A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts

Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of its organization. First, large regions of the genome, called gene deserts, are devoid of protein-coding sequences and have no obvious biological role. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) in all vertebrates. Most surprisingly, these structural features are strongly associated with genes that have essential functions during development. Among these, the vertebrate Iroquois (Irx) genes stand out on both fronts. Mammalian Irx genes are organized in two clusters (IrxA and IrxB) that span >1 Mb each with no other genes interspersed. Additionally, a large number of HCNRs exist within Irx clusters. We have systematically examined the enhancer activity of HCNRs from the IrxB cluster using transgenic Xenopus and zebrafish embryos. Most of these HCNRs are active in subdomains of endogenous Irx expression, and some are candidates to contain shared enhancers of neighboring genes, which could explain the evolutionary conservation of Irx clusters. Furthermore, HCNRs present in tetrapod IrxB but not in fish may be responsible for novel Irx expression domains that appeared after their divergence. Finally, we have performed a more detailed analysis on two IrxB ultraconserved non-coding regions (UCRs) duplicated in IrxA clusters in similar relative positions. These four regions share a core region highly conserved among all of them and drive expression in similar domains. However, inter-species conserved sequences surrounding the core, specific for each of these UCRs, are able to modulate their expression.     

Repetitive extragenic palindromic sequences in the Pseudomonas syringae pv. tomato DC3000 genome: extragenic signals for genome reannotation

Repetitive extragenic palindromic (REPs) sequences were first described in enterobacteriacea and later in Pseudomonas putida. We have detected a new variant (51 base pairs) of REP sequences that appears to be disseminated in more than 300 copies in the Pseudomonas syringae DC3000 genome. The finding of REP sequences in P. syringae confirms the broad presence of this type of repetitive sequence in bacteria. We analyzed the distribution of REP sequences and the structure of the clusters, and we show that palindromy is conserved. REP sequences appear to be allocated to the extragenic space, with a special preference for the intergenic spaces limited by convergent genes, while their presence is scarce between divergent genes. Using REP sequences as markers of extragenicity we re-annotated a set of genes of the P. syringae DC3000 genome demonstrating that REP sequences can be used for refinement of annotation of a genome. The similarity detected between virulence genes from evolutionarily distant pathogenic bacteria suggests the acquisition of clusters of virulence genes by horizontal gene transfer. We did not detect the presence of P. syringae REP elements in the principal pathogenicity gene clusters. This absence suggests that genome fragments lacking REP sequences could point to regions recently acquired from other organisms, and REP sequences might be new tracers for gaining insight into key aspects of bacterial genome evolution, especially when studying pathogenicity acquisition. In addition, as the P. syringae REP sequence is species-specific with respect to the sequenced genomes, it is an exceptional candidate for use as a fingerprint in precise genotyping and epidemiological studies.     

Coding exons function as tissue-specific enhancers of nearby genes

Enhancers are essential gene regulatory elements whose alteration can lead to morphological differences between species, developmental abnormalities, and human disease. Current strategies to identify enhancers focus primarily on noncoding sequences and tend to exclude protein coding sequences. Here, we analyzed 25 available ChIP-seq data sets that identify enhancers in an unbiased manner (H3K4me1, H3K27ac, and EP300) for peaks that overlap exons. We find that, on average, 7% of all ChIP-seq peaks overlap coding exons (after excluding for peaks that overlap with first exons). By using mouse and zebrafish enhancer assays, we demonstrate that several of these exonic enhancer (eExons) candidates can function as enhancers of their neighboring genes and that the exonic sequence is necessary for enhancer activity. Using ChIP, 3C, and DNA FISH, we further show that one of these exonic limb enhancers, Dync1i1 exon 15, has active enhancer marks and physically interacts with Dlx5/6 promoter regions 900 kb away. In addition, its removal by chromosomal abnormalities in humans could cause split hand and foot malformation 1 (SHFM1), a disorder associated with DLX5/6. These results demonstrate that DNA sequences can have a dual function, operating as coding exons in one tissue and enhancers of nearby gene(s) in another tissue, suggesting that phenotypes resulting from coding mutations could be caused not only by protein alteration but also by disrupting the regulation of another gene.     

