Members Of Multigene Family Research Articles

Polymorphic pseudogenes in the human genome - a comprehensive assessment

Background: Over the past decade, variations of the coding portion of the human genome have become increasingly evident. In this study, we focus on polymorphic pseudogenes, a unique and relatively unexplored type of pseudogene whose inactivating mutations have not yet been fixed in the human genome at the global population level. Thus, polymorphic pseudogenes are characterized by the presence in the population of both coding alleles and non-coding alleles originating from Loss-of-Function (LoF) mutations. These alleles can be found both in heterozygosity and in homozygosity in different human populations and thus represent pseudogenes that have not yet been fixed in the population. Results: A methodical cross-population analysis of 232 polymorphic pseudogenes, including 35 new examples, reveals that human olfactory signalling, drug metabolism and immunity are among the systems most impacted by the variable presence of LoF variants at high frequencies. Within this dataset, a total of 179 genes presented polymorphic LoF variants in all analysed populations. Transcriptome and proteome analysis confirmed that although these genes may harbour LoF alleles, when the coding allele is present, the gene remains active and can play a functional role in various metabolic pathways, including drug/xenobiotic metabolism and immunity. The observation that many polymorphic pseudogenes are members of multigene families argues that genetic redundancy may play a key role in compensating for the inactivation of one paralogue. Conclusions: The distribution, expression and integration of cellular/biological networks in relation to human polymorphic pseudogenes, provide novel insights into the architecture of the human genome and the dynamics of gene gain and loss with likely functional impact.

The Plasmodium falciparum histone methyltransferase PfSET10 is dispensable for the regulation of antigenic variation and gene expression in blood-stage parasites.

The malaria parasite Plasmodium falciparum employs antigenic variation of the virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1) to escape adaptive immune responses during blood infection. Antigenic variation of PfEMP1 occurs through epigenetic switches in the mutually exclusive expression of individual members of the multi-copy var gene family. var genes are located in perinuclear clusters of transcriptionally inactive heterochromatin. Singular var gene activation is linked to locus repositioning into a dedicated zone at the nuclear periphery and deposition of histone 3 lysine 4 di-/trimethylation (H3K4me2/3) and H3K9 acetylation marks in the promoter region. While previous work identified the putative H3K4-specific methyltransferase PfSET10 as an essential enzyme and positive regulator of var gene expression, a recent study reported conflicting data. Here, we used iterative genome editing to engineer a conditional PfSET10 knockout line tailored to study the function of PfSET10 in var gene regulation. We demonstrate that PfSET10 is not required for mutually exclusive var gene expression and switching. We also show that PfSET10 is dispensable not only for asexual parasite proliferation but also for sexual conversion and gametocyte differentiation. Furthermore, comparative RNA-seq experiments revealed that PfSET10 plays no obvious role in regulating gene expression during asexual parasite development and gametocytogenesis. Interestingly, however, PfSET10 shows different subnuclear localization patterns in asexual and sexual stage parasites and female-specific expression in mature gametocytes. In summary, our work confirms in detail that PfSET10 is not involved in regulating var gene expression and is not required for blood-stage parasite viability, indicating PfSET10 may be important for life cycle progression in the mosquito vector or during liver stage development.IMPORTANCEThe malaria parasite Plasmodium falciparum infects hundreds of millions of people every year. To survive and proliferate in the human bloodstream, the parasites need to escape recognition by the host's immune system. To achieve this, P. falciparum can change the expression of surface antigens via a process called antigenic variation. This fascinating survival strategy is based on infrequent switches in the expression of single members of the var multigene family. Previous research reported conflicting results on the role of the epigenetic regulator PfSET10 in controlling mutually exclusive var gene expression and switching. Here, we unequivocally demonstrate that PfSET10 is neither required for antigenic variation nor the expression of any other proteins during blood-stage infection. This information is critical in directing our attention toward exploring alternative molecular mechanisms underlying the control of antigenic variation and investigating the function of PfSET10 in other life cycle stages.

