Ribosomal Gene Family Research Articles

Dispersal from the Qinghai-Tibet plateau by a high-altitude butterfly is associated with rapid expansion and reorganization of its genome

Parnassius glacialis is a typical “Out of the QTP” alpine butterfly that originated on the Qinghai-Tibet Plateau (QTP) and dispersed into relatively low-altitude mountainous. Here we assemble a chromosome-level genome of P. glacialis and resequence 9 populations in order to explore the genome evolution and local adaptation of this species. These results indicated that the rapid accumulation and slow unequal recombination of transposable elements (TEs) contributed to the formation of its large genome. Several ribosomal gene families showed extensive expansion and selective evolution through transposon-mediated processed pseudogenes. Additionally, massive structural variations (SVs) of TEs affected the genetic differentiation of low-altitude populations. These low-altitude populations might have experienced a genetic bottleneck in the past and harbor genes with selective signatures which may be responsible for the potential adaptation to low-altitude environments. These results provide a foundation for understanding genome evolution and local adaptation for “Out of the QTP” of P. glacialis.

Chromosomal mapping of ribosomal clusters and telomeric sequences (TTAGG) n in nine species of lobsters (Crustacea, Decapoda)

Lobsters are ubiquitous, economically important decapod crustaceans with apparently conflicting evolutionary relationships. Here, we describe the chromosomal location of the major (45S rDNA) and minor (5S rDNA) ribosomal gene families in four species of Astacidea and five of Achelata, using two-color FISH. The major ribosomal family is located in 4–16 sites per diploid chromosome set, with Nephrops norvegicus (Nephropidae) showing the highest number described so far in Decapoda. The 5S rDNA is located in two sites in eight species; only in the crayfish Procambarus clarkii the 5S FISH signals were detected in four sites together with additional weaker signals. Furthermore, in N. norvegicus the minor ribosomal genes are syntenic with one major ribosomal cluster. Moreover, we located by two-color FISH the pentanucleotide (TTAGG) n telomeric repeat in the Nephropidae studied, showing the occurrence of a colocalization with 45S ribosomal sequences in Homarus gammarus. The comparison of chromosomal locations of repetitive sequences in Mediterranean, Atlantic, and South African lobster species as well as in marine and freshwater ones provides information on chromosomal evolution and cytotaxonomy of Decapoda.

Chromosome Mapping of 5S Ribosomal Genes in Indo-Pacific and Atlantic Muraenidae: Comparative Analysis by Dual Colour Fluorescence In Situ Hybridisation.

The Muraenidae is one of the largest and most complex anguilliform families. Despite their abundance and important ecological roles, morays are little studied, especially cytogenetically, and both their phylogenetic relationships and the taxonomy of their genera are controversial. With the aim of extending the karyology of this fish group, the chromosomal mapping of the 5S ribosomal gene family was performed on seven species belonging to the genera Muraena and Gymnothorax from both the Atlantic and Pacific oceans. Fluorescence in situ hybridisation (FISH) experiments were realized using species-specific 5S rDNA probes; in addition, two-colour FISH was performed to investigate the possible association with the 45S ribosomal gene family. Multiple 5S rDNA clusters, located either in species-specific or in possibly homoeologous chromosomes, were found. Either a syntenic or different chromosomal location of the two ribosomal genes was detected. Our results revealed variability in the number and location of 5S rDNA clusters and confirmed a substantial conservation of the number and location of the 45S rDNA.

Overexpression of the Selective Autophagy Cargo Receptor NBR1 Modifies Plant Response to Sulfur Deficit.

Plants exposed to sulfur deficit elevate the transcription of NBR1 what might reflect an increased demand for NBR1 in such conditions. Therefore, we investigated the role of this selective autophagy cargo receptor in plant response to sulfur deficit (-S). Transcriptome analysis of the wild type and NBR1 overexpressing plants pointed out differences in gene expression in response to -S. Our attention focused particularly on the genes upregulated by -S in roots of both lines because of significant overrepresentation of cytoplasmic ribosomal gene family. Moreover, we noticed overrepresentation of the same family in the set of proteins co-purifying with NBR1 in -S. One of these ribosomal proteins, RPS6 was chosen for verification of its direct interaction with NBR1 and proven to bind outside the NBR1 ubiquitin binding domains. The biological significance of this novel interaction and the postulated role of NBR1 in ribosomes remodeling in response to starvation remain to be further investigated. Interestingly, NBR1 overexpressing seedlings have significantly shorter roots than wild type when grown in nutrient deficient conditions in the presence of TOR kinase inhibitors. This phenotype probably results from excessive autophagy induction by the additive effect of NBR1 overexpression, starvation, and TOR inhibition.

