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PubMed2001

Aspartyl proteinase genes from apicomplexan parasites: evidence for evolution of the gene structure.

Aspartyl proteinases are a widely distributed family of enzymes. All vertebrate aspartyl proteinases share a conserved nine-exon gene structure, but in other organisms the structure of aspartyl proteinase genes varies considerably. The exon-intron patterns generally reflect phylogeny based on amino acid sequences. However, close comparison of these gene structures reveals some striking features, such as the conservation of intron positions and intron phases between aspartyl proteinases from nematodes and apicomplexans. Here, we discuss the implications of gene structure for the possible evolution of the aspartyl proteinase family, with particular reference to the plasmepsins of Plasmodium falciparum and eimepsin from Eimeria tenella.

Amino Acid Sequence↗
PubMed1993

Structural organization of the human gene encoding nuclear lamin A and nuclear lamin C.

We have determined the structural organization of the human gene that encodes nuclear lamins A and C, intermediate filament proteins of the nuclear lamina. Sequencing and restriction mapping show that the coding region spans approximately 24 kilobases. The 5'-proximal promoter region contains several GC-rich stretches, a CCAAT box, and a TATA-like element of sequence TATTA. The lamin A/C gene contains 12 exons. Alternative splicing within exon 10 gives rise to two different mRNAs that code for pre-lamin A and lamin C. Consequently, two proteins are generated, only one of which, pre-lamin A, can be modified by isoprenylation. The intron positions in the human lamin A/C gene are generally conserved in the previously characterized genes for Xenopus lamin LIII and mouse lamin B2, but different from those in a Drosophila lamin gene. In the regions coding for the central rod domains, the intron positions are also conserved when compared with the intron positions in the genes for most cytoplasmic intermediate filament proteins except those for nestin and neurofilaments. Analysis of the intron positions in these genes supports the hypothesis that the nuclear lamins and other intermediate filament proteins arose from a common ancestor.

Amino Acid Sequence↗
PubMed2005

Analysis of evolution of exon-intron structure of eukaryotic genes.

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.

Animals↗
PubMed2006

Analysis of ribosomal protein gene structures: implications for intron evolution.

Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs), which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be "conserved," i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.

Amino Acid Sequence↗
PubMed2005

New maximum likelihood estimators for eukaryotic intron evolution.

The evolution of spliceosomal introns remains poorly understood. Although many approaches have been used to infer intron evolution from the patterns of intron position conservation, the results to date have been contradictory. In this paper, we address the problem using a novel maximum likelihood method, which allows estimation of the frequency of intron insertion target sites, together with the rates of intron gain and loss. We analyzed the pattern of 10,044 introns (7,221 intron positions) in the conserved regions of 684 sets of orthologs from seven eukaryotes. We determined that there is an average of one target site per 11.86 base pairs (bp) (95% confidence interval, 9.27 to 14.39 bp). In addition, our results showed that: (i) overall intron gains are approximately 25% greater than intron losses, although specific patterns vary with time and lineage; (ii) parallel gains account for approximately 18.5% of shared intron positions; and (iii) reacquisition following loss accounts for approximately 0.5% of all intron positions. Our results should assist in resolving the long-standing problem of inferring the evolution of spliceosomal introns.

Animals↗
PubMed2005

A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro.

The splicing pattern of pre-mRNA is unpredictable in genes harboring a single-nucleotide change within the consensus sequence of a splice-donor site. In the dystrophin gene, a transition from G to A at the fifth position of intron-32 (4518+5G > A) has been reported as a polymorphism within the consensus sequence or a mutation identified in Duchenne muscular dystrophy (DMD). Here, we report both in vivo and in vitro evidence that shows inactivation of the splice-donor site caused by this mutation. In one Japanese DMD case, two novel dystrophin mRNAs were identified in the patient's lymphocytes, one with a 98 bp deletion of the 3' end of exon-32 (dys32-98) and the other with a 28 bp intron retained between exons 32 and 33 (dys32 + 28). Genomic sequencing disclosed a single-nucleotide change from G to A at the fifth position of intron-32 (4518+5G > A). To demonstrate in vitro the inactivation of this splice-donor site by this nucleotide change, mini-dystrophin genes comprising three exons harboring either normal or mutant intron-32 sequences were expressed in HeLa cells, and the splicing products were analyzed by reverse-transcription PCR amplification. A normal transcript consisting of three exons was obtained from the normal construct. From the mutant, we obtained one product containing a 98 bp deletion at the 3' end of exon-32, indicating complete inactivation of the native splice-donor site. Thus, both in vivo and in vitro experiments demonstrate that 4518+5G > A causes a splicing error leading to transcript termination; it did not behave like a silent polymorphism. Our results indicate that the in vitro splicing system is a powerful tool for determining the underlying mechanism of a disease-causing mutation in a splicing consensus sequence.

