A transmission ratio distortion and the ‘max-4’ ascus phenotype: Do both reflect the same Bateson-Dobzhansky-Muller Incompatibility emerging during trans-species introgression of translocations in Neurospora?

Over the last couple of decades, with the crisis of new antimicrobial arsenal, multidrug-resistant clinicalpathogens have been observed extensively. In clinical and medical settings, these persistent pathogens predominantly grow as complex heterogeneous structures enmeshed in a self-produced exopolysaccharide matrix,termed as biofilms. Since biofilms can rapidly form by adapting new environmental surroundings and havepotential effect on human health, it is critical to study them promptly and consistently. Biofilm infections arechallenging in the contamination of medical devices and implantations, food processing and pharmaceuticalindustrial settings, and in dental area caries, periodontitis and so on. The persistence of infections associatedwith biofilms has been mainly attributed to the increased antibiotic resistance offered by the cells growing inbiofilms. In fact, it is well known that this recalcitrance of bacterial biofilms is multifactorial, and there areseveral resistance mechanisms that may act in parallel in order to provide an enhanced level of resistance to thebiofilm. In combination, distinct resistance mechanisms significantly decrease our ability to control anderadicate biofilm-associated infections with current antimicrobial arsenal. In addition, various factors areknown to influence the process of biofilm formation, growth dynamics, and their heterogeneous responsetowards antibiotic therapy. The current review discusses the contribution of cellular and physiochemical factorson the growth dynamics of biofilm, especially their role in antibiotic resistance mechanisms of bacterialpopulation living in surface attached growth mode. A systematic investigation on the effects and treatment ofbiofilms may pave the way for novel therapeutic strategies to prevent and treat biofilms in healthcare andindustrial settings.

RNA-Seq, and ye shall find: Sexual-stage-specific A-to-I RNA editing in fungi.

Fusarium graminearum, a pathogen of wheat and barley, is a haploid homothallic ascomycete filamentous fungus (Goswami and Kistler 2004). It overwinters as saprophytic hyphae in plant debris and undergoes the sexual cycle in spring to produce fruiting bodies (perithecia) bearing the progeny ascospores. The genome sequence of the F. graminearum PH-1 strain was reported last year (King et al. 2015). This year, Jin-Rong Xu and colleagues in Northwest A&F University, China, and Purdue University, USA, re-sequenced the PH-1 genome and also performed RNA-Seq analysis on two independent biological replicates each of RNA from conidia, hyphae, and 8-day post-fertilization perithecia (Liu et al. 2016). Alignment of the two replicate perithecial RNA-Seq reads with the reference genome sequence revealed 23,041 and 19,764 single-nucleotide variants (SNVs), of which, respectively, 22,578 and 19,261 corresponded to A (adenosine)- to-G (guanosine) transitions, and 17,613 A-to-G transitions were common to both the replicates. Non- A-to-G variants were far fewer (463 and 503) and only 35.9% were common between the two perithecial replicates, suggesting that the non-A-to-G variants were false-positives. In sum, 26,056 A-to-G variants were identified as putative A-to-I RNA editing sites at which hydrolytic deamination at the C6 position of the purine ring of A produces I (inosine). Since I preferentially base-pairs with C (cytidine), an I within a transcript is read as G by the translation machinery. Also, during reverse transcription, I directs the incorporation of C; thus, it appears as a G in double-stranded cDNA. The conidial and hyphal RNA-Seq data showed only 68 and 112 A-to-G transitions and 335 and 452 non-A-to-G conversions, indicating that the A-to-I RNA editing is specific to the sexual stage. Xu and colleagues had initially set out to do a yeast two-hybrid experiment to identify proteins that interact with a protein kinase named Puk1 (perithecium unique kinase 1). Ascospores from the puk1 mutant have an abnormal morphology. Additionally, qRT-PCR showed that PUK1 transcription is markedly upregulated in perithecia, suggesting that PUK1 expression and function might be restricted to the sexual stage. Therefore, to generate the PUK1 ORF bait, they synthesized cDNA using RNA isolated from perithecial cultures of the PH-1 strain. Sequencing of the construct revealed that two tandem stop codons – UAG UAG – in the PUK1 ORF were changed to UGG UGG in the cDNA, presumably via A-to-I RNA editing. More than 90% of PUK1 reads in perithecial RNA-Seq showed the A-to-I editing, and experiments with site-specific mutant alleles showed that the editing was essential for PUK1 function. This was an unexpected discovery because fungi lack orthologs of the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes that in metazoans converts A to I in double-stranded RNA. Presumably, A-to-I RNA editing in fungi uses different enzymes than animals. This discovery motivated Xu and colleagues to expand the search for RNA editing genome-wide in transcriptomes from vegetative and sexual-stage tissues (conidia, hyphae, and perithecia). The percentage of reads with the A-to-G variant was taken as the RNA editing level at the site. Editing levels varied from 3% to 100%. Strikingly, 47% of genes bearing sites with editing levels >60% tended to be up-regulated or specifically expressed in perithecia compared to conidia and hyphae. A majority of the editing events resulted in amino acid substitutions, which suggested that Ato- I editing might be important for adaptation of protein functions during sexual reproduction. Editing events similar to those in PUK1 were found 69 other genes, including the rid (RIP defective) ortholog and genes important for meiosis (see below). All these genes displayed UAG-to-UGG change in exons that automated annotation had incorrectly predicted as introns.