Cytogenetics of Fraxinus mandshurica and F. quadrangulata: ploidy determination and rDNA analysis

Ashes (Fraxinus spp.) are important hardwood tree species in rural, suburban, and urban forests of the eastern USA. Unfortunately, emerald ash borer (EAB, Agrilus planipennis) an invasive insect pest that was accidentally imported from Asia in the late 1980s–early 1990s is destroying them at an alarming rate. All North American ashes are highly susceptible to EAB, although blue ash (F. quadrangulata) may have some inherent attributes that provide it some protection. In contrast Manchurian ash (F. mandshurica) is relatively resistant to EAB having coevolved with the insect pest in its native range in Asia. Given its level of resistance, Manchurian ash has been considered for use in interspecies breeding programs designed to transfer resistance to susceptible North American ash species. One prerequisite for successful interspecies breeding is consistency in chromosome ploidy level and number between the candidate species. In the current study, we cytologically determined that both Manchurian ash and blue ash are diploids (2n) and have the same number of chromosomes (2n = 2x = 46). We also characterized these species’ ribosomal gene families (45S and 5S rDNA) using fluorescence in situ hybridization (FISH). Both Manchurian and blue ash showed two 45S rDNA and one 5S rDNA sites, but blue ash appears to have an additional site of 45S rDNA. The 5S rDNA in both species is colocalized interstitially with one of the 45S rDNA sites. The copy number of these two ribosomal gene families in Manchurian ash were observed to be quite varied, which indicates the species are still undergoing evolutionary homogenization.

Knockdown of ribosomal protein S15A inhibits human kidney cancer cell growth in�vitro and in�vivo

Ribosomal protein S15A (RPS15A), a member of the ribosomal protein gene family, was demonstrated to be closely associated with tumorigenesis in multiple human malignancies. Nevertheless, the role of RPS15A in the progression of renal cell carcinoma (RCC) remains unknown. In the present study, by comparing the publicly available data from RCC tissues and reverse transcription-quantitative polymerase chain reaction results, it was identified that RPS15A was upregulated in RCC tissues and cell lines (P<0.001). Notably, knockdown of RPS15A suppressed 786-O cell proliferation (P<0.001) and promoted its apoptosis/necrotic (P=0.0001) in vitro. Additionally, tumour formation and growth of transfected 786-O cells were observed to be restrained in a mouse model (P<0.05). Subsequent to analysing the microarray data, 747 genes were differentially expressed in the RPS15A-knockdown 786-O cells. The enriched canonical pathways, diseases and functions of differentially expressed genes, and the interactive network of RPS15A in RCC were successfully constructed by ingenuity pathway analysis. Overall, the present results provided a preliminary experimental basis for RPS15A as a novel oncogene and potential therapeutic target in RCC.

XX/XY sex chromosome system and chromosome markers in the snake eel Ophisurus serpens (Anguilliformes: Ophichtidae)

ABSTRACTWe report evidence of an XX/XY sex chromosome system in the snake eel Ophisurus serpens (Anguilliformes: Ophichthidae). We characterized the male and female karyotypes by C-, replication- and HaeIII-bandings. The 45S and 5S ribosomal gene families were located using dual fluorescence in situ hybridization, which showed that the 5S rDNA sites were present on the X chromosome, beside an autosome pair. FISH with a telomeric peptide nucleic acid probe enabled recognition of Interstitial Telomeric Sequences (ITSs), likely remnants of chromosomal rearrangements, in five chromosome pairs, including the rDNA-bearing ones. Possible mechanisms of the origin of sex chromosomes in this species are discussed, considering the presence of a sex-linked marker and ITSs.