Nucleotide sequence analysis of hypervariable junctions of Haemophilus influenzae pilus gene clusters

Haemophilus influenzae pili are surface structures that promote attachment to human epithelial cells. The five genes that encode pili, hifABCDE, are found inserted in genomes either between pmbA and hpt (hif-1) or between purE and pepN (hif-2). We determined the sequence between the ends of the pilus clusters and bordering genes in a number of H. influenzae strains. The junctions of the hif-1 cluster (limited to biogroup aegyptius isolates) are structurally simple. In contrast, hif-2 junctions are highly diverse, complex assemblies of conserved intergenic sequences (including genes hicA and hicB) with evidence of frequent recombination. Variation at hif-2 junctions seems to be tied to multiple copies of a 23-bp Haemophilus intergenic dyad sequence. The hif-1 cluster appears to have originated in biogroup aegyptius strains from invasion of the hpt-pmbA region by a DNA template containing the hif-2 genes with termini in the hairpin loop of flanking intergenic dyad sequences. The pilus gene clusters are an interesting model of a mobile "pathogenicity island" not associated with a phage, transposon, or insertion element.     

Comparative analyses among the <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>, <Emphasis Type="Italic">Trichomonas tenax</Emphasis>, and <Emphasis Type="Italic">Tritrichomonas foetus</Emphasis> 5S ribosomal RNA genes

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers FJ492747, FJ492748, FJ492749, FJ492750, and FJ492751.     

Analysis of nucleotide sequence of Iranian maize mosaic virus confirms its identity as a distinct nucleorhabdovirus

The nucleotide sequence of the Iranian maize mosaic rhabdovirus (IMMV) was obtained using a random-PCR method (rPCR) followed by PCR with specific primers. Analysis of the complete nucleotide sequence of the IMMV genes and intergenic regions comprising a total of 12,381 nucleotides (including the partial sequences of leader and trailer regions) revealed six open reading frames (ORF) on the viral complementary RNA (vcRNA). On the basis of its similarities to other rhabdovirus sequences, the IMMV genome consists of 3'-leader-N-P-3-M-G-L-5'-trailer. The intergenic regions contained a characteristic consensus sequence, 3'-AAUUCUUUUUGGGUUU/G-5'. The IMMV gene products showed a high similarity to those of maize mosaic virus and taro vein chlorosis virus and a more distant relationship to other rhabdoviruses. Together with the biological, serological and morphological features described earlier, our molecular data provide evidence that IMMV is a distinct member of the genus Nucleorhabdovirus in the family Rhabdoviridae.     

Evolutionary Conservation of Regulatory Elements in Vertebrate Hox Gene Clusters

Comparisons of DNA sequences among evolutionarily distantly related genomes permit identification of conserved functional regions in noncoding DNA. Hox genes are highly conserved in vertebrates, occur in clusters, and are uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human, and mouse, which are separated by approximately 500 million years of evolution. In support of our approach, several identified putative regulatory elements known to regulate the expression of Hox genes were recovered. The majority of the newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac database). The regulatory intergenic regions located between the genes that are expressed most anteriorly in the embryo are longer and apparently more evolutionarily conserved than those at the other end of Hox clusters. Different presumed regulatory sequences are retained in either the Aα or Aβ duplicated Hox clusters in the fish lineages. This suggests that the conserved elements are involved in different gene regulatory networks and supports the duplication-deletion-complementation model of functional divergence of duplicated genes.     

Conservation and diversity of HMW1 and HMW2 adhesin binding domains among invasive nontypeable Haemophilus influenzae isolates

The pathogenesis of nontypeable Haemophilus influenzae (NTHi) begins with adhesion to the rhinopharyngeal mucosa. In almost 80% of NTHi clinical isolates, the HMW proteins are the major adhesins. The prototype HMW1 and HMW2 proteins, identified in NTHi strain 12, exhibit different binding specificities. The two binding domains have been localized in regions of maximal sequence dissimilarity (40% identity, 58% similarity). Two areas within these binding domains have been found essential for full level adhesive activity (designated the core-binding domains). To investigate the conservation and diversity of the HMW1 and HMW2 core-binding domains among isolates, PCR and DNA sequencing were used. First, we separately amplified the hmw1A-like and hmw2A-like structural genes in nine invasive NTHi isolates, discovering two new hmwA alleles, whose sequences are herein reported. Then, the hmw1A-like and hmw2A-like PCR products were used as the template in nested PCR to produce amplicons encompassing the encoding sequences of the two core-binding domains. In-depth sequence analysis was then performed among sequences of each group, with the support of specific computer programs. Overall, extensive sequence diversity among isolates was highlighted. However, similarity plots showed patterns consisting of peaks of relatively high similarity alternating with strongly divergent regions. The phylogenetic tree clearly indicated the HMW1-like and HMW2-like core-binding domain sequences as two clusters. Distinct sets of conserved amino acid motifs were identified within each group of sequences using the MEME/MOTIFSEARCH tool. Since HMW adhesins could represent candidates for future vaccines, identification of specific patterns of conserved motifs in otherwise highly variable regions is of great interest.     