Base Sequence↗
PubMed2004

Xpro: database of eukaryotic protein-encoding genes.

Xpro is a relational database that contains all the eukaryotic protein-encoding DNA sequences contained in GenBank with associated data required for the analysis of eukaryotic gene architecture. In addition to the information found in the GenBank records, which includes properties such as sequence, position, length and description about introns, exons and protein-coding regions, Xpro provides annotations on the splice sites and intron phases. Furthermore, Xpro validates intron positions using alignment information between the record's sequence and EST sequences found in dbEST. In the process of validation, alternative splicing information is also obtained and can be found in the database. The intron-containing genes in the Xpro are also classified as experimental or predicted based on the intron position validation and specific keywords in the GenBank records that are present in predicted genes. An Entrez-like query system, which is familiar to most biologists, is provided for accessing the information present in the database system. A non-redundant set of Xpro database contents is also obtained by cross-referencing to the Swiss-Prot/TrEMBL and Pfam databases. The database currently contains information for 493,983 genes--351,918 intron- containing genes and 142,065 intron-less genes. Xpro is updated for each new GenBank release and is freely available via the internet at http://origin.bic. nus.edu.sg/xpro.

Alternative Splicing↗
PubMed2002

Do introns favor or avoid regions of amino acid conservation?

Are intron positions correlated with regions of high amino acid conservation? For a set of ancient conserved proteins, with intronless prokaryotic but intron-containing eukaryotic homologs, multiple sequence alignments identified residues invariant throughout evolution. Intron positions between codons show no preferences. However, introns lying after the first base of a codon prefer conserved regions, markedly in glycines. Because glycines are in excess in conserved regions, this behavior could reflect phase-one introns entering glycine residues randomly in the ancestral sequences. Examination of intron positions within codons of evolutionarily invariable amino acids showed that roughly 50% of these introns are bordered by guanines at both 5'- and 3'-ends, 25% have a G only before the intron, and 5% have a G only after the intron, whereas about 20% are bordered by nonguanine bases.

Alternative Splicing↗
PubMed1993

On the ancient nature of introns.

We discuss some of the arguments for introns arising early or late in evolution. We outline the exon theory of genes and discuss the series of discoveries of introns in the gene (TPI) encoding triosephosphate isomerase (TPI) that have filled out a series of better fits to the Go plot, culminating in the 1986 prediction of an intron position that was finally discovered in 1992. We present a statistical argument that the 11-intron structure of TPI (based on attributing all of the introns to an ancestral gene and interpreting three cases of very close intron positions as examples of sliding) has a clear relationship to the protein structure. The exons of this 11-intron TPI are a better approximation to Mitiko Go's modules (Go, 1981) than are 99.9% of all alternative exon patterns corresponding to 11 introns placed randomly in the gene, and better than 96% of all alternative patterns in which the lengths of the exons are preserved while the introns are moved. We combine four tests relating exons to protein structure: (i) whether the exons are compact modules, (ii) whether the exons contain most of the close contacts in the protein, (iii) whether the exon configuration maximized buried surface area along the backbone, and (iv) whether the exons maximize their content of hydrogen bonds. On a joint measure for these tests, the native exon structure with 11 introns fits these tests better than 99.4% of all alternative structures obtained by permuting the exon lengths and intron positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

[New polymorphic sites in the structure of the human phenylalanine hydroxylase gene].

A previously unknown sequence of the human phenylalanine hydroxylase (PAH) gene intron 7 (GeneBank AN AF204239) has been reported. Screening of the group of phenylketonuria patients from Nobosibirsk region for polymorphic sites within intron 7 revealed single nucleotide substitutions at intron positions 332, 451, 574 and 791. Polymorphic site at intron position 791 corresponds to one of the eight restriction sites (MspI) utilized for haplotype construction. Analysis of the MspI allele frequencies in 29 phenylketonuria patients showed that the frequency of the MspI+ allele in this group was 79.4%. Polymorphic sites at nucleotide position +97 from the beginning of intron 10, and at nucleotide position -54 from the end of intron 5, were also described. The polymorphic sites revealed can be used as markers for identification of the PAH alleles in population genetic studies, and also serve for diagnostics of phenylketonuria (PKU). The presence of numerous nucleotide substitutions within the intronic sequences confirms highly polymorphic structure of the PAH gene.