Genetic diversity and phylogenetic relations of salmon trematode Nanophyetus japonensis

Nanophyetiasis is the severe zoonotic disease caused by parasitic worms from the genus Nanophyetus. Humans and carnivorous animals become infected when they ingest raw fish containing metacercariae, especially Pacific salmonids. Nanophyetiasis is detected in limited geographical areas which include the coastal regions of the North Pacific: the United States of America, Russian Federation and Japan. Despite the epidemiological significance, Nanophyetus species have not been well studied genetically. In this research, we for the first time explored genetic diversity of Nanophyetus japonensis from Japan in comparison with those of related species, N. salmincola from North America and N. schikhobalowi from the Russian Far East, based on sequence variation in the nuclear ribosomal gene family (18S, ITS1–5.8S–ITS2 and 28S) and mitochondrial nad1 gene, encoding subunit I of the respiratory chain NADH dehydrogenase. The results confirmed the independent species status for the compared flukes, demonstrated a greater genetic similarity of Asian species between themselves than each of them with the North American one, suggesting that N. japonensis and N. schikhobalowi are close sister species, and also revealed discrepancy between the levels of morphological and genetic differentiation.

Expression Profiling of Ribosomal Protein Gene Family in Dehydration Stress Responses and Characterization of Transgenic Rice Plants Overexpressing RPL23A for Water-Use Efficiency and Tolerance to Drought and Salt Stresses.

Our previous findings on the screening of a large-pool of activation tagged rice plants grown under limited water conditions revealed the activation of Ribosomal Protein Large (RPL) subunit genes, RPL6 and RPL23A in two mutants that exhibited high water-use efficiency (WUE) with the genes getting activated by the integrated 4x enhancers (Moin et al., 2016a). In continuation of these findings, we have comprehensively characterized the Ribosomal Protein (RP) gene family including both small (RPS) and large (RPL) subunits, which have been identified to be encoded by at least 70 representative genes; RP-genes exist as multiple expressed copies with high nucleotide and amino acid sequence similarity. The differential expression of all the representative genes in rice was performed under limited water and drought conditions at progressive time intervals in the present study. More than 50% of the RP genes were upregulated in both shoot and root tissues. Some of them exhibited an overlap in upregulation under both the treatments indicating that they might have a common role in inducing tolerance under limited water and drought conditions. Among the genes that became significantly upregulated in both the tissues and under both the treatments are RPL6, 7, 23A, 24, and 31 and RPS4, 10 and 18a. To further validate the role of RP genes in WUE and inducing tolerance to other stresses, we have raised transgenic plants overexpressing RPL23A in rice. The high expression lines of RPL23A exhibited low Δ13C, increased quantum efficiency along with suitable growth and yield parameters with respect to negative control under the conditions of limited water availability. The constitutive expression of RPL23A was also associated with transcriptional upregulation of many other RPL and RPS genes. The seedlings of RPL23A high expression lines also showed a significant increase in fresh weight, root length, proline and chlorophyll contents under simulated drought and salt stresses. Taken together, our findings provide a secure basis for the RPL gene family expression as a potential resource for exploring abiotic stress tolerant properties in rice.

Genetic divergence of human pathogens Nanophyetus spp. (Trematoda: Troglotrematidae) on the opposite sides of the Pacific Rim.

Human and animal nanophyetiasis is caused by intestinal flukes belonging to the genus Nanophyetus distributed on both North American and Eurasian coasts of Northern Pacific. In spite of the wide geographical distribution and medical and veterinary importance of these flukes, the intra-generic taxonomy of Nanophyetus spp. remains unresolved. The two most widely distributed nominal species, Nanophyetus salmincola and Nanophyetus schikhobalowi, both parasitizing humans and carnivorous mammals, were described from North America and eastern Eurasia, respectively. However, due to their high morphological similarity their interrelationships remained unclear and taxonomic status unstable. In this study, we explored genetic diversity of Nanophyetus spp. from the Southern Russian Far East in comparison with that of samples from North America based on the sequence variation of the nuclear ribosomal gene family (18S, internal transcribed spacers, ITS1-5·8S-ITS2 and 28S). High levels of genetic divergence in each rDNA region (nucleotide substitutions, indels, alterations in the secondary structures of the ITS1 and ITS2 transcripts) as well as results of phylogenetic analysis provided strong support for the status of N. salmincola and N. schikhobalowi as independent species.

