Genetic Inactivation of D-Amino Acid Oxidase Genes in Methylotrophic Yeast Hansenula Polymorpha

Cellular D-amino acid oxidases (ODA-FAD containing flavoproteins) are widely used in biotechnology. In this regard, it is of particular interest to investigate the functional role of the composition and regulation of individual ODA genes. The aim of this work was to investigate physiological and biochemical characteristics of ODA genes of strain DL1x Hansenula (O.) polymorpha in vivo using gene knockout methodology as well as to determine the nature of these genes expression and regulation of ODA activity as a function of nitrogen and carbon source composition in the cultivation medium. H. polymorpha is a thermotolerant methylotrophic yeast. They are used to study the mechanisms of peroxisomal biogenesis and degradation, regulation of methanol metabolism, nitrate assimilation and stress response. A serial dilution method has been used for rapid assessment of strain growth and stress tolerance. Vector design for genetic inactivation of ODA genes in H. polymorpha was performed using yeast vector pAM773. Selection of "knockout" cell clones was performed using PCR analysis. To complete the transformation process, H. polymorpha was deleted from the pAM773 vector and the obtained DNA was used in the experiment. In vitro cultivation of knockout strains of H. polymorpha was found to exhibit substratespecificity of the ODA. According to the authors, the HP2914 gene is important for D-alanine oxidation, while the gene complex 2400 and 2914 is important for ODA activation in the presence of D-Phe in the medium. It is likely that the gene complex 2082 and 2165 regulates ODA activation when cells are cultured in medium with D-Ala and D-Asp. It was found that in the absence of 2165, 2400, 2914 genes, increased ODA activity to D-Ala only was observed in cell culture medium. The presence of D-alanine combined with 1 % glycerol and 1 % methanol in the culture medium stimulated the activity of the three major ODAs of H. polymorpha through the expression of the HP2914 gene, while the presence of glucose and L-alanine in the culture medium suppressed their activity

Transcriptome-Wide Identification and Quantification of Caffeoylquinic Acid Biosynthesis Pathway and Prediction of Its Putative BAHDs Gene Complex in A. spathulifolius

The phenylpropanoid pathway is a major secondary metabolite pathway that helps plants overcome biotic and abiotic stress and produces various byproducts that promote human health. Its byproduct caffeoylquinic acid is a soluble phenolic compound present in many angiosperms. Hydroxycinnamate-CoA shikimate/quinate transferase is a significant enzyme that plays a role in accumulating CQA biosynthesis. This study analyzed transcriptome-wide identification of the phenylpropanoid to caffeoylquinic acid biosynthesis candidate genes in A. spathulifolius flowers and leaves. Transcriptomic analyses of the flowers and leaves showed a differential expression of the PPP and CQA biosynthesis regulated unigenes. An analysis of PPP-captive unigenes revealed a major duplication in the following genes: PAL, 120 unigenes in leaves and 76 in flowers; C3′H, 169 unigenes in leaves and 140 in flowers; 4CL, 41 unigenes in leaves and 27 in flowers; and C4H, 12 unigenes in leaves and 4 in flowers. The phylogenetic analysis revealed 82 BAHDs superfamily members in leaves and 72 in flowers, among which five unigenes encode for HQT and three for HCT. The three HQT are common to both leaves and flowers, whereas the two HQT were specialized for leaves. The pattern of HQT synthesis was upregulated in flowers, whereas HCT was expressed strongly in the leaves of A. spathulifolius. Overall, 4CL, C4H, and HQT are expressed strongly in flowers and CAA and HCT show more expression in leaves. As a result, the quantification of HQT and HCT indicates that CQA biosynthesis is more abundant in the flowers and synthesis of caffeic acid in the leaves of A. spathulifolius.

