MLVA typing of Brucella melitensis and B. abortus isolates of animal and human origin from India.

Brucellosis is a widely prevalent zoonotic disease of major public health significance. A collection of Brucella melitensis and Brucella abortus field isolates of animal and human origin were subjected to MLVA-15 typing followed by phylogeography studies. The MLVA-15 analysis of B. melitensis (n = 65) field isolates resulted in 48 different profiles. The panel I marker bruce45 was found to be most conserved, while the rest of the panel I markers showed low to moderate length polymorphism. Among the panel II markers, bruce04, bruce16 and bruce30 showed a high discriminatory index. The MLVA-15 typing of 13 B. abortus field isolates revealed 13 different genotypes with panel II markers showing higher discriminatory ability vis-à-vis panel I. The minimum spanning tree analysis (MST) in comparison with isolates from the international database revealed that all B. melitensis and B. abortus isolates from this study belonged to the 'Eastern Mediterranean' and the 'abortus C' lineage, respectively. The MLVA-15 typing could differentiate field isolates of B. abortus and B. melitensis originating from different regions, reaffirming the technique's potential of high resolution and suitability for local epidemiological studies. The MLVA scheme also has the advantage of comparison of local isolates with a worldwide database, allowing for phylogeographical studies.

Association mapping for important agronomic traits in wild and cultivated Vigna species using cross-species and cross-genera simple sequence repeat markers.

The genus Vigna is an agronomically important taxon, with many of its species inhabiting a wide range of environments and offering numerous useful genes for the improvement of the cultivated types. The present study aimed to detect the genomic regions associated with yield-attributing traits by genome-wide association mapping. A diverse panel of 98 wild and cultivated Vigna accessions (acc.) belonging to 13 different species was evaluated for yield and related traits during the kharif season of 2017 and 2018. The panel was also genotyped using 92 cross-genera and cross-species simple sequence repeat markers to study the population genetic structure and useful market-trait associations. The PCA and trait correlation established relationships amongst the traits during both seasons while 100-seed weight (HSW) had a positive correlation with pod length (PL), and days to first flowering (DFF) with days to maturity (DM). The population genetic structure analysis grouped different acc. into three genetically distinct sub-populations with SP-1 comprising 34 acc., SP-2 (24 acc.), and SP-3 (33 acc.) and one admixture group (7 acc.). Mixed linear model analysis revealed an association of 13 markers, namely, VR018, VR039, VR022, CEDG033, GMES0337, MBSSR008, CEDG220, VM27, CP1225, CP08695, CEDG100, CEDG008, and CEDG096A with nine traits. Seven of the aforementioned markers, namely, VR018 for plant height (PH) and terminal leaflet length (TLL), VR022 for HSW and pod length (PL), CEDG033 for DFF and DM, MBSSR008 for DFF and DM, CP1225 for CC at 30 days (CC30), DFF and DM, CEDG100 for PH and terminal leaflet length (TLL), and CEDG096A for CC30 and chlorophyll content at 45 days were associated with multiple traits. The marker CEDG100, associated with HSW, PH, and TLL, is co-localized in gene-encoding histone-lysine N-methyltransferase ATX5. Similarly, VR22, associated with PL and HSW, is co-located in gene-encoding SHOOT GRAVITROPISM 5 in mungbean. These associations may be highly useful for marker-assisted genetic improvement of mungbean and other related Vigna species.

Insights into the genetic diversity of an underutilized Indian legume, Vigna stipulacea (Lam.) Kuntz., using morphological traits and microsatellite markers.

Vigna stipulacea (Lam.) Kuntz., commonly known as Minni payaru is an underutilized legume species and has a great potential to be utilized as food crop. To evaluate and select the best germplasm to be harnessed in the breeding programme, we assessed the genetic diversity of V. stipulacea (94 accessions) conserved in the Indian National Genebank, based on morphological traits and microsatellite markers. Significant variation was recorded for the morphological traits studied. Euclidean distance using UPGMA method grouped all accessions into two major clusters. Accessions were identified for key agronomic traits such as, early flowering (IC331436, IC251436, IC331437); long peduncle length (IC553518, IC550531, IC553557, IC553540, IC550532, IC553564); and more number of seeds per pod (IC553529, IC622865, IC622867, IC553528). To analyse the genetic diversity among the germplasm 33 SSR primers were used anda total of 116 alleles were detected. The number of alleles varied from two to seven, with an average of 3.52 per loci. The polymorphic information content values varied from 0.20 to 0.74, with a mean of 0.40. The high number of alleles per locus and the allelic diversity in the studied germplasm indicated a relatively wider genetic base of V. stipulacea. Phylogenetic analysis clustered accessions into seven clades. Population structure analysis grouped them into five genetic groups, which were partly supported by PCoA and phylogenetic tree. Besides, PCoA and AMOVA also decoded high genetic diversity among the V. stipulacea accessions. Thus, morphological and microsatellite markers distinguished V. stipulacea accessions and assessed their genetic diversity efficiently. The identified promising accessions can be utilized in Vigna improvement programme through introgression breeding and/or can be used for domestication and enhanced utilization of V. stipulacea.