Whole-genome assembly of a hybrid Trypanosoma cruzi strain assembled with Nanopore sequencing alone.

Trypanosoma cruzi is the causative agent of Chagas disease, which causes 10,000 deaths per year. Despite the high mortality associated with Chagas, relatively few parasite genomes have been assembled to date, with genome assemblies unavailable even for some commonly used laboratory strains. This is at least partially due to T. cruzi's highly complex and highly repetitive genome, which defies investigation using traditional short-read sequencing methods. In this study, we have generated a high-quality whole-genome assembly of the hybrid Tulahuen strain, a commercially available type VI strain, using long-read Nanopore sequencing without short-read scaffolding. The assembled genome contains 25% repeat regions, 17% variable multigene family members, and 27% transposable elements (TEs) and is of comparable quality with T. cruzi genome assemblies that utilized both long- and short-read data. Notably, we find that regions with TEs are significantly enriched for multicopy surface proteins, and that surface proteins are, on average, closer to TEs than to other coding regions. This finding suggests that mobile genetic elements such as transposons may drive recombination within surface protein gene families. This work demonstrates the feasibility of Nanopore sequencing to resolve complex regions of T. cruzi genomes, and with these resolved regions, provides support for a possible mechanism for genomic diversification.

Unraveling Desmin's Head Domain Structure and Function.

Understanding the structure and function of intermediate filaments (IFs) is necessary in order to explain why more than 70 related IF genes have evolved in vertebrates while maintaining such dramatically tissue-specific expression. Desmin is a member of the large multigene family of IF proteins and is specifically expressed in myocytes. In an effort to elucidate its muscle-specific behavior, we have used a yeast two-hybrid system in order to identify desmin's head binding partners. We described a mitochondrial and a lysosomal protein, NADH ubiquinone oxidoreductase core subunit S2 (NDUFS2), and saposin D, respectively, as direct desmin binding partners. In silico analysis indicated that both interactions at the atomic level occur in a very similar way, by the formation of a three-helix bundle with hydrophobic interactions in the interdomain space and hydrogen bonds at R16 and S32 of the desmin head domain. The interactions, confirmed also by GST pull-down assays, indicating the necessity of the desmin head domain and, furthermore, point out its role in function of mitochondria and lysosomes, organelles which are disrupted in myopathies due to desmin head domain mutations.

The P. falciparum alternative histones Pf H2A.Z and Pf H2B.Z are dynamically acetylated and antagonized by PfSir2 histone deacetylases at heterochromatin boundaries.

The malaria parasite Plasmodium falciparum relies on variant expression of members of multi-gene families as a strategy for environmental adaptation to promote parasite survival and pathogenesis. These genes are located in transcriptionally silenced DNA regions. A limited number of these genes escape gene silencing, and switching between them confers variant fitness on parasite progeny. Here, we show that PfSir2 histone deacetylases antagonize DNA-interacting acetylated alternative histones at the boundaries between active and silent DNA. This finding implicates acetylated alternative histones in the mechanism regulating P. falciparum variant gene silencing and thus malaria pathogenesis. This work also revealed that acetylation of alternative histones at promoters is dynamically associated with promoter activity across the genome, implicating acetylation of alternative histones in gene regulation genome wide. Understanding mechanisms of gene regulation in P. falciparum may aid in the development of new therapeutic strategies for malaria, which killed 619,000 people in 2021.

Mechanistic insight into the role of AUXIN RESISTANCE4 in trafficking of AUXIN1 and LIKE AUX1-2.

