Fugu and human sequence comparison identifies novel human genes and conserved non-coding sequences

The compact genome of the pufferfish, Fugu rubripes, has been proposed as a 'reference' genome to aid in annotating and analysing the human genome. We have annotated and compared 85 kb of Fugu sequence containing 17 genes with its homologous loci in the human draft genome and identified three 'novel' human genes that were missed or incompletely predicted by the previous gene prediction methods. Two of the novel genes contain zinc finger domains and are designated ZNF366 and ZNF367. They map to human chromosomes 5q13.2 and 9q22.32, respectively. The third novel gene, designated C9orf21, maps to chromosome 9q22.32. This gene is unique to vertebrates, and the protein encoded by it does not contain any known domains. We could not find human homologs for two Fugu genes, a novel chemokine gene and a kinase gene. These genes are either specific to teleosts or lost in the human lineage. The Fugu-human comparison identified several conserved non-coding sequences in the promoter and intronic regions. These sequences, conserved during 450 million years of vertebrate evolution, are likely to be involved in gene regulation. The 85 kb Fugu locus is dispersed over four human loci, occupying about 1.5 Mb. Contiguity is conserved in the human genome between six out of 16 Fugu gene pairs. These contiguous chromosomal segments should share a common evolutionary history dating back to the common ancestor of mammals and teleosts. We propose contiguity as strong evidence to identify orthologous genes in distant organisms. This study confirms the utility of the Fugu as a supplementary tool to uncover and confirm novel genes and putative gene regulatory regions in the human genome.     

Identification of transposon-like elements in non-coding regions of tomato ACC oxidase genes

1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyses the terminal step in ethylene biosynthesis, is encoded by a small multigene family in tomato that is differentially expressed in response to developmental and environmental cues. In this study we report the isolation and sequencing of approximately 2 kb of 5′-flanking sequence of three tomato ACC oxidase genes (LEACO1, LEACO2, LEACO3) and the occurrence of class I and class II mobile element-like insertions in promoter and intron regions of two of them. The LEACO1 upstream region contains a 420-bp direct repeat which is present in multiple copies in the tomato genome and is very similar to sequences in the promoters of the tomato E4 and 2A11 genes. The region covering the repeats resembles the remnant of a retrotransposon. Two copies of a small transposable element, belonging to the Stowaway inverted repeat element family, have been found in the 5′-flanking sequence and the third intron of LEACO3. Received: 8 August 1996 / Accepted: 4 November 1996     

Complementary DNA sequence of a human cytoplasmic actin. Interspecies divergence of 3' non-coding regions

We have isolated and sequenced a cloned complementary DNA insert complementary to the messenger RNA of a cytoplasmic actin expressed in human epidermal cells. This provides the first cytoplasmic actin complementary DNA sequence for a vertebrate organism. The actin amino acid sequence predicted from this complementary DNA is identical to that of a bovine cytoplasmic actin and shows 98 and 85% homology with a Dictyostelium and a yeast actin, respectively. The complementary DNA sequence indicates that the 3' end of the mRNA contains an unusually long (greater than 400 nucleotides) 3' non-translated region. A comparison of this 3' non-coding region with those of recently determined actin complementary DNA sequences from other species reveals little or no homology among these sequences. Thus, these results indicate that although the actin amino acid sequences are extremely conserved, the non-coding regions of the mRNAs diverge rapidly.     

Hundreds of conserved non-coding genomic regions are independently lost in mammals

Conserved non-protein-coding DNA elements (CNEs) often encode cis-regulatory elements and are rarely lost during evolution. However, CNE losses that do occur can be associated with phenotypic changes, exemplified by pelvic spine loss in sticklebacks. Using a computational strategy to detect complete loss of CNEs in mammalian genomes while strictly controlling for artifacts, we find >600 CNEs that are independently lost in at least two mammalian lineages, including a spinal cord enhancer near GDF11. We observed several genomic regions where multiple independent CNE loss events happened; the most extreme is the DIAPH2 locus. We show that CNE losses often involve deletions and that CNE loss frequencies are non-uniform. Similar to less pleiotropic enhancers, we find that independently lost CNEs are shorter, slightly less constrained and evolutionarily younger than CNEs without detected losses. This suggests that independently lost CNEs are less pleiotropic and that pleiotropic constraints contribute to non-uniform CNE loss frequencies. We also detected 35 CNEs that are independently lost in the human lineage and in other mammals. Our study uncovers an interesting aspect of the evolution of functional DNA in mammalian genomes. Experiments are necessary to test if these independently lost CNEs are associated with parallel phenotype changes in mammals.     

Identification of a nucleotide sequence conserved in Lactococcus lactis bacteriophages

A genetic element which is conserved in the genomes of numerous Lactococcus lactis bacteriophage isolates has been identified and its nucleotide sequence determined. Approximately 95-99% of all L. lactis bacteriophages collected over a period of six years from two geographically distinct sources carry this conserved DNA fragment. Genetic variation in other regions of the genomes of these bacteriophages is exhibited by changes in the overall restriction patterns. The complete nt sequence for a 1.6-kb region from nine independent L. lactis bacteriophage isolates was determined and only five changes in the nt sequence were observed within a span of 1536 bp. This region has a single large 1356-bp open reading frame (ORF) coding for a 51-kDa protein. Three out of the five changes occur in a 187-bp region, 5' to this large ORF. The two additional changes are found within the 1356-bp ORF, which results in two amino acid substitutions that do not, however, change the net charge of the protein. The encoded protein is extremely charged and shares some homology with yeast translation initiation factor. In addition, there is a potential zinc-binding domain within this protein, similar to those observed in genes from bacteriophages T4 and T7.     