Transcriptome-wide Identification and Quantification of Caffeoylquinic Acid Biosynthesis Pathway and Prediction of their Putative BAHDs Gene Complex in A. spathulifolius

The phenylpropanoid pathway is a major secondary metabolite pathway that helps plants overcome biotic and abiotic stress and produces various by-products that promote human health. Its byproduct, chloroquinic acid (CQA), is a soluble phenolic compound present in many angiosperms. Hy-droxycinnamate-CoA shikimate/quinate transferase(BAHDs superfamily enzyme) is a significant en-zyme that plays a role in accumulating CQA biosynthesis. This study analyzed transcriptome-wide identification of the phenylpropanoid to chloroquinic acid biosynthesis candidate genes in A. spathulifolius flowers and leaves. Transcriptomic analyses of the flowers and leaves showed a differential expression of the PPP and CQA biosynthesis regulated unigenes. An analysis of PPP captive unigenes revealed the following: the major duplication of the key enzyme, PAL, 120 unigenes in leaves and 76 in flowers; the gene encoding C3’H, 169 unigenes in leaves and 140 unigenes in flowers; duplicated unigenes of 4CL, 41 in leaves and 27 in flowers. In addition, C4H unigenes had 12 unigenes in the leaves of A. spathulifolius and four in the flowers. The characterization of the BAHDs superfamily members identified 82 in leaves and 72 in flowers. Among them, phylogenetic analysis showed that five unigenes encoded HQT and three en-coded HCT in A. spathulifolius. The three HQT are common to both leaves and flowers, whereas the two HQT were specialized for leaves. The pattern of HQT synthesis was upregulated in flowers, whereas HCT was expressed strongly in the leaves of A. spathulifolius. Overall, 4CL, C4H, and HQT are expressed strongly in flowers, and caffeic acid and HCT show more expression in leaves. Therefore, CQA biosynthesis occurs in the flowers of A. spathulifolius rather than leaves.

High-throughput sequencing defines donor and recipient HLA B-cell epitope frequencies for prospective matching in transplantation

Compatibility for human leukocyte antigen (HLA) genes between transplant donors and recipients improves graft survival but prospective matching is rarely performed due to the vast heterogeneity of this gene complex. To reduce complexity, we have combined next-generation sequencing and in silico mapping to determine transplant population frequencies and matching probabilities of 150 antibody-binding eplets across all 11 classical HLA genes in 2000 ethnically heterogeneous renal patients and donors. We show that eplets are more common and uniformly distributed between donors and recipients than the respective HLA isoforms. Simulations of targeted eplet matching shows that a high degree of overall compatibility, and perfect identity at the clinically important HLA class II loci, can be obtained within a patient waiting list of approximately 250 subjects. Internal epitope-based allocation is thus feasible for most major renal transplant programs, while regional or national sharing may be required for other solid organs.

Expression, Interaction, and Role of Pseudogene Adh6-ps1 in Cancer Phenotypes

Pseudogenes have been classified as functionless and their annotation is an ongoing problem. The Adh6-ps1—a mouse pseudogene belonging to the alcohol dehydrogenase gene complex (Adh) was analyzed to review the conservation, homology, expression, and interactions and identify any role it plays in disease phenotypes using bioinformatics databases. Results showed that Adh6-ps1 have 2 transcripts (processed and unprocessed) which may have emerged from a transposition and duplication event, respectively, and that induced inversions (Uox gene, In(3)11Rk) involving gene complexes associated with Adh6-ps1 have been implicated in a diverse range of diseases. Adh6-ps1 is highly conserved in vertebrates particularly rodents and expressed in the liver. The top 5 MirRNA targets were Mir455, Mir511, Mir1903, Mir361, and Mir669o markers. While much is unknown about Mir1903 and Mir669o, the silencing of Mir455 and Mir511 is linked with hepatocellular carcinoma (HCC), and Mir361 is implicated in endometrial cancers. Given the identified MirRNA interactions with Adh6-ps1 and its expression in HCC and reproductive systems, it may well have a role in tumorigenesis and disease phenotypes. Nonetheless, further studies are required to establish these facts to add to the growing efforts to understand pseudogenes and their potential involvement in disease conditions.