AUXIN RESISTANCE4 (AXR4) regulates the trafficking of auxin influx carrier AUXIN1 (AUX1), a plasma-membrane protein that predominantly localizes to the endoplasmic reticulum (ER) in the absence of AXR4. In Arabidopsis (Arabidopsis thaliana), AUX1 is a member of a small multigene family comprising 4 highly conserved genes-AUX1, LIKE-AUX1 (LAX1), LAX2, and LAX3. We report here that LAX2 also requires AXR4 for correct localization to the plasma membrane. AXR4 is a plant-specific protein and contains a weakly conserved α/β hydrolase fold domain that is found in several classes of lipid hydrolases and transferases. We have previously proposed that AXR4 may either act as (i) a post-translational modifying enzyme through its α/β hydrolase fold domain or (ii) an ER accessory protein, which is a special class of ER protein that regulates targeting of their cognate partner proteins. Here, we show that AXR4 is unlikely to act as a post-translational modifying enzyme as mutations in several highly conserved amino acids in the α/β hydrolase fold domain can be tolerated and active site residues are missing. We also show that AUX1 and AXR4 physically interact with each other and that AXR4 reduces aggregation of AUX1 in a dose-dependent fashion. Our results suggest that AXR4 acts as an ER accessory protein. A better understanding of AXR4-mediated trafficking of auxin transporters in crop plants will be crucial for improving root traits (designer roots) for better acquisition of water and nutrients for sustainable and resilient agriculture.

Uncovering the role of Rad51 in homologous recombination-mediated antigenic diversification in the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum maintains the chronicity of infections through antigenic variation, a well-coordinated immune evasion mechanism. The most prominent molecular determinant of antigenic variation in this parasite includes the members of the var multigene family. Homologous recombination (HR)-mediated genomic rearrangements have been implicated to play a major role in var gene diversification. However, the key molecular factors involved in the generation of diversity at var loci are less known. Here, we tested the hypothesis that PfRad51 could carry out recombination between var genes that are not homologous but homeologous in nature. We employed the whole-genome sequencing (WGS) approach to investigate recombination events among var sequences over 100 generations and compared the rate of sequence rearrangement at the var loci in both PfRad51-proficient and -deficient parasite lines. This brief report provides evidence that the loss of the key recombinase function renders the parasite with inefficient HR and results in fewer recombination events among the var sequences, thereby impacting the diversification of the var gene repertoire.

Twist1‑mediated transcriptional activation of Claudin‑4 promotes cervical cancer cell migration and invasion.

Claudin-4, a member of the claudin multigene family, participates in events associated with mesenchymal-like activity of cancerous cells. Claudin-4 expression is upregulated in cervical cancer tissue compared with that in adjoining non-neoplastic tissue. However, the mechanisms that regulate Claudin-4 expression in cervical cancer are poorly understood. Moreover, whether Claudin-4 contributes to the migration and invasion of cervical cancer cells remains unclear. By western blotting, reverse transcription-qPCR, bioinformatics analysis, dual-luciferase reporter assay, chromatin immunoprecipitation assay, wound healing assay and Transwell migration/invasion assay, the present study confirmed that Claudin-4 was a downstream target of Twist1, a helix-loop-helix transcriptional factor, the activity of which has a positive correlation with Claudin-4 expression. Mechanistically, Twist1 directly binds to Claudin-4 promoter, resulting in the transactivation of expression. The depletion of the Twist1-binding E-Box1 domain on Claudin-4 promoter via CRISPR-Cas9 knockout system downregulates Claudin-4 expression and suppresses the ability of cervical cancer cells to migrate and invade by elevating E-cadherin levels and lowering N-cadherin levels. Following activation by transforming growth factor-β, Twist1 induces Claudin-4 expression, thus enhancing migration and invasion of cervical cancer cells. In summary, the present data suggested that Claudin-4 was a direct downstream target of Twist1 and served a critical role in promoting Twist1-mediated cervical cancer cell migration and invasion.

Disruption of a DUF247 Containing Protein Alters Cell Wall Polysaccharides and Reduces Growth in Arabidopsis.

Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughout Arabidopsis, particularly in vascular and developing tissues, and its disruption affects the expression of other gene members, indicating a regulatory control role within the gene family. Our results demonstrate that DUF247-1 is required for normal cell wall composition and structure and Arabidopsis growth.

Cucumis sativus PHLOEM PROTEIN 2-A1 like gene positively regulates salt stress tolerance in cucumber seedlings.

