A highly conserved microsatellite in the dystrophin gene of diverse mammalian species

The presence of a CA repeat within the 3'-untranslated region (UTR) of the dystrophin gene has been reported previously in several species. Because microsatellites showing high cross-species homology can be conveniently used as markers in those species for which detailed linkage maps have not yet been developed, we evaluated whether the CA repeat could be amplified from a wide variety of mammalian species. Using a single pair of canine-specific oligonucleotide primers, we successfully amplified the 3'-UTR from 18 different carnivore and six additional species (human, chimpanzee, goat, cow, rabbit and mouse) and show conservation of the CA repeat in the dystrophin gene from a wide range of evolutionarily diverse mammalian species.     

Purifying selection maintains highly conserved noncoding sequences in Drosophila

The majority of metazoan genomes consist of nonprotein-coding regions, although the functional significance of most noncoding DNA sequences remains unknown. Highly conserved noncoding sequences (CNSs) have proven to be reliable indicators of functionally constrained sequences such as cis-regulatory elements and noncoding RNA genes. However, CNSs may arise from nonselective evolutionary processes such as genomic regions with extremely low mutation rates known as mutation "cold spots." Here we combine comparative genomic data from recently completed insect genome projects with population genetic data in Drosophila melanogaster to test predictions of the mutational cold spot model of CNS evolution in the genus Drosophila. We find that point mutations in intronic and intergenic CNSs exhibit a significant reduction in levels of divergence relative to levels of polymorphism, as well as a significant excess of rare derived alleles, compared with either the nonconserved spacer regions between CNSs or with 4-fold silent sites in coding regions. Controlling for the effects of purifying selection, we find no evidence of positive selection acting on Drosophila CNSs, although we do find evidence for the action of recurrent positive selection in the spacer regions between CNSs. We estimate that approximately 85% of sites in Drosophila CNSs are under constraint with selection coefficients (N(e)s) on the order of 10-100, and thus, the estimated strength and number of sites under purifying selection is greater for Drosophila CNSs relative to those in the human genome. These patterns of nonneutral molecular evolution are incompatible with the mutational cold spot hypothesis to explain the existence of CNSs in Drosophila and, coupled with similar findings in mammals, argue against the general likelihood that CNSs are generated by mutational cold spots in any metazoan genome.     

Chickpea rhizobia symbiosis genes are highly conserved across multiple Mesorhizobium species

Chickpea has been considered as a restrictive host for nodulation by rhizobia. However, recent studies have reported that several Mesorhizobium species may effectively nodulate chickpea. With the purpose of investigating the evolutionary relationships between these different species with the ability of nodulating the same host, we analysed 21 Portuguese chickpea rhizobial isolates. Symbiosis genes nifH and nodC were sequenced and used for phylogenetic studies. Symbiotic effectiveness was determined to evaluate its relationship with symbiosis genes. The comparison of 16S rRNA gene-based phylogeny with the phylogenies based on symbiosis genes revealed evidence of lateral transfer of symbiosis genes across different species. Chickpea is confirmed as a nonpromiscuous host. Although chickpea is nodulated by many different species, they share common symbiosis genes, suggesting recognition of only a few Nod factors by chickpea. Our results suggest that sequencing of nifH or nodC genes can be used for rapid detection of chickpea mesorhizobia.     

Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated

Two catalase genes,cat1 andcat2, have been isolated from the castor bean genome. They were located in the same direction on a chromosome at a distance of 2.4 kb,cat1 being on the downstream side ofcat2. The two genes contained introns at the same positions except that one of the 7 introns incat1 is missing incat2 and the corresponding introns differed in size and sequence between the two genes. The translated regions of the two genes had the same number of nucleotides and exhibited 81.3% nucleotide sequence identity. In addition to introns, the nucleotide sequences of the 5-and 3-flanking regions are highly divergent between the two genes. In etiolated seedlings,cat1 mRNA was present abundantly in endosperms and cotyledons and only in a small amount in roots. Thecat1 mRNA could not be detected in hypocotyls. By contrast,cat2 mRNA is most abundant in hypocotyls and roots, while endosperms and cotyledons contained only low levels ofcat2 mRNA. Although neithercat1 norcat2 mRNA could be detected in dry seeds, both mRNAs showed temporal accumulation in the endosperm in response to germination. These results suggest that expression of two tightly linked catalase genes of castor bean,cat1 andcat2, are differentially regulated during development.     

A study of the distribution of phylogenetically conserved blocks within clusters of mammalian homeobox genes

Genome sequencing efforts of the last decade have produced a large amount of data, which has enabled whole-genome comparative analyses in order to locate potentially functional elements and study the overall patterns of phylogenetic conservation. In this paper we present a statistically based method for the characterization of these patterns in mammalian DNA sequences. We have applied this approach to the study of exceptionally well conserved homeobox gene clusters (Hox), based on an alignment of six species, and we have constructed a map of Hox cataloguing the conserved fragments, along with their locations in relation to the genes and other landmarks, sometimes showing unexpected layouts.     