Alleles↗
PubMed2001

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists.

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns.

Amino Acid Sequence↗
PubMed1996

Introns and protein revolution--an analysis of the exon/intron organisation of actin genes.

A catalogue of intron positions obtained from a large number of actin genes has been compiled with a view to understanding the possible origin of intervening sequences. Actins are ubiquitous proteins conserved in evolution and an analysis of their gene structures from various organisms has revealed that there may be at least 25 intron positions distributed at different positions in the coding regions. A comparison of intron positions from a wide range of organisms from that of yeast to human actins shows that introns could be more ancestral in origin. The conservation in the observed intron patterns within the different tissue types hints at a possible functional significance of introns in present day actin genes.

Actins↗
PubMed1985

Protein structures and split genes.

Exon-intron structures of eukaryotic genes were examined closely in their relation to primary and tertiary structures of the proteins they encode. Specific attention was given to the introns of genes encoding proteins having no repeats in their amino acid sequences. such introns have been shown to be located at sites corresponding to inter-domain or inter-module junctions of proteins identified in their three dimensional structures. "Modules," compact structural units in globular domains of proteins, are identified by drawing a distance map. Intron positions are found to correspond to intermodule junctions in various proteins whose X-ray crystallographic data are available: the globin family, CEWL, ovomucoid, cytochrome c, ADH, and trypsin-like serine proteinases. The good correspondence between intron positions and intermodule junctions excludes a mechanism of random insertion of introns, because the probability of intron insertion at each intermodule junction is extraordinarily small. Intron positions have been very stable and well conserved during evolution. However, at some inter-module junctions no introns are found. Modules in small proteins having no core modules buried in their interior have a character suitable for recruitment through their assembly into a stable domain; one side of them is rich in hydrophobic residues and the other in hydrophilic residues. Functionally important residues are scattered on different modules in the proteins examined. Based on these observations, the role of modules in the precellular period was conjectured: some of them might be functionally active by themselves but most modules might be only segments who could function as an active protein only in an assembly. The origin of introns might be traced back prior to the divergence of prokaryotes and eukaryotes.

Animals↗
PubMed1993

Structure of the gene encoding the human plasma membrane calcium pump isoform 1.

The complete structure of the gene for the human plasma membrane calcium ATPase isoform 1 (hPMCA1) has been elucidated. The protein is encoded by 21 exons present on overlapping clones covering more than 100 kilobases (kb) of DNA. An intron of over 35 kb separates the 5'-untranslated exon 1 from the exon containing the translational start codon. The entire putative promoter and 5'-flanking region is embedded in a CpG island and is characterized by the presence of numerous Sp1 factor-binding sequences and by the absence of a TATA box. In accordance with the ubiquitous tissue distribution of its mRNA these results suggest that the hPMCA1 gene is of the housekeeping type. No alternative splicing comparable to that identified in PMCA2 RNAs at site "A" and in PMCA3 RNAs close to site "C" seems to occur in hPMCA1 transcripts; however, a region in intron 6 shows significant resemblance to the site "A" alternatively spliced exons in PMCA2 and may represent a pseudoexon or a functional exon not yet detected in any PMCA1 mRNA. At six positions, intron interruptions in the hPMCA1 gene correlate with the boundaries of putative transmembrane domains in the protein, whereas most of the remaining intron positions do not show an obvious correlation with the proposed pump domain structure. The limited conservation of intron positions in different P-type pump genes indicates their early separation from a common ancestor.

Amino Acid Sequence↗
PubMed1994

The actin gene family in the oriental fruit fly Bactrocera dorsalis. Muscle specific actins.

The actin protein is a critical protein in eukaryotic cells. Four actin genes, constituting what appear to be a set of muscle specific actin genes, have been isolated from the genome of the oriental fruit fly Bactrocera dorsalis. DNA sequences have been determined for the coding as well as 3' and 5' flanking regions for each of these genes. These genes have also been characterized in terms of RNA expression patterns, and comparisons have been made to actin genes from other species. Consistent with other actins, there is a high degree of amino acid sequence conservation in the coding regions of these genes. However, even within the coding regions codon usage patterns in the oriental fruit fly are quite different from some other well characterized species. In addition, the DNA sequences in the intermediate 3' and 5' flanking regions exhibit virtually no detectable sequence homology both within and between species. In terms of introns, three of the four actin genes from the oriental fruit fly described here have a single intervening sequence. Two of these genes share the same intron position with the two muscle specific actin genes act79B and act88F from Drosophila melanogaster and with one muscle specific actin gene CcA1 from the Mediterranean fruit fly, Ceratitis capitata. Another gene from the oriental fruit fly shares the same intron position as the muscle specific actin gene act57B from D. melanogaster. Such conservation of intron positioning between species is highly unusual among previously characterized actin genes. Using unique sequences found in the 3' untranslated regions, gene specific probes have also been constructed. These have been used to detect the expression patterns of individual genes in a temporal and spatial manner. Each of the four genes examined here show differential patterns of expression. The patterns indicate that all four genes are most likely to encode muscle specific actins.

Actins↗
PubMed1989

Human testicular lactate dehydrogenase-C gene is interrupted by six introns at positions homologous to those of LDH-A (muscle) and LDH-B (heart) genes.

Human genomic clones containing parts of testis-specific lactate dehydrogenase-C gene of approximately 40 kilobases in length were isolated and characterized. The protein-coding sequence of human LDH-C gene is interrupted by six introns at positions homologous to those of mammalian LDH-A (muscle) and LDH-B (heart) genes, and exhibits 21%, 24% and 34% nucleotide differences with those of mouse LDH-C, human LDH-A and LDH-B genes, respectively.

Amino Acid Sequence↗
PubMed1995

An A-to-G mutation at the +3 position of intron 8 of the HEXA gene is associated with exon 8 skipping and Tay-Sachs disease.

Tay-Sachs disease (TSD) results from a deficiency of beta-hexosaminidase A (EC 3.2.1.52) activity. A child with late-infantile TSD was found to have two HEXA mutations, 986 + 3A-->G (A-->G at the +3 position of intron 8) and 533G-->A, associated with the variant B1 form of TSD. We were able to detect exon 8-deleted, but no correctly spliced HEXA mRNA, from the non-533G-->A allele in this patient. This suggests that 986 + 3A-->G results in missplicing and, together with 533G-->A, TSD.

Alleles↗
PubMed1991

Heterozygous mutation in the G+5 position of intron 33 of the pro-alpha 2(I) gene (COL1A2) that causes aberrant RNA splicing and lethal osteogenesis imperfecta. Use of carbodiimide methods that decrease the extent of DNA sequencing necessary to define an unusual mutation.

Cultured skin fibroblasts from a proband with osteogenesis imperfecta were found to synthesize normal and shortened alpha 2(I) chains of type I procollagen. A cDNA library was prepared using mRNA isolated from the proband's fibroblasts. Partial nucleotide sequencing of five clones demonstrated that two clones lacked the 54 base pairs (bp) of coding sequences found in exon 33 of the pro-alpha 2(I) gene (COL1A2). To reduce the amount of nucleotide sequencing required, heteroduplexes were prepared from two of the clones, one normal and the other lacking exon 33, and reacted with a water-soluble carbodiimide under conditions in which nonbase-paired G and T nucleotides are specifically modified by the reagent. Analysis of the heteroduplexes by immunoelectron microscopy suggested that the sequence variation near the codons of exon 33 was the only sequence difference in the cDNA clones. Amplification of cDNA from the proband by polymerase chain reaction gave products of two sizes, one of the expected size for the normal sequence and the other of the expected size for a product lacking the 54 bp in exon 33. To define the mutation in genomic DNA, a 1.6-kilobase region spanning exons 32 and 34 was amplified by the polymerase chain reaction and DNA heteroduplexes were prepared from the products. The heteroduplexes were treated with a water-soluble carbodiimide and then used as templates for primer extension under conditions in which extension terminates at the site of a carbodiimide-modified base. The results suggested a mismatch near the exon-intron boundary of exon 33 and a second mismatch near the 3' end of intron 33. Nucleotide sequencing of the polymerase chain reaction products revealed a single-base substitution in one allele that changed the moderately conserved G at position +5 of the 5' splice site of intron 33 to an A. In addition, there was an apparently neutral single-base substitution that placed both a G and T at position +661 of intron 33. The results provide only the third example of a mutation in the G at the +5 position of an intron that causes aberrant RNA splicing. Also, the results demonstrate that use of techniques involving carbodiimide modification of DNA heteroduplexes can reduce the amount of nucleotide sequencing necessary to define mutations in large and complex genes.

Adult↗