Conservation Of Intron Positions Research Articles

Phylogenetic analysis of teneurin genes and comparison to the rearrangement hot spot elements of E. coli

Teneurins are a novel family of transmembrane proteins conserved between invertebrates and vertebrates. There are two members in Drosophila, one in C. elegans and four members in mouse. Here, we describe the analysis of the genomic structure of the human teneurin-1 gene. The entire human teneurin-1 (TEN1) gene is contained in eight PAC clones representing part of the chromosomal locus Xq25. Interestingly, many X-linked mental retardation syndromes (XLMR) and non-specific mental retardation (MRX) are mapped to this region. The location of the human TEN1 together with the neuronal expression makes TEN1 a candidate gene for XLMR and MRX. We also identified large parts of the human teneurin-2 sequence on chromosome 5 and sections of human teneurin-4 at chromosomal position 11q14. Database searches resulted in the identification of ESTs encoding parts of all four human members of the teneurin family. Analysis of the genomic organization of the Drosophila ten-a gene revealed the presence of exons encoding a long form of ten-a, which can be aligned with all other teneurins known. Sequence comparison and phylogenetic trees of teneurins show that insects and vertebrates diverged before the teneurin ancestor was duplicated independently in the two phyla. This is supported by the presence of conserved intron positions between teneurin genes of man, Drosophila and C. elegans. It is therefore not possible to class any of the vertebrate teneurins with either Drosophila Ten-a or Ten-m. The C-terminal part of all teneurins harbours 26 repetitive sequence motifs termed YD-repeats. YD-repeats are most similar to the repeats encoded by the core of the rearrangement hot spot ( rhs) elements of Escherichia coli. This makes the teneurin ancestor a candidate gene for the source of the rhs core acquired by horizontal gene transfer.

Rose MADS-box genes ‘ MASAKO C1 and D1’ homologous to class C floral identity genes

We isolated AGAMOUS-like cDNA clones called MASAKO C1– C6 ( C4– C6 are 3′ or 5′ partial cDNA clones) and MASAKO D1 from wild rose ( Rosa rugosa Thunb. ex Murray). We found that MASAKO C1 was a homologue of AGAMOUS on the basis of sequence similarity, conservation of intron positions, and stamen- and carpel-specific expression within reproductive organs. MASAKO C1– C6 seem to have arisen via alternative splicing, and a possible function of one of the different mRNAs ‘ MASAKO C6’ is discussed in view of similarities between it and the ag- 4 variant 2 within Arabidopsis thaliana. MASAKO D1 was also thought to be a homologue of AGAMOUS based on similarity of their sequences, and on the tissue specific expression pattern with GAG2, which is an AGAMOUS homologue in ginseng. Two types of AGAMOUS homologue genes are possibly present in wild rose, as has been observed in tobacco, petunia, cucumber, and maize.

Characterization of a Glutathione S-transferase Gene Arranged in Tandem with the ATGST 1 Gene in Arabidopsis thaliana

Summary A novel glutathione S-transferase gene, designated ATGST 11 , is tandemly arranged with the ATGST 1 gene which is known to be regulated by various environmental stresses in the Arabidopsis genome. The ATGST 11 gene structure is very similar to the ATGST 1 gene with complete conservation of intron position and with a highly conserved nucleotide sequence, suggesting that they likely arose by duplication of a single primordial gene. The ATGST11 gene contains the entire transcriptional unit in 3 exons interrupted by two introns, which predicts a GST of 209 amino acids. The gene is proven to be constitutively transcribed at a very low level in root, stem, leaf and flower. Cis elements of the gene promoter, possibly responsible for the transcriptional regulation, may be mutated, and this suggests that gene transcription is not induced by environmental stimuli.

Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene [correction of glyceraldehyde-3-phosphate gene] and the use of its promoter for heterologous expression in Cryptococcus neoformans, a human pathogen.