The Sad paradox: Mutations with dominant and recessive phenotypes.

Robert Metzenberg (June 11, 1930 – July 15, 2007) was described as ‘a geneticist extraordinaire and “model human”’ (Selker 2008). Claudio Scazzocchio (28 July 1938 –), a colleague and friend of Bob Metzenberg, is since his retirement a visiting professor at Imperial College, London, ‘actively writing old work, spending quite a few hours a day in front of a computer; trying to learn some bioinformatics’ and ‘following quite closely everything that has to do with epigenetics of fungi’. In 2001, Metzenberg and colleagues reported the fascinating discovery, in Neurospora crassa, of meiotic silencing by unpaired DNA (MSUD). MSUD is a presumed RNAi-mediated dousing of the ascusexpression of any gene lacking a sequence homologue at the same allelic position on the homologous chromosome (e.g. the transposase gene of a transposable element inserted into a novel location; also see figure 1) (Shiu et al. 2001). Somewhat counter-intuitively, MSUD leads deficiencies (Df) to exert an ascus-dominant phenotype. In a Df × WT cross, genes uncovered by the Df on the WT chromosome remain unpaired in meiosis, and are silenced. Interestingly, the gene, suppressor of ascus dominance-1+ (sad-1+), which encodes a putative RNA-dependent RNA polymerase essential for MSUD, silences itself when opposite a deletion allele (Sad-1Δ), and, consequently, Sad-1Δ suppresses MSUD in heterozygous crosses (i.e. Sad-1Δ × WT) (figure 1). Such self-silencing is reminiscent of Bertrand Russell’s famous paradox about the village barber who shaves all those who don’t shave themselves: Who shaves the barber? Additionally, the homozygous Sad-1Δ × Sad-1Δ cross is infertile. In 2002, I wrote a ‘Commentary’ article describing the findings of Metzenberg and colleagues, and in it I also made reference to the paradox (Kasbekar 2002). Chancing upon my article last year, Scazzocchio graciously emailed his appreciation of it, and very soon a cordial correspondence developed between us. This correspondence brought to my possession three emails exchanged by Metzenberg and Scazzocchio in 2004, together with permission from Scazzocchio and Stan Metzenberg to use this exchange in any way I see fit. Nothing could be fitter than to publish this exchange in a new ‘Sidelights’ section in Journal of Biosciences. Interestingly, Scazzocchio also refers to Russell’s paradox in this scientific correspondence, and Metzenberg seems to suggest the barber shaves himself, but so incompletely as to create doubt about whether he shaved or merely trimmed his beard.