AST: an automated sequence-sampling method for improving the taxonomic diversity of gene phylogenetic trees.

A challenge in phylogenetic inference of gene trees is how to properly sample a large pool of homologous sequences to derive a good representative subset of sequences. Such a need arises in various applications, e.g. when (1) accuracy-oriented phylogenetic reconstruction methods may not be able to deal with a large pool of sequences due to their high demand in computing resources; (2) applications analyzing a collection of gene trees may prefer to use trees with fewer operational taxonomic units (OTUs), for instance for the detection of horizontal gene transfer events by identifying phylogenetic conflicts; and (3) the pool of available sequences is biased towards extensively studied species. In the past, the creation of subsamples often relied on manual selection. Here we present an Automated sequence-Sampling method for improving the Taxonomic diversity of gene phylogenetic trees, AST, to obtain representative sequences that maximize the taxonomic diversity of the sampled sequences. To demonstrate the effectiveness of AST, we have tested it to solve four problems, namely, inference of the evolutionary histories of the small ribosomal subunit protein S5 of E. coli, 16 S ribosomal RNAs and glycosyl-transferase gene family 8, and a study of ancient horizontal gene transfers from bacteria to plants. Our results show that the resolution of our computational results is almost as good as that of manual inference by domain experts, hence making the tool generally useful to phylogenetic studies by non-phylogeny specialists. The program is available at http://csbl.bmb.uga.edu/~zhouchan/AST.php.

Early evolutionary colocalization of the nuclear ribosomal 5S and 45S gene families in seed plants: evidence from the living fossil gymnosperm Ginkgo biloba

In seed plants, the colocalization of the 5S loci within the intergenic spacer (IGS) of the nuclear 45S tandem units is restricted to the phylogenetically derived Asteraceae family. However, fluorescent in situ hybridization (FISH) colocalization of both multigene families has also been observed in other unrelated seed plant lineages. Previous work has identified colocalization of 45S and 5S loci in Ginkgo biloba using FISH, but these observations have not been confirmed recently by sequencing a 1.8 kb IGS. In this work, we report the presence of the 45S-5S linkage in G. biloba, suggesting that in seed plants the molecular events leading to the restructuring of the ribosomal loci are much older than estimated previously. We obtained a 6.0 kb IGS fragment showing structural features of functional sequences, and a single copy of the 5S gene was inserted in the same direction of transcription as the ribosomal RNA genes. We also obtained a 1.8 kb IGS that was a truncate variant of the 6.0 kb IGS lacking the 5S gene. Several lines of evidence strongly suggest that the 1.8 kb variants are pseudogenes that are present exclusively on the satellite chromosomes bearing the 45S-5S genes. The presence of ribosomal IGS pseudogenes best reconciles contradictory results concerning the presence or absence of the 45S-5S linkage in Ginkgo. Our finding that both ribosomal gene families have been unified to a single 45S-5S unit in Ginkgo indicates that an accurate reassessment of the organization of rDNA genes in basal seed plants is necessary.

Genome size and cytogenetic characterization of three Algerian Retama species

Thirty-three populations belonging to the three Retama species, Retama monosperma, Retama raetam and Retama sphaerocarpa, were collected to study species differentiation using flow cytometry for 2C DNA assessment and molecular cytogenetics for karyotype organisation. All were 2n = 48. Genome size ranged from 1.76 to 1.97 pg and revealed significant intraspecific variation correlated to the geographic distribution of the populations. The number and position of the two ribosomal gene families 5S and 45S were determined by fluorescent in situ hybridization, revealing chromosome reorganisation between species. In R. raetam and R. monosperma, the minor 5S loci co-localised with 45S on the satellite chromosome pair. Fluorochrome banding identified GC- and AT-rich DNA regions. In R. monosperma a unique chromomycin positive GC-rich band was observed associated with the secondary constriction. In contrast, an original pattern showing two chromomycin positive bands localised at each side of the extended rDNA locus was observed in R. sphaerocarpa and R. raetam. The polymorphism revealed in our cytogenetic data allowed us to separate the group of R. raetam and R. monosperma from R. sphaerocarpa.

Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of chromosomal and genomic relationship levels between faba bean (Vicia faba) and its close wild relatives

The organisation of DNA sequences at chromosomal level in twelve Vicia species, more particularly among V. faba and its close wild relatives (V. narbonensis, V. hyaeniscyamus, V. johannis, V. galilaea, V. bithynica), has been performed using fl uorescence in situ hybridization (FISH). The four BamHI discrete size classes of 850, 900, 990 and 1750-bp of highly repetitive DNA isolated from V. faba, and the probes pTa 71 and pTa 794 containing the 18S-5.8S-26S rDNA and 5S rDNA, respectively, were used. The chromosomal location of the two ribosomal gene families distinguished V. faba from its close relatives. FISH to metaphase chromosomes in- dicates the presence of BamHI family sequences interspersed in both euchromatin and heterochromatin of the V. faba, V. narbonensis, V. hyaeniscyamus, V. galilaea, V. johannis and V. bithynica of taxonomic section Faba, and V. hybrida of section Hypechusa genomes; however, the level of hybridization clearly distinguished the V. faba genome from other species genomes. This analysis provides further evidence that V. faba should be considered as the most distinct. In the context of possible sources of germplasm for V. faba breeding, it is argued that none of the species can be considered as putative allies of faba bean. The repetitive sequences did not hybridize in situ with the chromosomal DNA of fiVicia species of section Vicia and section Cracca. The quantitative variation and/or complete absence of BamHI family repeats enabled differentiation among Vicia genomes.

Evolutionary Dynamics of the 5S rDNA Gene Family in the Mussel Mytilus: Mixed Effects of Birth-and-Death and Concerted Evolution

In higher eukaryotes, the gene family encoding the 5S ribosomal RNA (5S rRNA) has been used (together with histones) to showcase the archetypal example of a gene family subject to concerted evolution. However, recent studies have revealed conspicuous features challenging the predictions of this model, including heterogeneity of repeat units, the presence of functional 5S gene variants as well as the existence of 5S rDNA divergent pseudogenes lacking traces of homogenization. In the present work, we have broadened the scope in the evolutionary study of ribosomal gene families by studying the 5S rRNA family in mussels, a model organism which stands out among other animals due to the heterogeneity it displays regarding sequence and organization. To this end, 48 previously unknown 5S rDNA units (coding and spacer regions) were sequenced in five mussel species, leading to the characterization of two new types of units (referred to here as small-beta 5S rDNA and gamma-5S rDNA) coexisting in the genome with alpha and beta rDNA units. The intense genetic dynamics of this family is further supported by the first description of an association between gamma-5S rDNA units and tRNA genes. Molecular evolutionary and phylogenetic analyses revealed an extensive lack of homology among spacer sequences belonging to different rDNA types, suggesting the presence of independent evolutionary pathways leading to their differentiation. Overall, our results suggest that the long-term evolution of the 5S rRNA gene family in mussels is most likely mediated by a mixed mechanism involving the generation of genetic diversity through birth-and-death, followed by a process of local homogenization resulting from concerted evolution in order to maintain the genetic identities of the different 5S units, probably after their transposition to independent chromosomal locations.

Karyotype analysis of the pumpkinseed Lepomis gibbosus (Actinopterygii, Centrarchidae) by chromosomal banding and in situ hybridization

Summary Sunfish are widely distributed in their native North American freshwaters and in many other geographic regions, including Europe. In this work the cytogenetics of L. gibbosus were studied. In particular, the authors localized the heterochromatic regions and the major and minor ribosomal gene families. The nucleolar organizer region was localized terminally in the short arm of only one pair, a large acrocentric pair, using both FISH and silver staining. Furthermore, the 5S ribosomal gene family was also localized by FISH in only one pair, in the centromeric region of the smallest chromosome pair. NOR and 5S rDNA regions were both C-positive and CMA3 positive. The CMA3 positivity of the 5S ribosomal cluster is uncommon in fish, however, a similar situation has been found in M. salmoides, the only other centrarchid species studied with the same techniques. Moreover, the 5S ribosomal gene was sequenced and its molecular structure analysed.