Occurrence of protein structure elements in conserved sequence regions

Background  

Conserved protein sequence regions are extremely useful for identifying and studying functionally and structurally important regions. By means of an integrated analysis of large-scale protein structure and sequence data, structural features of conserved protein sequence regions were identified.     

Identification of four highly conserved regions in DNA-PKcs

The gene for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is responsible for severe combined immune deficiency (SCID) and its products associate with Ku70/Ku86 autoantigens, c-Abl, and other factors to exert its roles. Investigations to date suggest that DNA-PKcs comprises several regions which specifically interact with these known and other as yet unidentified factors. Nevertheless, the relationship between the structure and function of the DNA-PKcs molecule is poorly understood. Here we report cloning of the entire DNA-PKcs cDNA from chicken and Xenopus laevis. Comparative study of the DNA-PKcs polypeptides from four different vertebrates revealed at least three novel conserved regions in addition to the carboxyl-terminal region containing the phosphatidylinositol-3 kinase domain. We also demonstrated that the four vertebrates share the common genomic organization between a licensing factor, MCM4, and DNA-PKcs, both of which locate in a head-to-head manner within a few kilobase intervals. These data provide useful clues for the further genetic study of this huge multifunctional enzyme.     

Searching databases of conserved sequence regions by aligning protein multiple-alignments.

A general searching method for comparing multiple sequence alignments was developed to detect sequence relationships between conserved protein regions. Multiple alignments are treated as sequences of amino acid distributions and aligned by comparing pairs of such distributions. Four different comparison measures were tested and the Pearson correlation coefficient chosen. The method is sensitive, detecting weak sequence relationships between protein families. Relationships are detected beyond the range of conventional sequence database searches, illustrating the potential usefulness of the method. The previously undetected relation between flavoprotein subunits of two oxidoreductase families points to the potential active site in one of the families. The similarity between the bacterial RecA, DnaA and Rad51 protein families reveals a region in DnaA and Rad51 proteins likely to bind and unstack single-stranded DNA. Helix--turn--helix DNA binding domains from diverse proteins are readily detected and shown to be similar to each other. Glycosylasparaginase and gamma-glutamyltransferase enzymes are found to be similar in their proteolytic cleavage sites. The method has been fully implemented on the World Wide Web at URL: http://blocks.fhcrc.org/blocks-bin/LAMAvsearch.     

Evolutionary divergence and limits of conserved non-coding sequence detection in plant genomes

The discovery of regulatory motifs embedded in upstream regions of plants is a particularly challenging bioinformatics task. Previous studies have shown that motifs in plants are short compared with those found in vertebrates. Furthermore, plant genomes have undergone several diversification mechanisms such as genome duplication events which impact the evolution of regulatory motifs. In this article, a systematic phylogenomic comparison of upstream regions is conducted to further identify features of the plant regulatory genomes, the component of genomes regulating gene expression, to enable future de novo discoveries. The findings highlight differences in upstream region properties between major plant groups and the effects of divergence times and duplication events. First, clear differences in upstream region evolution can be detected between monocots and dicots, thus suggesting that a separation of these groups should be made when searching for novel regulatory motifs, particularly since universal motifs such as the TATA box are rare. Second, investigating the decay rate of significantly aligned regions suggests that a divergence time of ~100 mya sets a limit for reliable conserved non-coding sequence (CNS) detection. Insights presented here will set a framework to help identify embedded motifs of functional relevance by understanding the limits of bioinformatics detection for CNSs.     

Evolutionarily conserved regions in Caenorhabditis transposable elements deduced by sequence comparison.

In this paper we present the sequence of an intact Caenorhabditis briggsae transposable element, Tcb2. Tcb2 is 1606 base pairs in length and contains 80 base pair imperfect terminal repeats and a single open reading frame. We have identified blocks of T-rich repeats in the regions 150-200 and 1421-1476 of this element which are conserved in the Caenorhabditis elegans element Tc1. The sequence conservation of these regions in elements from different Caenorhabditis species suggests that they are of functional importance. A single open reading frame corresponding to the major open reading frame of Tc1 is conserved among Tc1, Tcb1, and Tcb2. Comparison of the first 550 nucleotides of the sequence among the three elements has allowed the evaluation of a model proposing an extension of the major open reading frame. Our data support the suggestion that Tc1 is capable of producing a 335 amino acid protein. A comparison of the sequence coding for the amino and carboxy termini of the 273 amino acid transposase from Caenorhabditis Tc1-like elements and Drosophila HB1 showed different amounts of divergence for each of these regions, indicating that the two functional domains have undergone different amounts of selection. Our data are not compatible with the proposal that Tc1-related sequences have been acquired via horizontal transmission. The divergence of Tc1 from the two C. briggsae elements, Tcb1 and Tcb2, indicated that all three elements have been diverging from each other for approximately the same amount of time as the genomes of the two species.