Genetic diversity and population genetic structure analysis of an extensive collection of wild and cultivated Vigna accessions.

Vigna is a large, pan-tropic and highly variable group of the legumes family which is known for its > 10 cultivated species having significant commercial value for their nutritious grains and multifarious uses. The wild vignas are considered a reservoir of numerous useful traits which can be deployed for introgression of resistance to biotic and abiotic stresses, seed quality and enhanced survival capability in extreme environments. Nonetheless, for their effective utilization through introgression breeding information on their genetic diversity, population structure and crossability is imperative. Keeping this in view, the present experiment was undertaken with 119 accessions including 99 wild Vigna accessions belonging to 19 species and 18 cultivated genotypes of Vigna and 2 of Phaseolus. Total 102 polymorphic SSRs were deployed to characterize the material at molecular level which produced 1758 alleles. The genotypes were grouped into four major clusters which were further sub-divided in nine sub-clusters. Interestingly, all cultivated species shared a single cluster while no such similarities were observed for the wild accessions as these were distributed in different groups of sub-clusters. The co-dominant allelic data of 114 accessions were then utilized for obtaining status of the accessions and their hybrid forms. The model-based population structure analysis categorized 114 accessions of Vigna into 6 genetically distinct sub-populations (K = 6) following admixture-model based simulation with varying levels of admixture. 91 (79.82%) accessions resembled their hierarchy and 23 (20.18%) accessions were observed as the admixture forms. Maximum number of accessions (25) were grouped in sub-population (SP) 6 and the least accessions were grouped in SP3 and SP5 (11 each). The population genetic structure, therefore, supported genetic diversity analysis and provided an insight into the genetic lineage of these species which will help in effective use of germplasm for development of cultivars following selective prebreeding activities.

Association Mapping for Yield Attributing Traits and Yellow Mosaic Disease Resistance in Mung Bean [Vigna radiata (L.) Wilczek].

Mung bean [Vigna radiata (L.) Wilczek] is an important short-duration grain legume widely known for its nutritional, soil ameliorative, and cropping system intensification properties. This study aims at evaluating genetic diversity among mung bean genotypes and detecting genomic regions associated with various yield attributing traits and yellow mosaic disease (YMD) resistance by association mapping. A panel of 80 cultivars and advanced breeding lines was evaluated for 10 yield-related and YMD resistance traits during kharif (monsoon) and summer seasons of 2018-2019 and 2019-2020. A total of 164 genome-wide simple sequence repeat (SSR) markers were initially screened, out of which 89 were found polymorphic which generated 317 polymorphic alleles with an average of 3.56 alleles per SSR locus. The number of alleles at each locus varied from 2 to 7. The population genetic structure analysis grouped different genotypes in three major clusters and three genetically distinct subpopulations (SPs) (i.e., SP-1, SP-2, and SP-3) with one admixture subpopulation (SP-4). Both cluster and population genetic structure analysis categorized the advanced mung bean genotypes in a single group/SP and the released varieties in other groups/SPs, suggesting that the studied genotypes may have common ancestral history at some level. The population genetic structure was also in agreement with the genetic diversity analysis. The estimate of the average degree of linkage disequilibrium (LD) present at the genome level in 80 mung bean genotypes unveiled significant LD blocks. Over the four seasons, 10 marker-trait associations were observed significant for YMD and four seed yield (SY)-related traits viz., days to flowering, days to maturity, plant height, and number of pods per plant using the mixed linear model (MLM) method. These associations may be useful for marker-assisted mung bean yield improvement programs and YMD resistance.

Misregulation of the IgH Locus in Thymocytes.

Functional antigen receptor genes are assembled by somatic rearrangements that are largely lymphocyte lineage specific. The immunoglobulin heavy chain (IgH) gene locus is unique amongst the seven antigen receptor loci in undergoing partial gene rearrangements in the wrong lineage. Here we demonstrate that breakdown of lineage-specificity is associated with inappropriate activation of the Eµ enhancer during T cell development by a different constellation of transcription factors than those used in developing B cells. This is reflected in reduced enhancer-induced epigenetic changes, eRNAs, formation of the RAG1/2-rich recombination center, attenuated chromatin looping and markedly different utilization of DH gene segments in CD4+CD8+ (DP) thymocytes. Additionally, CTCF-dependent VH locus compaction is disrupted in DP cells despite comparable transcription factor binding in both lineages. These observations identify multiple mechanisms that contribute to lineage-specific antigen receptor gene assembly.

