Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes-more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca(2+) signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes
Although often considered “minimal” organisms, mycoplasmas show a wide range of diversity with respect to host environment, phenotypic traits, and pathogenicity. Here we report the complete genomic sequence and proteogenomic map for the piscine mycoplasma Mycoplasma mobile, noted for its robust gliding motility. For the first time, proteomic data are used in the primary annotation of a new genome, providing validation of expression for many of the predicted proteins. Several novel features were discovered including a long repeating unit of DNA of ∼2435 bp present in five complete copies that are shown to code for nearly identical yet uniquely expressed proteins. M. mobile has among the lowest DNA GC contents (24.9%) and most reduced set of tRNAs of any organism yet reported (28). Numerous instances of tandem duplication as well as lateral gene transfer are evident in the genome. The multiple available complete genome sequences for other motile and immotile mycoplasmas enabled us to use comparative genomic and phylogenetic methods to suggest several candidate genes that might be involved in motility. The results of these analyses leave open the possibility that gliding motility might have arisen independently more than once in the mycoplasma lineage
Plasminogen (Plgn) is usually activated by proteolytic cleavage of Arg561-Val562. The new N-terminal amino group of Val562 forms a salt bridge with Asp740, creating the active protease plasmin (Pm). However, streptokinase (SK) binds to Plgn, generating an active protease in a poorly understood, nonproteolytic process. We hypothesized that the N-terminus of SK, Ile1, substitutes for the N-terminal Val562 of Pm, forming an analogous salt bridge with Asp740. SK initially forms an inactive complex with Plgn, which subsequently rearranges to create an active complex; this rearrangement is rate limiting at 4 degrees C. SK.Plgn efficiently hydrolyzes amide substrates at 4 degrees C, although DeltaIle1-SK. Plgn has no amidolytic activity. DeltaIle1-SK prevents formation of wild-type SK.Plgn. These results indicate that DeltaIle1-SK forms the initial inactive complex with plasminogen, but cannot form the active complex. However, when the experiment is performed at 37 degrees C, amidolytic activity is observed when DeltaIle1-SK is added to plasminogen. SDS-PAGE analysis demonstrates that the amidolytic activity results from the formation of DeltaIle1-SK.Pm. To further demonstrate that the activity of DeltaIle1-SK requires the conversion of Plgn to Pm, we characterized the reaction of SK with a mutant microplasminogen, Arg561Ala-microPlgn, that cannot be converted to microplasmin. Amidolytic activity is observed when Arg561Ala-microPlgn is incubated with wild-type SK at 37 degrees C; however, no amidolytic activity is observed in the presence of DeltaIle1-SK. These observations demonstrate that the amidolytic activity of DeltaIle1-SK at 37 degrees C requires the conversion of Plgn to Pm. Our findings indicate that Ile1 of SK is required for the nonproteolytic activation of Plgn by SK and are consistent with the hypothesis that Ile1 of SK substitutes for Val562 of Pm.
Plasminogen (Pg) activators such as streptokinase (SK) save lives by generating plasmin to dissolve blood clots. Some believe that the unique ability of SK to activate Pg in the absence of fibrin limits its therapeutic utility. We have found that SK contains an unusual NH 2 -terminal ''catalytic switch'' that allows Pg activation through both fibrin-independent and fibrin-dependent mechanisms. Unlike SK, a mutant (rSK⌬59) fusion protein lacking the 59 NH 2 -terminal residues was no longer capable of fibrinindependent Pg activation (k cat ͞K m decreased by >600-fold). This activity was restored by coincubation with equimolar amounts of the NH 2 -terminal peptide rSK1-59. Deletion of the NH 2 terminus made rSK⌬59 a Pg activator that requires fibrin, but not fibrinogen, for efficient catalytic function. The fibrin-dependence of the rSK⌬59 activator complex apparently resulted from selective catalytic processing of fibrinbound Pg substrates in preference to other Pg forms. Consistent with these observations, the presence (rSK) or absence (rSK⌬59) of the SK NH 2 -terminal peptide markedly altered fibrinolysis of human clots suspended in plasma. Like native SK, rSK produced incomplete clot lysis and complete destruction of plasma fibrinogen; in contrast, rSK⌬59 produced total clot lysis and minimal fibrinogen degradation. These studies indicate that structural elements in the NH 2 terminus are responsible for SK's unique mechanism of fibrin-independent Pg activation. Because deletion of the NH 2 terminus alters SK's mechanism of action and targets Pg activation to fibrin, there is the potential to improve SK's therapeutic efficacy.
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