Validation of an NSP-based (negative selection pattern) gene family identification strategy

BackgroundGene family identification from ESTs can be a valuable resource for analysis of genome evolution but presents unique challenges in organisms for which the entire genome is not yet sequenced. We have developed a novel gene family identification method based on negative selection patterns (NSP) between family members to screen EST-generated contigs. This strategy was tested on five known gene families in Arabidopsis to see if individual paralogs could be identified with accuracy from EST data alone when compared to the actual gene sequences in this fully sequenced genome.ResultsThe NSP method uniquely identified family members in all the gene families tested. Two members of the FtsH gene family, three members each of the PAL, RF1, and ribosomal L6 gene families, and four members of the CAD gene family were correctly identified. Additionally all ESTs from the representative contigs when checked against MapViewer data successfully identify the gene locus predicted.ConclusionWe demonstrate the effectiveness of the NSP strategy in identifying specific gene family members in Arabidopsis using only EST data and we describe how this strategy can be used to identify many gene families in agronomically important crop species where they are as yet undiscovered.

Molecular evolutionary analyses of the Arabidopsis L7 ribosomal protein gene family

Cytoplasmic ribosomal protein (r-protein) genes in Arabidopsis thaliana are encoded by 80 multigene families that contain between two and seven members. Gene family members are typically similar at the protein sequence level, with the most divergent members of any gene family retaining 94% identity, on average. However, three Arabidopsis r-protein families – S15a, L7 and P2 – contain highly divergent family members. Here, we investigated the organization, structure, expression and molecular evolution of the L7 r-protein family. Phylogenetic analyses showed that L7 r-protein gene family members constitute two distinct phylogenetic groups. The first group including RPL7B, RPL7C and RPL7D has homologs in plants, animals and fungi. The second group represented by RPL7A is found in plants but has no orthologs from other fully-sequenced eukaryotic genomes. These two groups may have derived from a duplication event prior to the divergence of animals and plants. All four L7 r-protein genes are expressed and all exhibit a differential expression in inflorescence and flowers. RPL7A and RPL7B are less expressed than the other genes in all tissues analyzed. Molecular characterization of nucleic and protein sequences of L7 r-protein genes and analysis of their codon usage did not indicate any functional divergence. The probable evolution of an extra-ribosomal function of group 2 genes is discussed.

Colocalization of the ribosomal gene families in Conger conger (Anguilliformes, Congridae)

The location of the major and minor ribosomal gene families in the conger eel (Conger conger) was investigated by in situ hybridization and CMA3 staining. The two gene families were localized in only one chromosome pair, on the short arm of pair 19, in an entirely CMA3‐positive region. Among the Anguilliformes, C. conger is the only species that shows the presence of the two gene families in the same chromosomal region.

Non-homogenized ITS regions in the parasitic nematode Cooperia oncophora.

Here, the validity of the assumption of concerted evolution of ribosomal regions in larval and adult Cooperia oncophora was assessed. In each of 4 individuals of this parasitic nematode, at least 78% of the sequences comprised different ITS variants. This implies that concerted evolution is not acting, which is corroborated by the scarcity of signatures of gene conversion and recombination. Mis-incorporation of nucleotides and illegitimate PCR-induced recombination turned out to be unlikely, and positions with substantial frequencies of alternative nucleotides corresponded to ambiguous positions in published ITS2 sequences of this and other Cooperia species based on direct sequencing. The ITS regions of each individual C. oncophora displayed a significant excess of unique mutations in agreement with expansion of the ribosomal gene family. Interesting corollaries of the inferred size changes of this gene family are genomic rearrangements that occur during larval development such as multiple rounds of endoduplication (in Rhabditidae), chromatin diminution (in Ascaris), and non-compensatory mutations on the secondary structure of the ITS2. It is yet unknown which process is important in trichostrongylids. Finally, although it can not be rigorously assessed in Cooperia, the ITS polymorphisms can readily be envisioned to affect phylogenetic reconstructions of closely related nematodes.