Lymphohematopoietic licence: Sterol C-14 reductase activity of lamin B receptor (Lbr) is essential for neutrophil differentiation.

Bertie Wooster, PG Wodehouse’s fictional character, proudly fancied himself a writer for having once contributed an article (‘What the Well-Dressed Man Is Wearing’) to Milady’s Boudoir, his Aunt Dahlia’s weekly magazine for women. My conceit of expertise in vertebrate lamin B receptor (Lbr) research is slightly less dubious. I co-authored two papers (Papavinasasundaram and Kasbekar 1994 and Prakash et al. 1999) that established that the C-terminal two-thirds of Lbr has sterol Δ14,15 reductase activity. An interloping article (Silve et al. 1998) reached pretty much the same conclusion (and to my chagrin, garnered the lion’s share of the citations). So the recent demonstration by Peter Gaines and coworkers that the Lbr sterol reductase regulates differentiation of neutrophils (Subramanian et al. 2012) filled me with proprietary pride, especially since neutrophils are the most abundant white blood cells in circulation and present the critical first line of defence against infectious microbes. Lbr is an integral protein of the vertebrate nuclear envelope inner membrane. Its N-terminal ~200 residues are hydrophilic, bind to B-type lamins, DNA and HP1-type chromatin proteins, and provide a substrate for p34cdc2, a key mitotic protein kinase. The ~420 residue hydrophobic, membrane-spanning, C-terminal domain (CTD) with sterol reductase activity anchors the nucleoplasmic domain to the inner nuclear membrane. Mutations in human LBR cause Pelger-Huët anomaly (PHA), a benign dominant disorder characterized by hyposegmentation of the neutrophil nucleus (Hoffmann et al. 2002). A spontaneously aborted fetus with Greenberg/HEM dysplasia was homozygous for LBR mutations, and peripheral blood neutrophils from the fetus’ mother displayed PHA (Waterham et al. 2003). Greenberg/HEM dysplasia and PHA reflect the pleiotropism of LBR mutations. In mouse, the Lbr gene is defined by the ichthyosis (ic) mutations (Shultz et al. 2003). Neutrophils from ic/ic mice display bilobed or ovoid nuclei typical of PHA. Additionally, ic/ic homozyogotes exhibit sparse hair, decreased body size and occasionally hydrocephalus and syndactyly. It was of interest to understand the functional significance of the Lbr protein’s sterol reductase activity, especially since KO mice for another locus, Tm7sf2, that encodes SR-1, a 418 residue protein with 58% identity with the Lbr CTD and possessing sterol C−14 reductase activity, do not display an observable phenotype (Bennati et al. 2006, 2008). First, a quick flashback to another 1994 paper: Tsai et al. (1994) had shown that transduction of normal mouse bone marrow cells with a retroviral vector harbouring a dominant-negative retinoic acid receptor (RARα403) could reproducibly immortalize lymphohematopoietic progenitors as stem-cell-factor-dependent clonal lines, designated as EML cells for their ability to subsequently undergo erythroid, myeloid and lymphoid differentiation in vitro. A 3-day treatment of EML cells with stem cell factor, IL-3, and high concentrations of all-trans retinoic acid, and then washing and switching them into GM-CSF, induced their differentiation into promyelocytes, designated as EPRO cells (EML-derived promyelocytes), that can be maintained in GM-CSF. Treatment of EPRO cells with high concentrations of retinoic acid in the presence of GM-CSF induced them to terminally differentiate into mature neutrophils with characteristic nuclear lobulation and respiratory burst response phenotypes. Several years later, Gaines et al. (2008) generated EML- and EPRO-like cells frombonemarrow of a C57BL/6J-Lbric-J/Lbric-J (ic/ic) mouse and a normal (+/ic) littermate, and found that neutrophils derived from EPRO-ic/ic cells exhibited nuclear hypolobulation identical to that seen in ichthyosis mice and displayed a deficient respiratory burst, whereas those from.

