Characterization of <Emphasis Type="Italic">EamaT1</Emphasis>, a member of <Emphasis Type="Italic">maT</Emphasis> family of transposable elements from the earthworm <Emphasis Type="Italic">Eisenia andrei</Emphasis> (Annelida,Oligochaeta)

The maT family is a unique clade within the Tc1-mariner superfamily, and their distribution is to date known as being limited to invertebrates. A novel transposon named EamaT1 is described from the genome of the earthworm Eisenia andrei. The full sized EamaT1 was obtained by degenerate and inverse PCR-based amplification. Sequence analysis of multiple copies of the EamaT1, which consisted of 0.9 and 1.4 kb elements, showed that the consensual EamaT1 with inverted terminal repeats (ITRs) of 69 bp was 1,422 bp long and flanked by a duplicated TA dinucleotide. The EamaT1 is present in approximately 120–250 copies per diploid genome but undergoes an inactivation process as a result of accumulating multiple mutations and is nonfunctional. The open reading frame (ORF) of the EamaT1 consensus encoding 356 amino acid sequences of transposase contained a DD37D signature and a conserved paired-like DNA binding motif for the transposition mechanism. The result of ITRs comparison confirmed their consensus terminal sequences (5′-CAGGGTG-3′) and AT-rich region on the internal bases for ITRs-transposase interaction.     

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5′-terminal untranslated region (5′-UTR) of 153 bp, and a 3′-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5′-UTR sequence contains an exon and an intron, and the 3′-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic–polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.     

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     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Characterization of Maltase Clusters in the Genus <Emphasis Type="Italic">Drosophila</Emphasis>

To reveal evolutionary history of maltase gene family in the genus Drosophila, we undertook a bioinformatics study of maltase genes from available genomes of 12 Drosophila species. Molecular evolution of a closely related glycoside hydrolase, the α-amylase, in Drosophila has been extensively studied for a long time. The α-amylases were even used as a model of evolution of multigene families. On the other hand, maltase, i.e., the α-glucosidase, got only scarce attention. In this study, we, therefore, investigated spatial organization of the maltase genes in Drosophila genomes, compared the amino acid sequences of the encoded enzymes and analyzed the intron/exon composition of orthologous genes. We found that the Drosophila maltases are more numerous than previously thought (ten instead of three genes) and are localized in two clusters on two chromosomes (2L and 2R). To elucidate the approximate time line of evolution of the clusters, we estimated the order and dated duplication of all the 10 genes. Both clusters are the result of ancient series of subsequent duplication events, which took place from 352 to 61 million years ago, i.e., well before speciation to extant Drosophila species. Also observed was a remarkable intron/exon composition diversity of particular maltase genes of these clusters, probably a result of independent intron loss after duplication of intron-rich gene ancestor, which emerged well before speciation in a common ancestor of all extant Drosophila species.     

Regulator of complement activation (RCA) gene cluster in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

Genome and expressed sequence tag information of Xenopus tropicalis suggested that short-consensus repeat (SCR)-containing proteins are encoded by three genes that are mapped within a 300-kb downstream of PFKFB2, which is a marker gene for the regulator of complement activation (RCA) loci in human and chicken. Based on this observation, we cloned the three cDNAs of these proteins using 3′- or 5′-RACE technique. Since their primary structures and locations of the proximity to the PFKFB2 locus, we named them amphibian RCA protein (ARC) 1, 2, and 3. Expression in human HEK293 or CHO cells suggested that ARC1 is a soluble protein of Mr ∼67 kDa, ARC2 is a membrane protein with Mr 44 kDa, and ARC3 a secretary protein with a putative transmembrane region. They were N-glycosylated during maturation. In human and chicken RCA clusters, the order in which genes for soluble, GPI-anchored, and membrane forms of SCR proteins are arranged is from the distant to proximity to the PFKFB2 gene. However, the amphibian ARC1, 2, and 3 resembled one another and did not reflect the same order found in human and chicken RCA genes. This may be due to self-duplication of ARCs to form a family, and it evolved after the amphibia separated from the ancestor of the amniotes, which possessed soluble, GPI-anchored, and membrane forms of SCR protein members. Taken together, frog possesses a RCA locus, but the constitution of the ARC proteins differs from that of the amniotes with a unique self-resemblance.     

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different <Emphasis Type="Italic">Solanum</Emphasis> species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1–4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613–884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Alternative splicing in the dyslexia-associated gene <Emphasis Type="Italic">KIAA0319</Emphasis>

The KIAA0319 gene in chromosome 6p22 has been strongly associated with developmental dyslexia. In this article we show a wide expression pattern of this gene in human adult brain by Northern blot analysis. We also performed RT-PCR analysis to detect alternative splicing variants in human brain. Most of the detected variants involve alternative splicing of the exons at the 5′ and the 3′ ends. Two main forms differing in the length of the 5′ UTR are detected at approximately the same rate. Two variants (B and C) lacking exon 19, which encodes the transmembrane domain, are the main alternative forms detected among those predicted to encode protein. These two variants could be secreted and might be involved in signaling functions. A similar RT-PCR analysis performed in mouse and rat adult brains showed that only some of the alternative splicing variants are equivalent to those found in the human gene. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The wheat D-genome HMW-glutenin locus: BAC sequencing,gene distribution,and retrotransposon clusters