PHLOEM PROTEIN 2-A1 like (PP2-A1) gene is a member of the PP2 multigene family, and the protein encoded by which has the function of stress defense. Based on our previous proteomic study of cucumber phloem sap, CsPP2-A1 protein expression was significantly enriched under salt stress. In this paper, we obtained CsPP2-A1 interfering (CsPP2-A1-RNAi) cucumber by Agrobacterium tumefaciens-mediated method. The phenotypic changes of wild-type (WT) cucumber, CsPP2-A1-overexpressing (OE) cucumber, and CsPP2-A1-RNAi cucumber under salt treatment were observed and compared. Furthermore, physiological indicators were measured in four aspects: osmoregulation, membrane permeability, antioxidant system, and photosynthetic system. The analysis of contribution and correlation for each variable were conducted by principal component analysis (PCA) and Pearson's correlation coefficient. The above results showed that CsPP2-A1-RNAi cucumber plants exhibited weaker salt tolerance compared to WT cucumber and CsPP2-A1-OE cucumber plants in terms of phenotype and physiological indicators in response to salt stress, while CsPP2-A1-OE cucumber always showed the robust salt tolerance. Together, these results indicated that CsPP2-A1 brought a salinity tolerance ability to cucumber through osmoregulation and reactive oxygen species (ROS) homeostasis. The results of the study provided evidence for the function of CsPP2-A1 in plant salt tolerance enhancement, and they will serve as a reference for future salt-tolerant cucumber genetic manipulation.

Comparative genomic and transcriptomic analyses of African swine fever virus strains

African swine fever (ASF) is the most devastating disease caused by the African swine fever virus (ASFV), impacting the pig industry worldwide and threatening food security and biodiversity. Although two vaccines have been approved in Vietnam to combat ASFV, the complexity of the virus, with its numerous open reading frames (ORFs), necessitates a more diverse vaccine strategy. Therefore, we focused on identifying and investigating the potential vaccine targets for developing a broad-spectrum defense against the virus. This study collected the genomic and/or transcriptomic data of different ASFV strains, specifically from in vitro studies, focusing on comparisons between genotypes I, II, and X, from the National Center for Biotechnology Information (NCBI) database. The comprehensive analysis of the genomic and transcriptomic differences between high- and low-virulence strains revealed six early genes, 13 late genes, and six short genes as potentially essential ORFs associated with high-virulence. In addition, many other ORFs (e.g., 14 multigene family members) are worth investigating. The results of this study provided candidate ORFs for developing ASF vaccines and therapies.

Evolutionary analysis of the LORELEI gene family in plants reveals regulatory subfunctionalization.

A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, reproduction, immunity, and stress responses in Arabidopsis (Arabidopsis thaliana). Genes encoding these proteins are members of multigene families in most angiosperms and could generate thousands of signaling complex variants. However, the links between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution by sampling land plant genomes and explored the function and expression of angiosperm LLGs. We found that LLG diversity within major land plant lineages is primarily due to lineage-specific duplication events, and that these duplications occurred both early in the history of these lineages and more recently. Our complementation and expression analyses showed that expression divergence (i.e. regulatory subfunctionalization), rather than functional divergence, explains the retention of LLG paralogs. Interestingly, all but one monocot and all eudicot species examined had an LLG copy with preferential expression in male reproductive tissues, while the other duplicate copies showed highest levels of expression in female or vegetative tissues. The single LLG copy in Amborella trichopoda is expressed vastly higher in male compared to in female reproductive or vegetative tissues. We propose that expression divergence plays an important role in retention of LLG duplicates in angiosperms.

A single point mutation in a member of FAD2 multigene family resulted in the generation of a high oleic line of Silybum marianum (L.) Gaertn.