A factor in a wild isolated Neurospora crassa strain enables a chromosome segment duplication to suppress repeat-induced point mutation.

Repeat-induced point mutation (RIP) is a sexual stage-specific mutational process of Neurospora crassa and other fungi that alters duplicated DNA sequences. Previous studies from our laboratory showed that chromosome segment duplications (Dps) longer than ~300 kbp can dominantly suppress RIP, presumably by titration of the RIP machinery, and that although Dps <200 kbp did not individually suppress RIP, they could do so in homozygous and multiply heterozygous crosses, provided the sum of the duplicated DNA exceeds ~300 kbp. Here we demonstrate suppression of RIP in a subset of progeny carrying the normally sub-threshold 154 kbp Dp(R2394) from a cross of T(R2394) to the wild isolated Carrefour Mme. Gras strain (CMG). Thus, the CMG strain contains a factor that together with Dp(R2394) produces a synthetic RIP suppressor phenotype. It is possible that the factor is a cryptic Dp that together with Dp(R2394) can exceed the size threshold for titration of the RIP machinery and thereby causes RIP suppression.

Translocations used to generate chromosome segment duplications in Neurospora can disrupt genes and create novel open reading frames.

In Neurospora crassa, crosses between normal sequence strains and strains bearing some translocations can yield progeny bearing a duplication (Dp) of the translocated chromosome segment. Here, 30 breakpoint junction sequences of 12 Dp-generating translocations were determined. The breakpoints disrupted 13 genes (including predicted genes), and created 10 novel open reading frames. Insertion of sequences from LG III into LG I as translocation T(UK8- 18) disrupts the eat-3 gene, which is the ortholog of the Podospora anserine gene ami1. Since ami1-homozygous Podospora crosses were reported to increase the frequency of repeat-induced point mutation (RIP), we performed crosses homozygous for a defi ciency in eat-3 to test for a corresponding increase in RIP frequency. However, our results suggested that, unlike in Podospora, the eat-3 gene might be essential for ascus development in Neurospora. Duplication–heterozygous crosses are generally barren in Neurospora; however, by using molecular probes developed in this study, we could identify Dp segregants from two different translocation–heterozygous crosses, and using these we found that the barren phenotype of at least some duplication–heterozygous crosses was incompletely penetrant.

Neurospora crassa fmf-1 encodes the homologue of the Schizosaccharomyces pombe Ste11p regulator of sexual development.

The Neurospora crassa fmf-1 mutation exerts an unusual 'perithecium-dominant' developmental arrest; fmf-1 x fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. We localized fmf-1 to the LG IL genome segment between the centromere-proximal breakpoint of the chromosome segment duplication Dp(IL)39311 and the centromere. By mapping crossovers with respect to RFLP markers in this region we further localized fmf-1 to an approximately 34-kb-genome segment. Partial sequencing of this segment revealed a point mutation in the gene NCU 09387.1, a homologue of the Schizosaccharomyces pombe ste11+ regulator of sexual development. The fmf-1 mutation did not complement a NCU 09387.1 deletion mutation, and transformation with wild-type NCU 09387.1 complemented fmf-1. S. pombe Ste11 protein (Ste11p) is a transcription factor required for sexual differentiation and for the expression of genes required for mating pheromone signalling in matP and matM cells. If FMF-1 also plays a corresponding role in mating pheromone signalling in Neurospora, then protoperithecia in an fmf-1 x fmf-1+ cross would be unable to either send or receive sexual differentiation signals and thus become arrested in development.

Translesion DNA polymerases Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are not essential for repeat-induced point mutation in Neurospora crassa.

Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) in Neurospora crassa. Additionally, we demonstrate that the upr-1 gene, which encodes the catalytic subunit of Pol zeta, is a highly polymorphic locus in Neurospora.