A splicing site mutation in <Emphasis Type="Italic">BrpFLC1</Emphasis> and repressed expression of <Emphasis Type="Italic">BrpFLC</Emphasis> genes are associated with the early flowering of purple flowering stalk

FLOWERING LOCUS C (FLC), which encodes a MADS-box domain protein, is a flowering repressor involved in the key position of Arabidopsis (Arabidopsis thaliana) flowering network. In Brassica species, several FLC homologues are involved in flowering time like Arabidopsis FLC. Here, we report the analysis of splicing variation in BrpFLC1 and the expression of BrpFLC homologues associated with early flowering of Purple Flowering Stalk (Brassica campestris L. ssp. chinensis L. var. purpurea Bailey). It was indicated that a splice site mutation happened in intron 6 with G to A at the 5′ splice site. Three alternative splicing patterns of BrpFLC1, including the entire exon 6 excluded and 24 bp or 87 bp of intron 6 retained, were identified in Purple Flowering Stalk. But there was only one normal splicing pattern in Pakchoi (Brassica campestris ssp. chinensis var. communis). Northern blotting and semi-quantitative RT-PCR revealed that the expression levels of the three FLC homologues in Purple Flowering Stalk were lower than that in Pakchoi. However, the expression levels of downstream genes, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT), were higher in Purple Flowering Stalk. These results suggest that a natural splicing site mutation in BrpFLC1 gene and repressed expression of all BrpFLC genes contribute significantly to flowering time variation in Purple Flowering Stalk.     

The cytochrome oxidase II gene in mitochondria of the sugar-beetBeta vulgaris L.

We have cloned and analyzed the sugar-beet mitochondrial gene for cytochrome oxidase subunit II (coxII). The sugar-beet and its deduced amino acid sequence were compared to its homologouscoxII gene sequences from both monocot and dicot plants. It was found to be highly conserved (89–95%) compared to homologue in other plant species. The 780 bp coding sequence of the sugar beetcoxII gene is interrupted at position 383 by a 1463 bp intron. This intron contains an additional 107 bp sequence that is not found in any of the plantcoxII genes studied thus far. The structure of the intron suggests that a large intron existed in an ancestralcoxII gene before monocots and dicots diverged in evolution. Three CGG codons in the sugar-beetcoxII coding sequence align with conserved tryptophan residues in the homologous gene of other species, suggesting that RNA editing takes place also in sugar-beet mitochondria. In 13 out of 24 codons ofcoxII mRNA that were found to be edited in four other plants, the sugar-beet gene already utilizes the edited codons. This phenomenon may indicate that the mitochondrial genome in sugar-beet is phylogenetically more archaic relative to these plants. An additional sequence of 279 bp that is identical to the first exon ofcoxII was identified in the mtDNA of the sugar-beet. This pseudo-gene is transcribed and its existence in the mitochondrial genome is unexplained.     

The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization, evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5 border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3 acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

Cloning and characterization of an allene oxide cyclase,PpAOC3, in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Allene oxide cyclase (AOC) is a key enzyme in the octadecanoid pathway of flowering plants that synthesizes 12-oxo-phytodienoic acid (OPDA), which is a biosynthetic precursor of the signal molecule jasmonic acid (JA). A database search of the Physcomitrella patens genome revealed the presence of an AOC gene unique from the two previously reported AOC genes, PpAOC1 and PpAOC2. After cloning the identified AOC gene, designated PpAOC3, the obtained cDNA sequence (897 bp) was larger than the predicted AOC gene (765 bp) in the database because a speculated intron was not fully deleted. Although PpAOC3 did not display high similarity with AOC proteins from other species, recombinant PpAOC3 exhibited AOC activity and translocated to chloroplasts, as is observed for other AOC proteins. Notably, the expression profile of PpAOC3 differed from the other PpAOCs, as its expression in protonemata was higher than that in gametophores. Although the function of oxylipins such as OPDA and JA remains elusive in P. patens, further characterization of the enzymes in the octadecanoid pathway and the role of oxylipin will aid in the elucidation of physiological processes in this model bryophyte.     

