of mitochondrial sequences amongst the 2292 sequences from the normalized library. Our ESTs include homologues that may modulate haemostatic, immune and inflammatory responses of the hosts. Other ESTs probably represent important components of the highly efficient secretory pathways for salivary proteins and concomitantly transmitted pathogens.

EST library sequencing of genes expressed during early limb regeneration in the fiddler crab and transcriptional responses to ecdysteroid exposure in limb bud explants.

We have constructed directional and randomly primed cDNA libraries from mRNAs isolated during progressive stages of fiddler crab (Uca pugilator) limb regeneration. Data from these libraries are being assembled into an on-line database (http://www.genome.ou.edu/crab.html) that is both BLAST and keyword searchable; the data set is also available through GenBank. The first characterized library was made from mRNA isolated 4 days post-autotomy, when the first sign of morphological differentiation, cuticle secretion, is observed. Analysis of 1698 cDNA clones led to assignment of 473 contigs and 417 singlets, for a total of 890 sequences. Of these, ∼86% showed no assignments to characterized genes on database searching, while 14% could be assigned to a known ortholog in the COG (Clusters of Orthologous Groups) database. BLAST searches to specific protein domains in the Gene Ontology database led to assignments for ∼40% of the assembled sequences. Sequence similarity searches of other crustacean EST databases produced hits to 13-30% of the Uca query sequences. The ESTs include several genes that may be potentially ecdysteroid-responsive, such as homologs to chaperone proteins and cuticle protein genes, as well as homologs to arthropod proteins involved in retinoid/terpenoid metabolism. We have tested 3 potential candidate genes for their ability to be induced by ecdysteroid in limb bud explants; an arthropodial cuticle protein gene, and the nuclear receptor genes EcR and RXR. A subset of early blastemal limb buds (8 days post autotomy) show a positive response to ecdysteroid by 1-1.5 h, followed by a decrease in transcript abundance at longer periods of sustained incubation. Later stage buds (12 days post autotomy-late premolt) show decreases in steady-state mRNA levels by 1.5 h, or are completely refractory to ecdysteroid exposure.

PCR-Based Identification of Postmortem Microbial Contaminants—A Preliminary Study

Investigation of postmortem blood can reveal the presence of significant ethanol levels. However, in some instances it cannot easily be determined if the source of ethanol is from ingestion or from postmortem endogenous fermentation by contaminating microbes. Described here is a robust polymerase chain reaction (PCR)-based method for detecting the presence of common ethanol producing microbial contaminants in human blood. A set of DNA primers were designed for use in PCR to amplify and detect the genomic DNA from humans and three test microorganisms Escherichia coli, Proteus vulgaris, and Candida albicans. A rapid and reproducible protocol was developed for isolating genomic DNA from mixed human blood-microorganism samples that yields a suitable template for PCR. The organism-specific primer pairs can detect the presence of the target microorganisms in human blood at concentrations as low as 10 colony forming units/mL. The PCR products readily can be detected after agarose gel electrophoresis. This method provides an additional means of rapidly identifying microbial contaminants in postmortem blood samples.