Silybum marianum (L.) Gaertn. has been recently valorised as oilseed crop for its productivity and for the chemical composition of its oil. However, a high oleic acid oil (HO) line, which would be useful for cooking but also for biodiesel production and other industrial applications, was not found in its natural germplasm. To address this issue, an ethyl methanesulfonate-induced mutagenesis programme was implemented, and the screening of the mutagenized population led to the identification of a HO mutant line with a much higher oleic acid oil content with respect to the wild type (WT). Since Fatty Acid Desaturase 2 (FAD2) genes are responsible for the determination of oleic acid levels in vegetable oils of other species, the present research focused on the characterization of this gene family as the possible target of mutagenesis. An extremely large multigene family consisting of twenty-one unique and complete FAD2 orthologous sequences was identified in S. marianum. Sequence comparison of all the SmFAD2 genes between the WT and HO genotypes revealed a single nucleotide polymorphism (SNP) in SmFAD2–1, the only fruit-specific gene, resulting in a non-synonymous substitution in the predicted protein in the high oleic genotype. The SNP and the HO phenotype were found to perfectly cosegregate in the F2 progeny derived from the HO x WT crosses, suggesting the direct implication of this gene in the determination of oleic and linoleic acid fruit content in S. marianum fruits.

Intracellular Localization and Gene Expression Analysis Provides New Insights on LEA Proteins' Diversity in Anhydrobiotic Cell Line.

Simple SummaryPolypedilum vanderplanki (sleeping chironomid) is widely known for its ability to withstand complete desiccation in a state of anhydrobiosis. The genome of this insect contains a number of hugely expanded paralogous gene groups, including 27 genes that encode late embryogenesis abundant (LEA) proteins. An important question regarding such paralogous genes is whether they are functionally specialized or not. Previously, we found that PvLEA proteins in C-terminal fusions with green fluorescent protein (AcGFP1) have four distinct localization types in mammalian cells. In the current paper, we studied PvLEA expression and localization in both N- and C-terminal fusions with AcGFP1 in anhydrobiotic Pv11 cells, derived from P. vanderplanki. We found that all but two PvLea genes are expressed in Pv11 cells and are upregulated during anhydrobiosis-inducing trehalose treatment similarly to the larvae of P. vanderplanki during the real induction of anhydrobiosis. We found that the localization of PvLEA proteins in N-terminal fusions with AcGFP1 is highly uniform in Pv11 cells and the Sf9 insect cell line. We observed an inconsistency of PvLEA localization between different cell cultures and between N- and C-terminal fusions, that needs to be taken into account when using PvLEA in the engineering of anhydrobiotic cell lines.Anhydrobiosis, an adaptive ability to withstand complete desiccation, in the nonbiting midge Polypedilum vanderplanki, is associated with the emergence of new multimember gene families, including a group of 27 genes of late embryogenesis abundant (LEA) proteins (PvLea). To obtain new insights into the possible functional specialization of these genes, we investigated the expression and localization of PvLea genes in a P. vanderplanki-derived cell line (Pv11), capable of anhydrobiosis. We confirmed that all but two PvLea genes identified in the genome of P. vanderplanki are expressed in Pv11 cells. Moreover, PvLea genes are induced in Pv11 cells in response to anhydrobiosis-inducing trehalose treatment in a manner highly similar to the larvae of P. vanderplanki during the real induction of anhydrobiosis. Then, we expanded our previous data on PvLEA proteins localization in mammalian cells that were obtained using C-terminal fusions of PvLEA proteins and green fluorescent protein (GFP). We investigated PvLEA localization using N- and C-terminal fusions with GFP in Pv11 cells and the Sf9 insect cell line. We observed an inconsistency of PvLEA localization between different fusion types and different cell cultures, that needs to be taken into account when using PvLEA in the engineering of anhydrobiotic cell lines.

Genome-wide identification of expansin in Fragaria vesca and expression profiling analysis of the FvEXPs in different fruit development.