Orthologues of a plant-specific At-4/1 gene in the genus Nicotiana and the structural properties of bacterially expressed 4/1 protein

Arabidopsis thaliana At-4/1 is the protein of unknown function capable of polar localization in plant cells and intercellular trafficking. In this work, we cloned cDNAs and chromosomal genes of At-4/1 orthologues from several Nicotiana species. Similarly to the 4/1 genes of A. thaliana and Oryza sativa, Nicotiana 4/1 genes have eight exons and seven introns but are considerably longer due to their larger introns. The allotetraploid genome of Nicotiana tabacum, which is known to consist of the ‘S genome’ originated from Nicotiana sylvestris and the ‘T genome’ derived from Nicotiana tomentosiformis, encodes two 4/1 genes. The T genome-encoded 4/1 gene, but not that of the S genome, contains a SINE-like transposable element in its intron 2. The 4/1 genes of Nicotiana hesperis and Nicotiana benthamiana lack such an element in the intron 2, but possess a related SINE-like sequence in their intron 4. Collectively, the sequence analysis data provide an insight into the organization of 4/1 genes in flowering plants and the patterns of evolution in the genus Nicotiana. The Nicotiana 4/1 proteins and those of other flowering plants show a significant level of sequence similarity. Computer-assisted analysis was further used to compare their predicted secondary structures. Several algorithms confidently predicted the presence of several coiled-coil domains occupying similar positions in different 4/1 proteins. Analysis of circular dichroism spectra carried out for bacterially expressed N. tabacum 4/1 protein (Nt-4/1) and its N- and C-terminally truncated mutants confirmed that the secondary structure of Nt-4/1 is generally alpha-helical. The C-terminal region of Nt-4/1 was found to undergo a partial proteolysis in Escherichia coli cells. Differential scanning calorimetry of Nt-4/1 protein and its mutants revealed three calorimetric domains most probably corresponding to the N-terminal, central, and C-terminal structural domains of the protein.     

Partial molecular characterization,expression pattern,polymorphism and association analysis of porcine <Emphasis Type="Italic">SKP2</Emphasis> gene

As a component of E3 ubiquitin protein ligases called SCFs, SKP2 protein belongs to a member of FBLs protein which is the biggest eukaryotic subfamily of F-BOX proteins with 12 members. In this study, we cloned and sequenced partial cDNA, intron 1 and intron 6 of porcine SKP2 gene. The partial cDNA is 1,402 bp long and has an open reading frame of 1,272 bp which encodes 424 putative amino acids. The deduced protein comprises a conserved F-BOX domain at position from the 90th to 140th amino acid. The phylogenetic tree indicated that porcine SKP2 has the closest genetic relationship with bovine SKP2 than other selected animal species. Quantitative RT-PCR analysis displayed that the tissue expression level of porcine SKP2 fluctuated remarkably in a large range, and it expressed in thymus with the highest level and in longissimus dorsi muscle with the lowest level. Two SNPs were identified, meanwhile, further polymorphism analysis with Cfr42I showed that AA genotype was in dominance absolutely among four kinds of unrelated Chinese indigenous miniature and one introduced Landrace pig breeds. In addition, association analysis with immune traits and blood parameters revealed that the SNP Cfr42I in intron 1 was significantly associated with red cell distribution width of neonate piglets at 0 day (P = 0.027).     

Unravelling proximate cues of mass flowering in the tropical forests of South‐East Asia from gene expression analyses

Elucidating the physiological mechanisms of the irregular yet concerted flowering rhythm of mass flowering tree species in the tropics requires long‐term monitoring of flowering phenology, exogenous and endogenous environmental factors, as well as identifying interactions and dependencies among these factors. To investigate the proximate factors for floral initiation of mast seeding trees in the tropics, we monitored the expression dynamics of two key flowering genes, meteorological conditions and endogenous resources over two flowering events of Shorea curtisii and Shorea leprosula in the Malay Peninsula. Comparisons of expression dynamics of genes studied indicated functional conservation of FLOWERING LOCUS T (FT) and LEAFY (LFY) in Shorea. The genes were highly expressed at least 1 month before anthesis for both species. A mathematical model considering the synergistic effect of cool temperature and drought on activation of the flowering gene was successful in predicting the observed gene expression patterns. Requirement of both cool temperature and drought for floral transition suggested by the model implies that flowering phenologies of these species are sensitive to climate change. Our molecular phenology approach in the tropics sheds light on the conserved role of flowering genes in plants inhabiting different climate zones and can be widely applied to dissect the flowering processes in other plant species.     