The GPD gene encoding glyceraldehyde-3-phosphate dehydrogenase was isolated from Cryptococcus neoformans, a heterobasidiomycetous yeast that is pathogenic to humans. The gene contains 11 introns, differing from the conserved intron positions found in the GPD genes of Basidiomycetes. The predicted amino-acid sequence of this gene is extremely similar to that reported from GPD proteins of other basidiomycetes. The promoter region of the C. neoformans GPD gene was similar to those of other basidiomycetes. Plasmid constructs containing up to 1600 base pairs upstream of the native GPD open reading frame were used to express either the native URA5 gene in a ura5 mutant or the heterologous hphI gene (a bacterial gene that confers resistance to the aminoglycoside hygromycin) in a wild-type strain of C. neoformans. Transformation frequencies resulting from the plasmid-borne Gpdp::URA5 gene were at levels similar to those of the native URA5, which suggested that all the sequences necessary for proper expression were present. Transformation frequencies using the Gpdp::hphI gene constructs were poor. However, addition of DNA sequences flanking the 3'-end of an native C. neoformans gene significantly improved the transformation frequencies resulting from the expression of the heterologous hphI gene.

Amplification Primers That Target Locus-Specific Introns in Actinopterygian Fishes

The study of interspecific and intraspecific phylogeny has benefited from the development universal amplification primers that target specific regions of the mitochondrial genome. Unfortunately, analogous primer sets for nuclear-encoded regions have not always proven reliable in targeting single loci across a wide variety of organisms. We describe a strategy for developing sets of oligonucleotide primers that flank conserved intron positions within single loci. We demonstrate this approach through development of two sets of amplification primers that target polymorphic, single-locus intron regions from a large number of actinopterygian fishes. HE study of inter- and intraspecific phylog

Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis.

Advances in elucidating the molecular processes controlling flower initiation and development have provided unique opportunities to investigate the developmental genetics of non-flowering plants. In addition to providing insights into the evolutionary aspects of seed plants, identification of genes regulating reproductive organ development in gymnosperms could help determine the level of homology with current models of flower induction and floral organ identity. Based upon this, we have searched for putative developmental regulators in conifers with amino acid sequence homology to MADS-box genes. PCR cloning using degenerate primers targeted to the MADS-box domain revealed the presence of over 27 MADS-box genes within black spruce (Picea mariana), including several with extensive homology to either AP1 or AGAMOUS, both known to regulate flower development in Arabidopsis. This indicates that like angiosperms, conifers contain a large and diverse MADS-box gene family that probably includes regulators of reproductive organ development. Confirmation of this was provided by the characterization of an AGAMOUS-like cDNA clone called SAG1, whose conservation of intron position and tissue-specific expression within reproductive organs indicate that it is a homologue of AGAMOUS. Functional homology with AGAMOUS was demonstrated by the ability of SAG1 to produce homeotic conversions of sepals to carpels and petals to stamens when ectopically expressed in transgenic Arabidopsis. This suggests that some of the genetic pathways controlling flower and cone development are homologous, and antedate the 300-million-year-old divergence of angiosperms and gymnosperms.

The Arabidopsis SHAGGY-related protein kinase (ASK) gene family: structure, organization and evolution.

Higher plants contain a multigene family encoding proteins that share a highly conserved catalytic protein kinase domain about 70% identical to SHAGGY protein kinase (SGG) and glycogen synthase kinase-3 (GSK-3), respectively, from Drosophila and mammals. In this study we have characterized the structure and evolution of the Arabidopsis SHAGGY-related protein kinase (ASK) gene family. At least ten ASK genes are present per haploid genome of Arabidopsis. The genomic sequences of five ASK genes show a strikingly high conservation of intron positions and exon lengths. Phylogenetic analyses suggested that the Arabidopsis gene family contains at least three ancient classes of genes that diverged early in land plant evolution. The different classes may reflect specificity of substrates and/or biological functions. Eight out of the ten predicted ASK genes were mapped and shown to be dispersed over the five Arabidopsis chromosomes. A tentative model for the organization and evolution of the Arabidopsis ASK genes is presented.