Strawberry is a highly efficient and economical horticultural crop plant, and strawberry fruits are easy to soften after ripening and decay after harvest, which severely impacts the economic benefits. Expansins are plant cell-wall loosening proteins involved in the process of fruit softening, loosening cell walls and reducing fruit firmness. In this study, 35 FvEXPs genes were identified in the F. vesaca genome. These genes were divided into four subfamilies (27 FvEXPAs, 5 FvEXPBs, 1 FvEXLAs, and 2 FvEXLBs) and were unevenly distributed on 7 chromosomes. Gene structure and motif analysis showed the conserved structure and motif in same subgroup, however, the different motifs and structures may reveal functional divergence of multigene family members of FvEXPs in different developmental stages of fruits. The expression profiling by RNA-seq and qRT-PCR analysis revealed that the FvEXP genes have distinct expression patterns among different stages of strawberry development and ripening. Among them, 3 genes (FvEXPA9, FvEXPA12, and FvEXPA27) were highly expressed in the ripening stage, FvEXPA9 and FvEXPA12 were especially highly expressed in turning stage, whereas FvEXPA27 was especially highly expressed in red stage. Our study provides a better understanding of the FvEXP genes, which may benefit strawberry biotechnological breeding and genetic modification for improving fruit quality and delaying fruit softening.

Differential Effect of Deleting Members of African Swine Fever Virus Multigene Families 360 and 505 from the Genotype II Georgia 2007/1 Isolate on Virus Replication, Virulence, and Induction of Protection.

ABSTRACTAfrican swine fever virus multigene family (MGF) 360 and 505 genes have roles in suppressing the type I interferon response and in virulence in pigs. The role of the individual genes is poorly understood. Different combinations of these genes were deleted from the virulent genotype II Georgia 2007/1 isolate. Deletion of five copies of MGF 360 genes, MGF360-10L, -11L, -12L, -13L, and -14L, and three copies of MGF505-1R, -2R, and -3R reduced virus replication in macrophages and attenuated virus in pigs. However, only 25% of the immunized pigs were protected against challenge. Deletion of MGF360-12L, -13L, and -14L and MGF505-1R in combination with a negative serology marker, K145R (GeorgiaΔK145RΔMGF(A)), reduced virus replication in macrophages and virulence in pigs, since no clinical signs or virus genome in blood were observed following immunization. Four of six pigs were protected after challenge. In contrast, deletion of MGF360-13L and -14L, MGF505-2R and -3R, and K145R (GeorgiaΔK145RΔMGF(B)) did not reduce virus replication in macrophages. Following immunization of pigs, clinical signs were delayed, but all pigs reached the humane endpoint. Deletion of genes MGF360-12L, MGF505-1R, and K145R reduced replication in macrophages and attenuated virulence in pigs since no clinical signs or virus genome in blood were observed following immunization. Thus, the deletion of MGF360-12L and MGF505-1R, in combination with K145R, was sufficient to dramatically attenuate virus infection in pigs. However, only two of six pigs were protected, suggesting that deletion of additional MGF genes is required to induce a protective immune response. Deletion of MGF360-12L, but not MGF505-1R, from the GeorgiaΔK145R virus reduced virus replication in macrophages, indicating that MGF360-12L was most critical for maintaining high levels of virus replication in macrophages.IMPORTANCE African swine fever has a high socioeconomic impact and no vaccines to aid control. The African swine fever virus (ASFV) has many genes that inhibit the host’s interferon response. These include related genes that are grouped into multigene families, including MGF360 and 505. Here, we investigated which MGF360 and 505 genes were most important for viral attenuation and protection against genotype II strains circulating in Europe and Asia. We compared viruses with deletions of MGF genes. Deletion of just two MGF genes in combination with a third gene, K145R, a possible marker for vaccination, is sufficient for virus attenuation in pigs. Deletion of additional MGF360 genes was required to induce higher levels of protection. Furthermore, we showed that the deletion of MGF360-12L, combined with K145R, impairs virus replication in macrophages in culture. Our results have important implications for understanding the roles of the ASFV MGF genes and for vaccine development.