A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a rapid and robust method for determining and studying the function of plant genes or expressed sequence tags (ESTs). However, only a few plant species are amenable to VIGS. There is a need for a systematic study to identify VIGS-efficient plant species and to determine the extent of homology required between the heterologous genes and their endogenous orthologs for silencing. Two approaches were used. First, the extent of phytoene desaturase (PDS) gene silencing was studied in various Solanaceous plant species using Nicotiana benthamiana NbPDS sequences. In the second approach, PDS sequences from a wide range of plant species were used to silence the PDS gene in N. benthamiana. The results showed that tobacco rattle virus (TRV)-mediated VIGS can be performed in a wide range of Solanaceous plant species and that heterologous gene sequences from far-related plant species can be used to silence their respective orthologs in the VIGS-efficient plant N. benthamiana. A correlation was not always found between gene silencing efficiency and percentage homology of the heterologous gene sequence with the endogenous gene sequence. It was concluded that a 21-nucleotide stretch of 100% identity between the heterologous and endogenous gene sequences is not absolutely required for gene silencing.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.     

Intracellular targetting signals of polymeric immunoglobulin receptors are highly conserved between species

A rat liver cDNA library, constructed in the plasmid expression vector pUEX, was immunoscreened using a rabbit polyclonal antiserum raised against rat liver Golgi membrane proteins. A sub-set of isolated clones were shown to encode the rat polymeric immunoglobulin receptor (pIgR). DNA sequence analysis of these clones provided the complete coding sequence of rat pIgR. Subsequent alignment of rat, rabbit and human predicted amino acid sequences demonstrated that the greatest degree of homology between the three pIgRs lies in their cytoplasmic tails; a region previously shown to be important for correct targetting and trancytosis of rabbit pIgR [(1984) Nature 308, 37-43].     

A comparative genomic analysis of plant hormone related genes in different species

Plant hormones are small molecules that play important roles throughout the life span of a plant,known as auxin,gibberellin,cytokinin,abscisic acid,ethylene,jasmonic acid,salicylic acid,and brassinosteroid.Genetic and molecular studies in the model organism Arabidopsis thaliana have revealed the individual pathways of various plant hormone responses.In this study,we selected 479 genes that were convincingly associated with various hormone actions based on genetic evidence.By using these 479 genes as queries,a genome-wide search for their orthoiogues in several species(microorganisms,plants and animals) was performed.Meanwhile,a comparative analysis was conducted to evaluate their evolutionary relationship.Our analysis revealed that the metabolisms and functions of plant hormones are generally more sophisticated and diversified in higher plant species.In particular,we found that several phytohormone receptors and key signaling components were not present in lower plants or animals.Meanwhile,as the genome complexity increases,the orthologne genes tend to have more copies and probably gain more diverse functions.Our study attempts to introduce the classification and phylogenic analysis of phytohormone related genes,from metabolism enzymes to receptors and signaling components,in different species.     

A comparative genomic analysis of plant hormone related genes in different species

Plant hormones are small molecules that play important roles throughout the life span of a plant,known as auxin,gibberellin,cyto-kinin,abscisic acid,ethylene,jasmonic acid,salicylic acid,and brassinosteroid.Genetic and molecular studies in the model organism Arabidopsis thaliana have revealed the individual pathways of various plant hormone responses.In this study,we selected 479 genes that were convincingly associated with various hormone actions based on genetic evidence.By using these 479 genes as querie...     

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

Summary Five specific transposon-induced nodulation defective (Nod⁻) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod⁻ mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod⁻ on all their known plant hosts, (b), they could not induce root hair curling (Hac⁻) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.     

The two genes for the small subunit of RuBP Carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii

Summary Ribulose bisphosphate carboxylase-oxygenase (Rubisco) is a key enzyme in the photosynthetic fixation of CO₂ by the chloroplast. The synthesis of the enzyme is an example of the cooperation between the chloroplast and the nucleocytoplasmic compartments, as it is assembled from subunits encoded in the two respective genomes. I have used a synthetic oligonucleotide probe to isolate the nuclear Rubisco small subunit genes (rbcS) directly from a genomic library of Chlamydomonas reinhardtii DNA. They constitute only a small family: there are two rbcS genes, and an additional related sequence, in the C. reinhardtii genome. All three are clustered within 11kb at a single locus, and should thus be particularly well suited for genetic manipulation. The pattern of expression of rbcS RNA is dependent on the growth conditions.     

A highly permeable species boundary between two anadromous fishes

Meristic identification, mitochondrial DNA and a suite of microsatellite markers were employed to estimate the incidence of hybridization in wild populations of anadromous Allis shad Alosa alosa and twaite shad Alosa fallax in southern Irish riverine and estuarine waters. It was shown that 16% of the fishes examined were misclassified using meristic count of gill rakers. Next, a significant proportion of fishes that were robustly assigned to a species using nuclear markers were shown to possess the mtDNA of the other. The genomes of A. alosa and A. fallax in Ireland are extensively introgressed, which suggests a complex history of hybridization between these species, which can only partially be explained by recent man-made habitat changes.     

The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not

The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.     

Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases

Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes. Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity). The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid. We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation. We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences. Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained.     

Evolutionary growth process of highly conserved sequences in vertebrate genomes

Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage.     

Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.