PARl' I SYNTHESIS OF L AMINO ACID OXIDASE BY A SERINE OR GLYCINE R~RING STRAIN OF NEIJROOPORA PARl' II STUDIES CONCERNING MUIJrIPLE ELEX TROPHORErIC FORMS OF TYROOINASE IN NEIJROOPORA Thesis by Joyce[.]
One marine bacterial strain, R3, has been newly isolated from the intertidal zone of Dinghai sea area. Measurements of a-keto acids and H 2 O 2 existing in fermentation supernatant were carried out to show that R3 can produce L-aminoacidoxidase (LAAO) with a broad substrate specificity. Physiological and biochemical analysis showed that it can grow great at the conditions with sodium chloride concentration of 1.5% - 3%, temperature of 15˚C - 35˚C and pH of 6 - 7. In addition, molecular identification of 16S rDNA was performed to show that R3 was proximal to Pseudoalteromonas spp. with the highest identity of 98.5% to Pseudoalteromonas rubra. Therefore, it was designated as Pseudoalteromo- nas sp. R3. Further studies are required to arrive at a better understanding of this LAAO and secure an application.
The alternative oxidase transfers electrons from ubiquinol to molecular oxygen, providing a mechanism for bypassing the later steps of the standard cytochrome-mediated electron transport chain. The enzyme is found in an array of organisms and in many cases is known to be produced in response to perturbations of the standard chain. Alternative oxidase is encoded in the nucleus but functions in the inner mitochondrial membrane. This implies the existence of a retrograde regulation pathway for communicating from the mitochondrion to the nucleus to induce alternative oxidase expression. Previous studies on alternative oxidase in fungi and plants have shown that a number of genes are required for expression of the enzyme, but the identity of these genes has remained elusive. By gene rescue we have now shown that the aod-2 and aod-5 genes of Neurosporacrassa encode transcription factors of the zinc-cluster family. Electrophoretic mobility shift assays show that the DNA-binding domains of the AOD2 and AOD5 proteins act in tandem to bind a sequence element in the alternative oxidase gene promoter that is required for expression. Both proteins contain potential PAS domains near their C terminus, which are found primarily in proteins involved in signal transduction.
D ojcinovic et al. 2005; Z arkovic et al. 2005; C lifton et al. 2006; R hoads et al. 2006). In addition, the majority of plant alternative oxidases have two conserved cyste- ine residues in the N terminus of the protein, which are involved in the post-translational regulation of alterna- tive oxidase activity (U mbach et al. 2006). Disulphide bond formation between cysteine residues on adjacent subunits of an alternative oxidase homodimer results in an inactive complex. Conversely, the enzyme is activated if the reduced forms of these cysteine residues interact with a -keto acids. In trypanosomes, the half-life of alternative oxidase mRNA dramatically changes during differentiation, indicating post-transcriptional regula- tion (C haudhuri et al. 2002). Nuclear run-on and transcript analysis studies from Magnaporthe grisea showed that the alternative oxidase gene is consti- tutively transcribed. In noninducing conditions, the transcript is actively degraded by one or more factors that are sensitive to cycloheximide (Y ukioka et al.
Coexpression of Lifeact-TagRFP and ␤ -tubulin–GFP re- vealed distinct but coordinated recruitment of F-actin and mi- crotubules during different stages of cell polarization and tip extension during colony initiation. Previous studies using mi- crotubule-depolymerizing drugs showed that germ tube emer- gence (but not elongation) can be achieved in N. crassa without microtubules (7). Our data showing that polarization of F-actin always preceded polarization of microtubules further rein- forces the notion that germ tube emergence and elongation is a two-step process (9, 21) that first involves F-actin to establish a polarized bud and maintain tip polarity but subsequently requires microtubules for further extension. In contrast, CATs are thinner than germ tubes, show determinate growth (45), and do not require microtubules to facilitate cell fusion (46). Consistently, we observed the dynamic rearrangement of actin organization during CAT formation and fusion, suggesting a predominant role for actin in these processes. This notion is supported by findings in Ustilago maydis, where it has been demonstrated that cell-cell recognition and cell-cell fusion ex- clusively depend on F-actin during all stages of polar growth whereas microtubules are required only for long-distance growth of hyphae (19). The function of CATs is to connect cells that are less than 10 to 12 m apart, i.e., CATs do not need to extend further than 5 to 6 m. The F-actin cytoskel- eton is apparently sufficient to support this short-distance growth and to facilitate fusion. Our observations suggest that recruitment of both cytoskeletal elements occurs in a distinct but coordinated manner and might influence which protrusion is being formed and maintained at any point in time.
