Many proteins form complexes that function in reaction pathways. Therefore, to understand protein function, it is necessary to reconstitute complexes and pathways in vitro. However, it is not straightforward to achieve full activity in reconstituted systems. To address this problem, we present a yeast system named "in saccharo" analysis, which uses budding yeast for simultaneous expression and analysis of many kinds of non-host proteins, such as human proteins. For this purpose, vectors that can accommodate many genes are required. Here, we describe the construction of a chromosome vector by insertion of unique barcode sequences (BCs) into the ribosomal RNA gene repeat (rDNA). Each unit of the rDNA has a BC that is used as the target for integration of an external gene. Because rDNA is naturally capable of maintaining many repetitive copies, the vector is expected to retain the numerous external genes that may be required for reconstitution of functional protein complexes and reaction pathways. Consistent with this prediction, we were able to clone three human genes that form the RNA silencing pathway, which has no functional equivalent in budding yeast, and to demonstrate functionality in this in saccharo analysis system.
The pathogenesis of pheochromocytoma and paraganglioma (PCPG) catecholamine-producing tumors is exceedingly complicated. Here, we sought to identify important genes affecting the prognosis and survival rate of patients suffering from PCPG. We analyzed 95 samples obtained from two microarray data series, GSE19422 and GSE60459, from the Gene Expression Omnibus (GEO) repository. First, differentially expressed genes (DEGs) were identified by comparing 87 PCPG tumor samples and eight normal adrenal tissue samples using R language. The GEO2R tool and Venn diagram software were applied to the Database for Annotation, Visualization and Integrated Discovery (DAVID) to analyze Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO). We further employed Cytoscape with the Molecular Complex Detection (MCODE) tool to make protein–protein interactions visible for the Search Tool for Retrieval of Interacting Genes (STRING). These procedures resulted in 30 candidate DEGs, which were subjected to Kaplan–Meier analysis and validated by Gene Expression Profiling Interactive Analysis (GEPIA) to determine their influence on overall survival rate. Finally, we identified ALDH3A2 and AKR1B1, two genes in the glycerolipid metabolism pathway, as being particularly enriched in PCPG tumors and correlated with T and B tumor-infiltrating immune cells. Our results suggest that these two DEGs are closely associated with the prognosis of malignant PCPG tumors.
The melanocortin-1 receptor gene (MC1R) controls production of the pigments eumelanin and pheomelanin. Changes in MC1R lead to variation in coat color in mammals, which can range from entirely black (melanism) to yellowish. In this study, we report a case of a wild-caught Norway rat (Rattus norvegicus) from Sado Island, Japan with a yellowish coat color. Upon sequencing the whole coding region of the Mc1r gene (954 bp), we found a 1-bp deletion at site 337 (c.337del), indicative of a frameshift mutation, which was characterized as a severe loss-of-function or null mutation. A spectrophotometer was used to measure coat color, revealing that the rat had a distinctly lighter coat, based on lightness score, than mice with homozygous similar loss-of-function mutations. This implies that loss-of-function mutations can yield different phenotypes in murine rodents. The loss-of-function-mutant rat exhibited a contrasting coat pattern consisting of darker and lighter colors along its dorsal and ventral sides, respectively. Similar patterns have been observed in homozygous MC1R-deficient mutants in other mammals, implying that the countershading pattern can still be expressed despite the absence of MC1R in the melanocyte.
Patchouli, Pogostemon cablin (Blanco) Benth., is a traditional Chinese medicinal plant from the order Lamiales. It is considered a valuable herb due to its essential oil content and range of therapeutic effects. This study aimed to explore the evolutionary history of repetitive sequences in the patchouli genome by analyzing tandem repeats and transposable elements (TEs). We first retrieved genomic data for patchouli and four other Lamiales species from the GenBank database. Next, the content of tandem repeats with different period sizes was identified. Long terminal repeats (LTRs) were then identified with LTR_STRUC. Finally, the evolutionary landscape of TEs was explored using an in-house PERL program. The analysis of repetitive sequences revealed that tandem repeats constitute a higher proportion of the patchouli genome compared to the four other species. Analyses of TE families showed that most of the repetitive sequences in the patchouli genome are TEs, and that recently inserted TEs make up a comparatively larger proportion than older ones. Our analyses of LTR retrotransposons in their host genome indicated the existence of ancient LTR retrotransposon expansion, and the escape of these elements from natural selection revealed their ages. Our identification and analyses of repetitive sequences should provide new insights for further investigation of patchouli evolution.
The onset of Sjögren's syndrome (SS) is hidden, early diagnosis is difficult, and the disorder seriously endangers the physical and mental health of affected people. This study aims to identify potential biomarkers of SS and to investigate the characteristics of immune cell infiltration. We used four SS gene expression profile data series from the Gene Expression Omnibus database, and applied bioinformatics analysis and machine learning algorithms to screen two biomarkers, SELL (L-selectin) and IFI44 (interferon-induced protein 44), from 101 differentially expressed genes. The two-gene model comprising SELL and IFI44 showed good diagnostic ability for SS in the training set (AUC = 0.992) and verification set (AUC = 0.917). Analysis of infiltrating immune cells in SS identified naive B cells, resting CD4 memory T cells, activated CD4 memory T cells, gamma delta T cells, M0 macrophages, M1 macrophages, plasma cells, CD8 T cells, activated NK cells and monocytes as candidate participants in the SS process. Furthermore, SELL was associated with M2 macrophages, activated CD4 memory T cells, gamma delta T cells, resting NK cells and plasma cells, while IFI44 was associated with activated mast cells, resting NK cells, resting mast cells and CD8 T cells. This study demonstrates that SELL and IFI44 can serve as good diagnostic markers for SS and may also be new diagnostic and therapeutic targets for SS.
Nuclear microsatellite markers were developed for Geranium thunbergii, an herbaceous plant characterized by petal color polymorphism. Utilizing RNA sequencing data obtained by next-generation sequencing techniques, we developed and characterized 19 polymorphic microsatellite markers with two to 12 alleles in the nuclear genome. These markers will be used to reveal the genetic structure and demographic history of G. thunbergii in the Japanese archipelago, which will elucidate the genetic background of flower color polymorphism among populations.