The RAG-2 Inhibitory Domain Gates Accessibility of the V(D)J Recombinase to Chromatin.

Accessibility of antigen receptor loci to RAG is correlated with the presence of H3K4me3, which binds to a plant homeodomain (PHD) in the RAG-2 subunit and promotes V(D)J recombination. A point mutation in the PHD, W453A, eliminates binding of H3K4me3 and impairs recombination. The debilitating effect of the W453A mutation is ameliorated by second-site mutations that locate an inhibitory domain in the interval from residues 352 through 405 of RAG-2. Disruption of the inhibitory domain stimulates V(D)J recombination within extrachromosomal substrates and at endogenous antigen receptor loci. Association of RAG-1 and RAG-2 with chromatin at the IgH locus in B cell progenitors is dependent on recognition of H3K4me3 by the PHD. Strikingly, disruption of the inhibitory domain permits association of RAG with the IgH locus in the absence of H3K4me3 binding. Thus, the inhibitory domain acts as a gate that prohibits RAG from accessing the IgH locus unless RAG-2 is engaged by H3K4me3.

Sequential Enhancer Sequestration Dysregulates Recombination Center Formation at the IgH Locus.

Immunoglobulin heavy-chain (IgH) genes are assembled by DNA rearrangements that juxtapose a variable (VH), a diversity (DH), and a joining (JH) gene segment. Here, we report that in the absence of intergenic control region 1 (IGCR1), the intronic enhancer (Eµ) associates with the next available CTCF binding site located close to VH81X via putative heterotypic interactions involving YY1 and CTCF. The alternate Eµ/VH81X loop leads to formation of a distorted recombination center and altered DH rearrangements and disrupts chromosome conformation that favors distal VH recombination. Cumulatively, these features drive highly skewed, Eµ-dependent recombination of VH81X. Sequential deletion of CTCF binding regions on IGCR1-deleted alleles suggests that they influence recombination of single proximal VH gene segments. Our observations demonstrate that Eµ interacts differently with IGCR1- or VH-associated CTCF binding sites and thereby identify distinct roles for insulator-like elements in directing enhancer activity.

Chromatin Interactions in the Control of Immunoglobulin Heavy Chain Gene Assembly.

Expression of antibody heavy chain occurs via precisely timed developmental activation of the immunoglobulin heavy chain (IgH) gene locus during B cell development. IgH locus activation permits coordinated gene rearrangements that assemble variable (VH), diversity (DH), and joining (JH) gene segments into functional genes. Chromosomal conformation changes and epigenetic mechanisms play critical roles in ensuring rearrangement fidelity while minimizing hazardous consequences of broken DNA ends generated during recombination. In this review, we summarize the current status of regulatory mechanisms that underpin effective IgH gene assembly. For this, the germline locus is divided into two parts: a 2.4Mb 5' part that contains all VH gene segments and a 300kb 3' domain that contains DH and JH gene segments, as well as exons that encode IgH isotypes. Structural features of each part are discussed individually, followed by consideration of how the two parts come together to complete IgH recombination. Throughout we emphasize current insights, propose plausible mechanisms, and highlight key questions for future studies.

Functional interaction of the Ras effector RASSF5 with the tyrosine kinase Lck: critical role in nucleocytoplasmic transport and cell cycle regulation.

RASSF5 is a member of the Ras association domain family, which is known to be involved in cell growth regulation. Expression of RASSF5 is extinguished selectively by epigenetic mechanism(s) in different cancers and cell lines, and reexpression usually suppresses cell proliferation and tumorigenicity. To date, the mechanism regulating RASSF5 nuclear transport and its role in cell growth regulation remains unclear. Using heterokaryon assay, we have demonstrated that RASSF5 shuttles between the nucleus and the cytoplasm, and its export from the nucleus is sensitive to leptomycin B, suggesting that RASSF5 is exported from the nucleus by a CRM-1-dependent export pathway. We further demonstrate that RASSF5 contains a hydrophobic-rich nuclear export signal (NES) towards the C-terminus and two nuclear localization signals-one each at the N-terminus and the C-terminus. Combination of mutational and immunofluorescence analyses suggests that the functional NES residing between amino acids 260 and 300 in the C-terminus is necessary for the efficient export of RASSF5 from the nucleus. In addition, substitution of conserved hydrophobic residues within the minimal NES impaired RASSF5 export from the nucleus. Furthermore, exchange of proline residues within the putative Src homology 3 binding motifs altered the export of RASSF5 from the nucleus despite the presence of functional NES, suggesting that multiple domains independently modulate the nucleocytoplasmic transport of RASSF5. Interestingly, the present investigation provided evidence that RASSF5 interacts with the tyrosine kinase Lck through its C-terminal Src homology 2 binding motif and showed that Lck-mediated phosphorylation is critical for the efficient translocation of RASSF5 into the nuclear compartment. Interestingly, our data demonstrate that wild type and nuclear export defective (DeltaNES) mutant of RASSF5 but not the import defective mutant of accumulate the cells at G1/S phase and induce apoptosis. Furthermore, the Lck-interaction-defective mutant of RASSF5 induces apoptosis without altering cell cycle progression, suggesting that RASSF5 induces apoptosis independent of cell cycle arrest. Together, our data demonstrate that interaction with Lck is critical for RASSF5 phosphorylation, which in turn regulates the cell growth control activity of RASSF5. Finally, we have shown that RASSF5 encodes four splice variants and is translocated to the nucleus by the classical nuclear import pathway. One of the splice variants, RASSF5C, was found to be localized in the cytoplasm and translocated into the nucleus upon leptomycin B treatment despite the absence of N-terminal nuclear localization signal, suggesting that distribution of RASSF5 variants in different cellular compartments may be critical for Ras-dependent cell growth regulation. Collectively, the present investigation provided evidence that Lck-mediated phosphorylation regulates the nucleocytoplasmic shuttling and cell growth control activities of RASSF5.

Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2.

Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates nucleo-cytoplasmic transport and cell growth arrest activity of RASSF2. Taken together, the present study suggests that active transport between nucleus and cytoplasm may constitute an important regulatory mechanism for RASSF2 function.

Nuclear transport of Ras-associated tumor suppressor proteins: different transport receptor binding specificities for arginine-rich nuclear targeting signals.

Ras proteins regulate a wide range of biological processes by interacting with a variety of effector proteins. In addition to the known role in tumorigensis, the activated form of Ras exhibits growth-inhibitory effects by unknown mechanisms. Several Ras effector proteins identified as mediators of apoptosis and cell-cycle arrest also exhibit properties normally associated with tumor suppressor proteins. Here, we show that Ras effector RASSF5/NORE-1 binds strongly to K-Ras but weakly to both N-Ras and H-Ras. RASSF5 was found to localize both in the nucleus and the nucleolus in contrast to other Ras effector proteins, RASSF1C and RASSF2, which are localized in the nucleus and excluded from nucleolus. A 50 amino acid residue transferable arginine-rich nucleolar localization signal (NoLS) identified in RASSF5 is capable of interacting with importin-beta and transporting the cargo into the nucleolus. Surprisingly, similar arginine-rich signals identified in RASSF1C and RASSF2 interact with importin-alpha and transport the heterologous cytoplasmic proteins to the nucleus. Interestingly, mutation of arginine residues within these nuclear targeting signals prevented interaction of Ras effector proteins with respective transport receptors and abolished their nuclear translocation. These results provide evidence for the first time that arginine-rich signals are able to recognize different nuclear import receptors and transport the RASSF proteins into distinct sub-cellular compartments. In addition, our data suggest that the nuclear localization of RASSF5 is critical for its cell growth control activity. Together, these data suggest that the transport of Ras effector superfamily proteins into the nucleus/nucleolus may play a vital role in modulating Ras-mediated cell proliferation during tumorigenesis.

A novel lysine-rich domain and GTP binding motifs regulate the nucleolar retention of human guanine nucleotide binding protein, GNL3L.

A variety of G-proteins and GTPases are known to be involved in nucleolar function. We describe here a new evolutionarily conserved putative human GTPase, guanine nucleotide binding protein-like 3-like (GNL3L). Genes encoding proteins related to GNL3L are present in bacteria and yeast to metazoa and suggests its critical role in development. Conserved domain search analysis revealed that the GNL3L contains a circularly permuted G-motif described by a G5-G4-G1-G2-G3 pattern similar to the HSR1/MMR1 GTP-binding protein subfamily. Highly conserved and critical residues were identified from a three-dimensional structural model obtained for GNL3L using the crystal structure of an Ylqf GTPase from Bacillus subtilis. We demonstrate here that GNL3L is transported into the nucleolus by a novel lysine-rich nucleolar localization signal (NoLS) residing within 1-50 amino acid residues. NoLS identified here is necessary and sufficient to target the heterologous proteins to the nucleolus. We show for the first time that the lysine-rich targeting signal interacts with the nuclear transport receptor, importin-beta and transports GNL3L into the nucleolus. Interestingly, depletion of intracellular GTP blocks GNL3L accumulation into the nucleolar compartment. Furthermore, mutations within the G-domains alter the GTP binding ability of GNL3L and abrogate wild-type nucleolar retention even in the presence of functional NoLS, suggesting that the efficient nucleolar retention of GNL3L involves activities of both basic NoLS and GTP-binding domains. Collectively, these data suggest that GNL3L is composed of distinct modules, each of which plays a specific role in molecular interactions for its nucleolar retention and subsequent function(s) within the nucleolus.