植物交配系统与分子进化理论及其应用研究

<p indent="0mm">With the transition of plant mating system from outcrossing to selfing, both the flower traits and genomic structure develop into selfing syndromes. Here we systematically discussed the theoretical basis and the molecular mechanisms that form these characteristics, and analyzed the basic theory of the interactions between mating system and different evolutionary processes. Selfing results in decrease in the effective population size, mutation rate, recombination rate and selection efficacy, but increase in genetic drift effects and linkage disequilibrium. These theories are effective in explaining the patterns of the relative rate of nonsynonymous to synonymous mutations, the change of the number of transposable elements, the genetic correlation between multigene family members and the pattern of genomic structure. Although molecular markers are often employed to score mating system, studies of mating system and molecular evolution have been mainly in different research fields. Some questions of biological significance could arise from an integration of these two research fields, such as the mode of driving or reinforcing molecular mechanism of speciation that the mating system operates, the different models of molecular evolution between populations of selfing versus outcrossing system, and the distinct genomic structure between populations of the same species with different mating systems. Clarification of these questions aids in our understanding of the role that mating system plays in affecting molecular evolution within and between species.

CD4 T Cell Responses to Theileria parva in Immune Cattle Recognize a Diverse Set of Parasite Antigens Presented on the Surface of Infected Lymphoblasts.

Parasite-specific CD8 T cell responses play a key role in mediating immunity against Theileria parva in cattle (Bos taurus), and there is evidence that efficient induction of these responses requires CD4 T cell responses. However, information on the antigenic specificity of the CD4 T cell response is lacking. The current study used a high-throughput system for Ag identification using CD4 T cells from immune animals to screen a library of ∼40,000 synthetic peptides representing 499 T. parva gene products. Use of CD4 T cells from 12 immune cattle, representing 12 MHC class II types, identified 26 Ags. Unlike CD8 T cell responses, which are focused on a few dominant Ags, multiple Ags were recognized by CD4 T cell responses of individual animals. The Ags had diverse properties, but included proteins encoded by two multimember gene families: five haloacid dehalogenases and five subtelomere-encoded variable secreted proteins. Most Ags had predicted signal peptides and/or were encoded by abundantly transcribed genes, but neither parameter on their own was reliable for predicting antigenicity. Mapping of the epitopes confirmed presentation by DR or DQ class II alleles and comparison of available T. parva genome sequences demonstrated that they included both conserved and polymorphic epitopes. Immunization of animals with vaccine vectors expressing two of the Ags demonstrated induction of CD4 T cell responses capable of recognizing parasitized cells. The results of this study provide detailed insight into the CD4 T cell responses induced by T. parva and identify Ags suitable for use in vaccine development.

PRAMEF2-mediated dynamic regulation of YAP signaling promotes tumorigenesis

PRAMEF2 is a member of the PRAME multigene family of cancer testis antigens, which serve as prognostic markers for several cancers. However, molecular mechanisms underlying its role in tumorigenesis remain poorly understood. Here, we report that PRAMEF2 is repressed under conditions of altered metabolic homeostasis in a FOXP3-dependent manner. We further demonstrate that PRAMEF2 is a BC-box containing substrate recognition subunit of Cullin 2-based E3 ubiquitin ligase complex. PRAMEF2 mediates polyubiquitylation of LATS1 kinase of the Hippo/YAP pathway, leading to its proteasomal degradation. The site for ubiquitylation was mapped to the conserved Lys860 residue in LATS1. Furthermore, LATS1 degradation promotes enhanced nuclear accumulation of the transcriptional coactivator YAP, resulting in increased expression of proliferative and metastatic genes. Thus, PRAMEF2 promotes malignant phenotype in a YAP-dependent manner. Additionally, elevated PRAMEF2 levels correlate with increased nuclear accumulation of YAP in advanced grades of breast carcinoma. These findings highlight the pivotal role of PRAMEF2 in tumorigenesis and provide mechanistic insight into YAP regulation.

Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host.

Trypanosoma brucei ssp., unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of T. brucei show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing. T. brucei brucei EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks. We present a genome assembly of EATRO1125, including contigs for the intermediate chromosomes and minichromosomes that serve as repositories of VSG genes. In addition, de novo transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes. Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus. Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified. Finally, RNA-Seq data were used to create an optimised annotation file with 5' and 3' untranslated regions accurately mapped for 9302 genes. We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.