A SINE Family Widely Distributed in the Plant Kingdom and its Evolutionary History

The distribution and evolution of Au SINE in plants were examined. Au SINE is a short interspersed element first identified in Aegilops umbellulata, a close relative of wheat. The Au SINE was previously found in species such as wheat, maize, tobacco, and tomato, but not in rice. In this study, we first searched public databases, and next examined the presence of Au in a broad range of plant species by PCR using internal primers of Au. Although Au is likely to be absent from many species including rice, it was identified in many Gramineae, Solanaceae, and Fabaceae species, and also in a basal angiosperm species, Asimina triloba. Phylogenetic studies suggest that Au SINE originated before the divergence of monocots and eudicots. Au SINE sequences of Asimina, Triticum, Zea, Nicotiana, Lotus, Medicago, and Glycine were aligned and compared. Although sequences of Au were highly conserved among distantly related species, every Au element in Glycine had a 16 bp deletion and its 3′ end differed from sequences of other species. This type of Au could only be found in G. max, and not in other species including other Fabaceae species such as M.␣truncatula and L. japonicus. This is the first report of a plant SINE family present in multiple lineages, and the evolution of Au SINE in the plant kingdom, especially in Gramineae and Fabaceae is discussed. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

The mitochondrial coxII intron has been lost in two different lineages of dicots and altered in others

The central part of the mitochondrial coxII gene was amplified from 38 different dicots and two monocots using polymerase chain reaction. In 30 of the 40 plants studied, the amplified coxll gene-fragment contains an intron, ranging from 930 bp in Capsicum (pepper) in Solanaceae to 1,635 bp in Ampelamus albidans (climbing milkweed) in Asclepiadaceae. The composition of this intron varies as revealed by Southern hybridizations using oligonucleotide probes specific to the coxII intron-regions in maize, wheat, and rice. In the Apocynaceae, Calharanthus roseus (Madagascar periwinkle) and Vinca minor (common periwinkle) lack the coxII intron, while other members of the same family (various Mandevilla species, Nerium oleander and Apocynum cannabinum) and members of the closely related Asclepiadaceae (Asclepias incarnata, Ampelamus albidans and Asclepias tuberosa) retain the intron. Analysis of these data suggest a selective loss of the coxII intron from a plant, ancestral to both Catharanthus and Vinca, after the divergence of the Asclepiadaceae and Apocynaceae. The remaining eight plants from the Brassicaceae, Cucurbitaceae, Fabaceae, and Onagraceae lacking the intron fall into a single group or clade using the phylogenetic tree proposed by Chase et al. (Annals of the Missouri Botanical Garden: 80: 528–580, 1993) based on sequence of the chloroplast rbcL gene.     

A three‐marker DNA barcoding approach for ecological studies of xerothermic plants and herbivorous insects from central Europe

The DNA barcoding technique developed for species identification has recently been adapted for ecological studies (e.g. host plant identification). Comprehensive barcode databases, covering most species inhabiting areas, habitats or communities of interest are essential for reliable and efficient identification of plants. Here we present a three‐barcode (plastid rbcL and matK genes and the trnL intron) database for xerothermic plant species from central Europe. About 85% of the xerothermic plant species (126 out of c. 150) known to be associated with xerothermic habitats were collected and barcoded. The database contains barcodes for 117 (rbcL and trnL) and 96 (matK) species. Interspecific nucleotide distances were in the ranges 0–17.9% (0–3.2% within genera) for rbcL, 0–44.4% (0–3.1%) for trnL and 0–52.5% (0–10.9%) for matK. Blast‐searching of each sequence in the database against the entire database showed that species‐level identification is possible for 89.6% (rbcL), 98.4% (trnL) and 96.4% (matK) of examined plant species. The utility of the presented database for identification of host plants was demonstrated using two insect species associated with xerothermic habitats: the oligophagous leaf‐beetle Cheilotoma musciformis (for which two host plants in Fabaceae were identified) and the polyphagous weevil Polydrusus inustus (which was found to feed on 14 host plants, mostly Rosaceae, Asteraceae and Fabaceae). The developed database will be useful in various applications, including biodiversity, phylogeography, conservation and ecology. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 576–592.     