Evolution and phylogeny of the corticotropin-releasing factor (CRF) family of peptides: Expansion and specialization in the vertebrates

New sequence data on CRF family members from a number of genomes has led to the modification of our understanding of CRF evolution in the Metazoa. The corticotropin-releasing factor (CRF) family of peptides include four paralogous lineages in jawed vertebrates; CRF, urotensin-I/urocortin/sauvagine, urocortin 2 (Ucn2) and urocortin 3 (Ucn3). CRF and the urotensin-I/urocortin/sauvagine group represent a gene duplication from one lineage, whereas Ucns 2 and 3 are the result of a gene duplication in the other paralogous lineage. Both paralogous lineages are the result of a gene duplication from a single ancestral peptide that occurred after the divergence of the tunicates from the ancestor that led to the evolution of chordates and vertebrates. The presence of a single CRF-like peptide in tunicates and insects suggests that a single CRF-like ancestor was present before the separation of deuterostomes and protostomes. Currently there is no strong evidence that indicates that CRF-like peptides were present in metazoan taxa that evolved before this time although the structural similarity between some CRF peptides in insects, tunicates and vertebrates with the calcitonin family of peptides hints that prior to the formation of deuterostomes and protostomes the ancestral peptide possessed both CRF and calcitonin-like structural attributes. Here, we show evidences of conservation of CRF-like function dating back to early prokaryotes. This ancestral CRF–calcitonin-like peptide may have initially resulted from a horizontal gene transfer event from prokaryotes to a protistan species that later gave rise to the metazoans.     

Comparison of human chromosome 21 conserved nongenic sequences (CNGs) with the mouse and dog genomes shows that their selective constraint is independent of their genic environment

The analysis of conservation between the human and mouse genomes resulted in the identification of a large number of conserved nongenic sequences (CNGs). The functional significance of this nongenic conservation remains unknown, however. The availability of the sequence of a third mammalian genome, the dog, allows for a large-scale analysis of evolutionary attributes of CNGs in mammals. We have aligned 1638 previously identified CNGs and 976 conserved exons (CODs) from human chromosome 21 (Hsa21) with their orthologous sequences in mouse and dog. Attributes of selective constraint, such as sequence conservation, clustering, and direction of substitutions were compared between CNGs and CODs, showing a clear distinction between the two classes. We subsequently performed a chromosome-wide analysis of CNGs by correlating selective constraint metrics with their position on the chromosome and relative to their distance from genes. We found that CNGs appear to be randomly arranged in intergenic regions, with no bias to be closer or farther from genes. Moreover, conservation and clustering of substitutions of CNGs appear to be completely independent of their distance from genes. These results suggest that the majority of CNGs are not typical of previously described regulatory elements in terms of their location. We propose models for a global role of CNGs in genome function and regulation, through long-distance cis or trans chromosomal interactions.     

Comparative genomic analysis of the MHC: the evolution of class I duplication blocks, diversity and complexity from shark to man