We then studied the conservation patterns of the 9730 N.
crassa protein-coding genes and the 1478 lincRNA genes by using BLAST (https://blast.ncbi.nlm.nih.gov)  and analyzed the sequence homologies in four Ascomycota spe- cies, Sordaria macrospora, Chaetomium thermophilum, As- pergillus niger and Saccharomyces cerevisiae, and a vertebrate with a small genome, the pufferfish Takifugu rubripes. In stark contrast to coding sequences, Neurospora lincRNAs are weakly conserved. While 87% of the Neuros- pora coding genes shared homologies in the phylogenetic- ally closest relative Sordaria macrospora, only 3% of the lincRNA genes were conserved between these two species and less than 1% in other species (Fig. 2b). Despite this rapid evolution, we observed strong homologies between Neurosporacrassa and Sordaria macrospora for 13 lincR- NAs (Additional file 3: Table S1). On close inspection, we found that 9 of these Neurospora lincRNAs evolved from protein-coding into noncoding gene sequences by acquiring frame disruptions. Such a mechanism is reported for the human Xist gene involved in X-chromosome inactivation [45, 46]. In addition, in 4 cases open reading frames were detected on the opposite strand. These RNAs may be lncRNAs that are antisense to putative coding mRNAs and their sense partners might not be expressed in our experi- mental setup. These instances are further discussed in the following section. Low degree sequence conservation of lincRNAs is also reported between zebrafish and human  and between mouse and human [48, 49]. These studies along with ours suggest that evolution of lincRNA genes is distinct from protein-coding genes and they emerge within particular lineages or species. Despite the weak sequence constraint, large numbers of lincRNAs in many species imply unconventional functional contribution by these RNAs to gene regulation.
When the cytochrome-mediated mitochondrial electron transport chain of Neurosporacrassa is disrupted, an alternative oxidase encoded by the nuclear aod-1 gene is induced. The alternative oxidase donates electrons directly to oxygen from the ubiquininol pool and is insensitive to chemicals such as antimycin A and KCN that affect the standard electron transport chain. To facilitate isolation of mutants affecting regulation of aod-1, a reporter system containing the region upstream of the aod-1 coding sequence fused to the coding sequence of the N. crassa tyrosinase gene (T) was transformed into a strain carrying a null allele of the endogenous T gene. In the resulting reporter strain, growth in the presence of chloramphenicol, an inhibitor of mitochondrial translation whose action decreases the level of mitochondrial translation prod- ucts resulting in impaired cytochrome-mediated respiration, caused induction of both alternative oxidase and tyrosinase. Conidia from the reporter strain were mutagenized, plated on medium containing chloram- phenicol, and colonies that did not express tyrosinase were identified as potential regulatory mutants.
Though evolved to be resistant to degradation, the plant cell wall can be broken down by a variety of micro- organisms. One important example is Neurosporacrassa, a fungus prospering in burnt grasslands. N. crassa secretes cellulases and hemicellulases to degrade ligno- cellulosic material, thereby producing primarily shorter chain carbohydrates that can be consumed for its sur- vival. Cellodextrin and xylodextrin utilization pathways were previously identified as major strategies used by N. crassa and other fungi to utilize complex biomass [2, 3]. In both cases, secreted enzymes first break down the cellulose and hemicellulose to soluble cellodextrins and xylodextrins, respectively. These are then transported into the cells by cellodextrin and xylodextrin transporters and—in the case of xylodextrins—reduced before they are further processed to monomeric sugars by intracel- lular hydrolases.