Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans.

Background: Switchgrass (Panicum virgatum L.) is a C4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. Results: The expression of a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Conclusion: Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.

Insertional mutagenesis in mice deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 reveals cancer gene interactions and correlations with tumor phenotypes.

The cyclin dependent kinase (CDK) inhibitors p15, p16, p21, and p27 are frequently deleted, silenced, or downregulated in many malignancies. Inactivation of CDK inhibitors predisposes mice to tumor development, showing that these genes function as tumor suppressors. Here, we describe high-throughput murine leukemia virus insertional mutagenesis screens in mice that are deficient for one or two CDK inhibitors. We retrieved 9,117 retroviral insertions from 476 lymphomas to define hundreds of loci that are mutated more frequently than expected by chance. Many of these loci are skewed toward a specific genetic context of predisposing germline and somatic mutations. We also found associations between these loci with gender, age of tumor onset, and lymphocyte lineage (B or T cell). Comparison of retroviral insertion sites with single nucleotide polymorphisms associated with chronic lymphocytic leukemia revealed a significant overlap between the datasets. Together, our findings highlight the importance of genetic context within large-scale mutation detection studies, and they show a novel use for insertional mutagenesis data in prioritizing disease-associated genes that emerge from genome-wide association studies.

Primary upper tract urothelial carcinoma with thyroid-like features.

We present a unique case of primary urothelial carcinoma with both histological and immunohistochemical features similar to thyroid papillary carcinoma. Following surgical resection of the primary tumor and localized metastatic lymphadenectomy, the patient was treated with a course of adjuvant chemotherapy. No evidence of primary thyroid carcinoma was noted. The patient was without recurrence after a 6 month follow-up.

The npgA/ cfwA gene encodes a putative 4'-phosphopantetheinyl transferase which is essential for penicillin biosynthesis in Aspergillus nidulans.

Non-ribosomal peptide synthetases, polyketides and fatty acid synthetases have a modular organisation of multi-enzymatic activities. In all of them, the acyl or peptidyl carrier proteins have 4'-phosphopantetheine (P-pant) as an essential prosthetic group. This is added by 4'-phosphopantetheinyl transferases (PPTases) that derive the P-pant group from coenzyme A. While many PPTases of varying specificity have now been isolated from a number of bacteria, a filamentous fungal PPTase has yet to be characterised. Through database searching of the Aspergillus fumigatus genome sequence against Sfp from Bacillus subtilis, we identified a unique sequence which appears to encode a PPTase, as deduced from conserved residues considered important in PPTases. The PPTase candidate was used to search the NCBI data base and an unexpected homologue in A. nidulans was identified as npgA. Mutations in this gene (cfwA/ npgA) were identified previously as leading to defects in growth and pigmentation. To test whether the temperature-sensitive cfwA2 mutation impairs penicillin biosynthesis, which is dependent on the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, bioassays with B. calidolactis were set up at permissive and non-permissive temperatures. The cfwA2 mutant did not produce penicillin at the non-permissive temperature. Since no other PPTase homologue has been detected in the A. fumigatus genome to date, the data suggest that a single enzyme may be able to transfer the cofactor to a broad range of enzymes with acyl or peptidyl carrier protein domains.

Cloning and expression analysis of the pcbAB-pcbC beta-lactam genes in the marine fungus Kallichroma tethys.

Here we report the identification of the beta-lactam biosynthesis genes pcbAB and pcbC from a cosmid genomic DNA library of the marine fungus Kallichroma tethys. A BLAST homology search showed that they share high sequence identity with the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetases and isopenicillin N synthases, respectively, of various fungal and bacterial beta-lactam producers, while phylogenetic analysis indicated a close relationship with homologous genes of the cephalosporin-producing pyrenomycete Acremonium chrysogenum. Expression analysis by reverse transcription-PCR suggested that both genes are highly regulated and are expressed in the late growth phase of K. tethys cultures. Complementation of an Aspergillus nidulans strain deficient in ACV synthetase suggested that at least pcbAB is functional, although attempts to isolate active antibiotic from K. tethys were unsuccessful.

Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice.

We have used large-scale insertional mutagenesis to identify functional landmarks relevant to cancer in the recently completed mouse genome sequence. We infected Cdkn2a(-/-) mice with Moloney murine leukemia virus (MoMuLV) to screen for loci that can participate in tumorigenesis in collaboration with loss of the Cdkn2a-encoded tumor suppressors p16INK4a and p19ARF. Insertional mutagenesis by the latent retrovirus was synergistic with loss of Cdkn2a expression, as indicated by a marked acceleration in the development of both myeloid and lymphoid tumors. We isolated 747 unique sequences flanking retroviral integration sites and mapped them against the mouse genome sequence databases from Celera and Ensembl. In addition to 17 insertions targeting gene loci known to be cancer-related, we identified a total of 37 new common insertion sites (CISs), of which 8 encode components of signaling pathways that are involved in cancer. The effectiveness of large-scale insertional mutagenesis in a sensitized genetic background is demonstrated by the preference for activation of MAP kinase signaling, collaborating with Cdkn2a loss in generating the lymphoid and myeloid tumors. Collectively, our results show that large-scale retroviral insertional mutagenesis in genetically predisposed mice is useful both as a system for identifying genes underlying cancer and as a genetic framework for the assignment of such genes to specific oncogenic pathways.

Sequencing the Aspergillus fumigatus genome.

Aspergillus fumigatus is the most common mould pathogen of human beings and unusually causes both invasive disease in immunocompromised patients and allergic disease in patients with atopic immune systems. 4% of patients dying in modern European teaching hospitals have invasive aspergillosis and it is the leading infectious cause of death in leukaemia and bone marrow transplant patients. Until 2001, only two licensed antifungal drugs were available to treat aspergillosis-amphotericin B and itraconazole. Its 28-30Mb genome is being sequenced in an international collaboration, with the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA) as the two main centres. A whole-genome shotgun approach was adopted and initiated in 2001 with an expected completion date in 2003. The complete sequence will permit identification of pathways specific to pathogenic Aspergillus species, help identify new targets for antifungal drugs, and enable investigations into the basic biology of fungi. Numerous secondary metabolic pathways with biotechnological applications and pharmacological properties are found in the Aspergilli and the genome sequence will facilitate research in this area.