Summary: The major histocompatibility complex (MHC) genomic region is composed of a group of linked genes involved functionally with the adaptive and innate immune systems. The class I and class II genes are intrinsic features of the MHC and have been found in all the jawed vertebrates studied so far. The MHC genomic regions of the human and the chicken (B locus) have been fully sequenced and mapped, and the mouse MHC sequence is almost finished. Information on the MHC genomic structures (size, complexity, genic and intergenic composition and organization, gene order and number) of other vertebrates is largely limited or nonexistent. Therefore, we are mapping, sequencing and analyzing the MHC genomic regions of different human haplotypes and at least eight nonhuman species. Here, we review our progress with these sequences and compare the human MHC structure with that of the nonhuman primates (chimpanzee and rhesus macaque), other mammals (pigs, mice and rats) and nonmammalian vertebrates such as birds (chicken and quail), bony fish (medaka, pufferfish and zebrafish) and cartilaginous fish (nurse shark). This comparison reveals a complex MHC structure for mammals and a relatively simpler design for nonmammalian animals with a hypothetical prototypic structure for the shark. In the mammalian MHC, there are two to five different class I duplication blocks embedded within a framework of conserved nonclass I and/or nonclass II genes. With a few exceptions, the class I framework genes are absent from the MHC of birds, bony fish and sharks. Comparative genomics of the MHC reveal a highly plastic region with major structural differences between the mammalian and nonmammalian vertebrates. Additional genomic data are needed on animals of the reptilia, crocodilia and marsupial classes to find the origins of the class I framework genes and examples of structures that may be intermediate between the simple and complex MHC organizations of birds and mammals, respectively.     

Differentiation of two biovars of Ureaplasma urealyticum based on the 16S-23S rRNA intergenic spacer region

The 16S-23S rRNA intergenic spacer regions of 14 strains representing the 14 serovars of Ureaplasma urealyticum were amplified by PCR and sequenced for genetic differentiation between the two biovars Parvo and T960. Although the spacer region of the Parvo and T960 biovars comprised 302 nucleotides and lacked spacer tRNA genes, 15 nucleotides were different between the two biovars. The four nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 1, 3, 6, and 14 in the Parvo biovar were found to be identical. Similarly, the 10 nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 2, 4, 5, and 7 to 13 in the T960 biovar were found to be identical. The nucleotide sequence of the T960 biovar contains multiple restriction sites for restriction endonuclease SspI, which allows differentiation of the T960 biovar from the Parvo biovar.     

Immunoglobulin light chain (IgL) genes in zebrafish: Genomic configurations and inversional rearrangements between (V(L)-J(L)-C(L)) gene clusters

In mammals, Immunoglobulin light chain (IgL) are localized to two chromosomal regions (designated kappa and lambda). Here we report a genome-wide survey of IgL genes in the zebrafish revealing (V(L)-J(L)-C(L)) clusters spanning 5 separate chromosomes. To elucidate IgL loci present in the zebrafish genome assembly (Zv6), conventional sequence similarity searches and a novel scanning approach based on recombination signal sequence (RSS) motifs were applied. RT-PCR with zebrafish cDNA was used to confirm annotations, evaluate VJ-rearrangement possibilities and show that each chromosomal locus is expressed. In contrast to other vertebrates in which IgL exon usage has been studied, inversional rearrangement between (V(L)-J(L)-C(L)) clusters were found. Inter-cluster rearrangements may convey a selective advantage for editing self-reactive receptors and poise zebrafish by virtue of their extensive numbers of V(L), J(L) and C(L) to have greater potential for immunoglobulin gene shuffling than traditionally studied mice and human models.     

Ancient haplotypes resulting from extensive molecular rearrangements in the wheat A genome have been maintained in species of three different ploidy levels

Plant genomes, in particular grass genomes, evolve very rapidly. The closely related A genomes of diploid, tetraploid, and hexaploid wheat are derived from a common ancestor that lived <3 million years ago and represent a good model to study molecular mechanisms involved in such rapid evolution. We have sequenced and compared physical contigs at the Lr10 locus on chromosome 1AS from diploid (211 kb), tetraploid (187 kb), and hexaploid wheat (154 kb). A maximum of 33% of the sequences were conserved between two species. The sequences from diploid and tetraploid wheat shared all of the genes, including Lr10 and RGA2 and define a first haplotype (H1). The 130-kb intergenic region between Lr10 and RGA2 was conserved in size despite its activity as a hot spot for transposon insertion, which resulted in >70% of sequence divergence. The hexaploid wheat sequence lacks both Lr10 and RGA2 genes and defines a second haplotype, H2, which originated from ancient and extensive rearrangements. These rearrangements included insertions of retroelements and transposons deletions, as well as unequal recombination within elements. Gene disruption in haplotype H2 was caused by a deletion and subsequent large inversion. Gene conservation between H1 haplotypes, as well as conservation of rearrangements at the origin of the H2 haplotype at three different ploidy levels indicate that the two haplotypes are ancient and had a stable gene content during evolution, whereas the intergenic regions evolved rapidly. Polyploidization during wheat evolution had no detectable consequences on the structure and evolution of the two haplotypes.