A bacterial-artificial-chromosome (BAC) clone from the genome of Triticum tauschii, the D-genome ancestor of hexaploid bread wheat, was sequenced and the presence of the two paralogous x- and y-type high-molecular-weight (HMW) glutenin genes of the Glu-D1 locus was confirmed. These two genes occur in the same orientation, are 51,893 bp apart, and the separating DNA includes a 31,000-bp cluster of retrotransposons. A second retrotransposon cluster of 32,000 bp follows the x-type HMW-glutenin gene region. Each HMW-glutenin gene is found within a region of mainly unique DNA sequence which includes multiple additional genes including an active endosperm globulin gene not previously reported in the Triticeae family, a leucine-rich-repeat (LRR) type gene truncated at the 5′ end of the BAC, a kinase gene of unknown activity, remnants of a paralogous second globulin gene, and genes similar to two hypothetical rice genes. The newly identified globulin genes are assigned to a locus designated Glo-2. Comparison to available orthologous regions of the wheat A and B genomes show rapid sequence divergences flanking the HMW-glutenin genes, and the absence of two hypothetical and unknown genes found 5′ to the B-genome x-type ortholog. The region surrounding the Glu-D1 locus is similar to other reported Triticeae BAC sequences; i.e. small gene islands separated by retrotransposon clusters. Electronic Publication     

Identification of a novel human MAST4 gene, a new member of human microtubule associated serine/threonine kinase family

Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here, we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64, 63, 59, and 39% identical amino acid residues with MAST1, MAST2, MAST3, and MASTL, respectively. RT-PCR analysis revealed a relatively high expression level of MAST4 in most normal human tissues, with the exception of in testis, small intestine, colon, and peripheral blood leukocyte. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 808–815. The text was submitted by the authors in English. The nucleotide sequences reported in this paper have been submitted to GenBank under accession number: AY830839. These two authors contributed equally to this paper.     

Molecular cloning and characterization of the <Emphasis Type="Italic">Dicer-like</Emphasis> 2 gene from <Emphasis Type="Italic">Brassica rapa</Emphasis>

Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3′ end of BrDCL2, clones with three different lengths of 3′ untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain.     

Heterogeneity of the internal structure of PDR1, a family of Ty1/copia-like retrotransposons in pea

We characterised the extent of heterogeneity among PDR1 elements, a Ty1/copia-like retrotransposon family in pea, by restriction mapping and PCR with primers designed to amplify four functional domains. The data suggest that two main subfamilies of PDR1 differ in the size of their 5′-region. There are also sequence variants and rearranged copies which include a wide range of deletions of different sizes and deletions combined with insertions of host DNA, or inversions of various regions of the retrotransposon. A deletion hot-spot has been found at nucleotide position 394, where buffer sequences of 26 bp and 38 bp containing microsatellite motifs have been generated. There is more heterogeneity in the gag domain of PDR1 than in other functional domains, and the extent and pattern of this diversity was assessed among 56 Pisum accessions. We found a higher rate of rearrangement and sequence variation within the gag domain of PDR1 in P. fulvum and P. abyssinicum accessions than would be expected from the degree of insertion site polymorphism. A neighbour-joining phylogenetic tree constructed for gag sequences has a similar branching pattern to the equivalent insertion site tree, implying that the PDR1 family and its gag domain have coevolved with the pea genome. Combining both trees revealed clear and distinct subgroups among the Pisum ssp. Received: 17 March 1999 / Accepted: 20 July 1999     

Evolution of Duplicated <Emphasis Type="BoldItalic">reggie</Emphasis> Genes in Zebrafish and Goldfish

Invertebrates, tetrapod vertebrates, and fish might be expected to differ in their number of gene copies, possibly due the occurrence of genome duplication events during animal evolution. Reggie (flotillin) genes code for membrane-associated proteins involved in growth signaling in developing and regenerating axons. Until now, there appeared to be only two reggie genes in fruitflies, mammals, and fish. The aim of this research was to search for additional copies of reggie genes in fishes, since a genome duplication might have increased the gene copy number in this group. We report the presence of up to four distinct reggie genes (two reggie-1 and two reggie-2 genes) in the genomes of zebrafish and goldfish. Phylogenetic analyses show that the zebrafish and goldfish sequence pairs are orthologous, and that the additional copies could have arisen through a genome duplication in a common ancestor of bony fish. The presence of novel reggie mRNAs in fish embryos indicates that the newly discovered gene copies are transcribed and possibly expressed in the developing and regenerating nervous system. The intron/exon boundaries of the new fish genes characterized here correspond with those of human genes, both in location and phase. An evolutionary scenario for the evolution of reggie intron-exon structure, where loss of introns appears to be a distinctive trait in invertebrate reggie genes, is presented. Received: 24 January 2001 / Accepted: 27 July 2001