Structure of the human CD94 C-type lectin gene.

The genomic structure of the human CD94 gene was obtained by analyzing genomic clones obtained from two different libraries. The CD94 gene contains six exons separated by five introns. The carbohydrate-recognition domain (CRD) is encoded by three exons, and the conservation of intron positions within the CRD indicated that CD94 is closely related to group V of C-type lectins. Primer extension and S1 nuclease protection assays showed that initiation of transcription in the CD94 gene is heterogeneous, but restricted to a 60 base pair region around the major initiation site enclosed within a putative initiator element (TTA+1TTCA). The study of the promoter region of CD94 may help to understand the selective expression of this C-type lectin glycoprotein on NK cells and subsets of cytotoxic T cells.

Exon–Intron Organization of the Human Type 2 Desmocollin Gene (DSC2): Desmocollin Gene Structure Is Closer to “Classical” Cadherins Than to Desmogleins

The cadherins are a superfamily of calcium-dependent glycoproteins that are cell adhesion molecules. Two families of cadherins, the desmocollins (Dsc) and desmogleins (Dsg), are found only in the desmosome type of cell–cell junction. They are each present in at least three different isoforms with differing spatial and temporal distributions and are specified by two clusters of closely linked genes on human chromosome 18q12.1. The human DSC2 gene, coding for the most widely distributed form of the desmocollins, has been found to consist of more than 32 kb of DNA. By using PCR we have determined the exon–intron organization. The gene is arranged into 17 exons ranging in size from 46 to 258 bp; exon 16 is alternatively spliced, giving rise to the a and b forms of the protein. This has revealed a remarkable degree of conservation of intron position with other cadherins. The desmocollin exon–intron organization is more similar to the so-called classical cadherins than to the desmogleins, especially in the cytoplasmic domain. Intron 1 is the largest in DSC2, as it is in the desmogleins, in contrast to the classical cadherins, where intron 2 is extremely large; this latter intron is missing from the desmogleins.

TheDfur2Gene ofDrosophila melanogaster:Genetic Organization, Expression During Embryogenesis, and Pro-Protein Processing Activity of Its Translational Product Dfurin2

The gene structure and expression of the Dfur2 gene of Drosophila melanogaster, which encodes the subtilisin-like serine endoprotease Dfurin2, was studied. The Dfur2 gene is very compact in contrast to the related Dfur1 gene, which has an estimated size of over 100 kbp. The 6-kb Dfur2 mRNA is encoded by 16 exons dispersed over a genomic region of about 9 kbp. The exon/intron organization shows conservation of intron positions not only in comparison with Dfur1, but also with the related mammalian genes FUR, PC1/PC3, PC2, and PC4. This conservation supports the hypothesis that all genes belonging to the family of subtilisin-like pro-protein processing enzymes are evolutionary related by descent from a common ancestral gene. In primer extension experiments, Dfur2 transcription initiation sites were identified in the presumed Dfur2 promoter region. This region was found to contain general RNA polymerase II promoter elements like a potential TATA box, a potential CAP signal, and several potential CCAAT boxes. Also, several sequence motifs putatively corresponding to binding sites for Drosophila transcription factors like zeste, bicoid, and engrailed were found to be present. RNA in situ hybridization experiments on Drosophila embryos revealed presumably maternal Dfur2 expression until the syncytial blastoderm (stage 5 of embryogenesis), no expression during gastrulation (stage 9), transient expression in a subset of neurons in the central nervous system of stage 12-13 embryos, and, from stage 13 onwards, expression in the developing tracheal tree. In a vaccinia expression system, the endoprotease Dfurin2 not only cleaved wild-type precursor of von Willebrand factor (pro-vWF) with pro-region cleavage site R-S-K-R decreases, but also, although to a lesser extent, pro-vWF mutants in which the P2 (vWFK-2A) or P4 (vWFR-4A) basic residue with respect to the pro-region cleavage site had been mutated. This cleavage specificity resembles that of human furin. The cleavage of pro-vWF by Dfurin2 shows that the previously reported lack of cleavage of the precursor of the beta A-chain of activin-A by Dfurin2 in this vaccinia expression system is substrate determined.

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.

Structure and expression of the Aplysia californica calmodulin gene

We have isolated and characterized the calmodulin (CaM) gene of the marine mollusc Aplysia californica. Its primary structure, deduced from the nucleotide sequence of the gene, is highly conserved, containing only three amino acid substitutions compared to the CaM of higher vertebrates. The structural gene consists of four exons that encode the protein, 3′ non-translated mRNA and at least one additional exon encoding 5′ non-translated mRNA and the initiator methionine. A comparison of the exon/intron structure of CaM genes from several organisms shows conservation of intron position, suggesting that introns have been both inserted and deleted during evolution. Northern analysis of RNA prepared from several Aplysia tissues reveals the presence of several CaM mRNA transcripts with the greatest expression observed in neural and muscle tissue. One of these transcripts is abundant only in mRNA prepared from nervous tissue.

Conservation of intron position indicates separation of major and variant H2As is an early event in the evolution of eukaryotes

Genomic clones of Drosophila and Tetrahymena histone H2A variants were isolated using the corresponding cDNA clones (van Daal et al. 1988; White et al. 1988). The site corresponding to the initiation of transcription was defined by primer extension for both Drosophila and Tetrahymena genomic sequences. The sequences of the genomic clones revealed the presence of introns in each of the genes. The Drosophila gene has three introns: one immediately following the initiation codon, one between amino acids 26 and 27 (gln and phe), and one between amino acids 64 and 65 (glu and val). The Tetrahymena gene has two introns, the positions of which are identical to the first two introns of the Drosophila gene. The chicken H2A.F variant gene has been recently sequenced and it contains four introns (Dalton et al. 1989). The first three of these are in the same positions as the introns in the Drosophila gene. The fourth intron interrupts amino acid 108 (gly). In all cases the sizes and the sequences of the introns are divergent. However, the fact that they are in conserved positions suggests that at least two of the introns were present in the ancestral gene. A phylogenetic tree constructed from the sequences of the variant and major cell cycle-regulated histone H2A proteins from several species indicates that the H2A variant proteins are evolutionarily separate and distinct from the major cell cycle-regulated histone H2A proteins. The ancestral H2A gene must have duplicated and diverged before fungi and ciliates diverged from the rest of the eukaryote lineage. In addition, it appears that the variant histone H2A proteins analyzed here are more conserved than the major histone H2A proteins.

Structure and expression of the human gene encoding plasminogen activator inhibitor, PAI-1

The gene ( pail) encoding human plasminogen activator inhibitor, PAI-1 was cloned from a λEMBL3 genomic library and was found to span approx. 12 kb and to contain eight introns. Primer extension and S1 nuclease analyses both showed the transcription start point to be located 142 nt upstream from the start codon. The 5'-flanking region was sequenced and found to contain a TATA box, but no CAAT sequence. When a fragment containing 730 nt of 5'-untranslated region was placed upstream from a promoterless cat gene, it was shown to function as a promoter when transfected into COS cells. Northern-blot analysis was consistent with low level expression of the endogenous pail gene in COS cells. When the pail gene structure was compared to those of other members of the serine-protease-inhibitor encoding gene family, little conservation of intron positions was observed.

Nucleotide sequence of the carboxy-terminal portion of a lodgepole pine actin gene

We have determined the nucleotide sequence of the carboxy-terminal portion of an actin gene (PAc1-A) isolated from Pinuscontorta var. latifolia (Engelm.). Pairwise comparisons of both nucleotide and deduced amino acid sequences were made among PAc1-A, the soybean actins SAc3 and SAc1, maize actin MAc1, chicken β-actin, and yeast β-actin. Of the other actins SAc3 was most similar to the PAc1-A amino acid sequence (91.3% identity) and yeast actin the least similar (78.3% identity). The intron in PAc1-A is present at the same location as the third intron found in MAc1, SAc1, and SAc3 actin genes. This conservation of intron position is unusual when compared with nonplant actin genes.

Structural organization of the mouse cytosolic malate dehydrogenase gene: Comparison with that of the mouse mitochondrial malate dehydrogenase gene

We cloned and characterized a mouse cytosolic malate dehydrogenase (cMDHase) (EC 1.1.1.37) gene, which is about 14 × 10 3 base-pairs long and is interrupted by eight introns. The 5′ and 3′ flanking regions and the exact sizes and boundaries of the exon blocks, including the transcription-initiation sites, were determined. The 5′ end of the gene lacks the TATA and CAAT boxes characteristic of eukaryotic promoters, but contains G + C-rich sequences, one putative binding site for a cellular transcription factor, Sp1, and at least two major transcription-initiation sites. The sequences around the transcription-initiation sites are compatible with the formation of a number of potentially stable stem-loop structures. We compared structural organization of the mouse cMDHase gene with that of the previously characterized mouse mitochondrial MDHase (mMDHase) gene, and found that the conservation of intron positions spreads across much of the two genes. This result suggests that a common ancestral gene for the cytosolic MDHase and the mitochondrial MDHase was broken up by introns, before the divergence. We also compared the nucleotide sequence of the promoter region of the mouse cytosolic MDHase gene with that of the other three mouse genes coding for isoenzymes participating in the malate-aspartate shuttle, i.e. mitochondrial MDHase, cytosolic and mitochondrial aspartate aminotransferases (cAspATase and mAspATase). We found that highly conserved regions are present in the promoter region of the cAspATase gene.

Comparison of the cis-acting control regions of two coordinately controlled genes involved in ethanol utilization in Aspergillus nidulans

The alcA and aldA genes of Aspergillus nidulans are regulated in exactly the same manner, being subject to positive control by the product of the alcR gene. We report the complete nucleotide sequence of the alcA gene and its 5′ non-coding region, preliminary localization of the region involved in the regulation of alcA expression, and a detailed comparison of this region to the 5′ non-coding region of aldA (Pickett et al., 1987). The 5′ flanking regions of the genes contain six similar sequence elements. Three of these elements are located upstream from the messenger RNA start points and one is related to a sequence element found in the region responsible for ethanol induction of the yeast ADH2 gene (Beier et al., 1985). The other homologous elements are located within the messenger RNA leader and may be associated with selection of messenger RNA start points. The amino acid sequence of alcohol dehydrogenase I (348 residues) shows a significant level of homology with analogous sequences in other organisms. Gene alcA contains introns which are similar in size and structure to other fungal introns. We discuss the positions of the introns in alcA of A. nidulans with particular reference to the conservation of intron position in and the evolutionary assembly of enzymes which possess NAD-binding domains.

The sequence of a type II keratin gene expressed in human skin: conservation of structure among all intermediate filament genes.

We report here the coding sequence of the gene for a 56-kDa type II keratin (designated K6b). Using a subclone specific for a unique 3' noncoding region of the encoded mRNA, we have shown that this gene is one of at least two 56-kDa keratin genes expressed in abundance in human epidermis. Segmenting the coding portion of this gene are eight introns, six of which are identically positioned with those of a distantly related type III intermediate filament gene (vimentin), and five of which are identically positioned with those of a distantly related type I gene (50-kDa keratin). These results indicate a common ancestral origin for all three classes of intermediate filament genes. All of the highly conserved intron positions are located within, but do not demarcate, the four central alpha-helical domains common to all intermediate filament polypeptides, suggesting that these genes were probably not created piecemeal by recombination-mediated linkage of separate structural domains as they presently are known.