Genomic Structure and Chromosomal Localization of Mouse Cyclin G1 Gene

Mouse genomic DNA harboring the full coding sequence of cyclin G1 was cloned and analyzed. The locations of five coding exons and the intron–exon boundary sequences were found to be conserved between the mouse and the human genes. Two putative binding sites for thep53tumor suppressor gene product were found around the first exon: one was located in the 5′ regulatory region, and the other was in the first intron. The mouse cyclin G1 gene was mapped to bands A5 to B1 of chromosomes 11 (11A5–B1) by FISH using genomic DNA clone as a biotinylated probe. The location of mouse cyclin G1 is syntenic to that of its human homologue, which we previously mapped to 5q32–q34 of chromosome 5. An additional faint signal was detected on chromosome 4 (4B1–C2), probably indicating the presence of a cyclin G1-related gene or pseudogene in the mouse genome.     

Expression of a polyubiquitin promoter isolated from Gladiolus

A polyubiquitin promoter (GUBQ1) including its 5′UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5′ and 3′ intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5′UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5′UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.     

The evolution of protamine P1 genes in dasyurid marsupials

We report the complete DNA sequences of the protamine P1 gene and flanking regions for 13 species of the marsupial family Dasyuridae. The structure of the protamine locus is conserved in dasyurids and consists of two exons (of lengths 142–151 and 47 bp) separated by an intron (208–240 bp). A key feature of the dasyund intron is a 38–40 by duplication found in all species examined to date. This duplication apparently predates the radiation of modern dasyurid lineages and may be homologous to a similar feature in the marsupial mole (Notoryctes). Sequences from a species of Planigale demonstrate that this genus is unique among marsupials in possessing cysteine residues in its protamine P1 molecules. Cysteines may provide enhanced chemical stability for condensed sperm nuclei, a physiological feature that would converge on the common eutherian pattern. Phylogenetic analysis of the protamine genes yields a tree that is largely congruent with previous molecular systematic studies in two areas: (1) There are three main dasyurid lineages corresponding to the Sminthopsinae, Dasyurinae, and Phascogalinae; (2) Dasyurinae and Phascogalinae are sister groups. This study is the first estimate of dasyurid relationships based on a nuclear DNA sequence. Correspondence to: J.D. Retief     

Distribution and evolution of introns in drosophila amylase genes

While the two amylase genes of Drosophila melanogaster are intronless, the three genes of D. pseudoobscura harbor a short intron. This raises the question of the common structure of the Amy gene in Drosophila species. We have investigated the presence or absence of an intron in the amylase genes of 150 species of Drosophilids. Using polymerase chain reaction (PCR), we have amplified a region that surrounds the intron site reported in D. pseudoobscura and a few other species. The results revealed that most species contain an intron, with a variable size ranging from 50 to 750 bp, although the very majoritary size was around 60–80 bp. Several species belonging to different lineages were found to lack an intron. This loss of intervening sequence was likely due to evolutionarily independent and rather frequent events. Some other species had both types of genes: In the obscura group, and to a lesser extent in the ananassae subgroup, intronless copies had much diverged from intron-containing genes. Base composition of short introns was found to be variable and correlated with that of the surrounding exons, whereas long introns were all A-T rich. We have extended our study to non-Drosophilid insects. In species from other orders of Holometaboles, Lepidoptera and Hymenoptera, an intron was found at an identical position in the Amy gene, suggesting that the intron was ancestral. Received: 23 October 1995 / Accepted: 5 March 1996     

Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron

Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3′ end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals.