<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gypsy homologous sequences inDrosophila subobscura (gypsyDS)

Summary Characterization of sequences homologous to theDrosophila melanogaster gypsy transposable element was carried out inDrosophila subobscura (gypsyDS). They were found to be widely distributed among natural populations of this species. From Southern blot and in situ analyses, these sequences appear to be mobile in this species.GypsyDS sequences are located in both euchromatic and heterochromatic regions. A completegypsyDS sequence was isolated from aD. subobscura genomic library, and a 1.3-kb fragment which aligns with the ORF2 of theD. melanogaster gypsy element was sequenced. Comparisons of this sequence in three species (D. subobscura, D. melanogaster, and D. virilis) indicate that there is greater similarity between theD. subobscura-D. virilis sequences than betweenD. subobscura andD. melanogaster. Molecular divergence ofgypsy sequences betweenD. virilis andD. subobscura is estimated at 16 MY, whereas the most likely divergence time of these two species is more than 60 MY. These data strongly suggest thatgypsy sequences have been horizontally transferred between these species.Offprint requests to: T.M. Alberola     

Concordant divergence in proteins and mitochondrial DNA between two vole species in the genusClethrionomys

The bank vole (Clethrionomys glareolus) and the northern red-backed vole (C. rutilus) are two closely related species where interspecific crosses result in fertile female but sterile male offspring. Mitochondrial DNA (mtDNA) fromC. rutilus has passed the species barrier and is found inC. glareolus from northern Fennoscandia. The present report shows that the genetic distance between the two species, calculated from enzyme data (Nei'sD), is 0.64. Isoelectric focusing of muscle proteins resolved around 55 bands, of which each species had 6 or 7 bands not present in the other species. Sequence divergence of mtDNA from the two species is 13.9%. A comparison between protein and mtDNA distances in other species pairs reveals a high correlation between the two measures, indicating that differences in mtDNA between taxa are not random when compared to divergence in protein-coding nuclear genes. The relationship between genetic divergence in proteins and that in mtDNA betweenClethrionomys glareolus andC. rutilus is similar to that found in other species pairs. It is also shown that despite large differences on the protein level it is still, in some cases, possible for species pairs to produce fertile hybrid females.This study was sponsored by the Swedish Natural Science Research Council, the Erik Philip-Sörensen Foundation, and the Nilsson-Ehle Foundation.     

New Resources for Marine Genomics: Bacterial Artificial Chromosome Libraries for the Eastern and Pacific Oysters (Crassostrea virginica and C. gigas)

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at     

Characteristics of NADPH oxidase genes (Nox2, p22, p47, and p67) and Nox4 gene expressed in blood cells of juvenile Ciona intestinalis

To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox), p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4, p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively. The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic phox proteins in C. intestinalis interact is different from that in human.     

Structure and the evolutionary implication of the triplicated complement factor B genes of a urochordate ascidian, Ciona intestinalis

To elucidate the evolution of the complement system and MHC class III region, we analyzed the complement factor B (Bf) genes of a urochordate ascidian, Ciona intestinalis. Three different cDNA species, termed CiBf-1, CiBf-2 and CiBf-3, were identified. The deduced amino-acid sequences all contained the usual domains of vertebrate Bf and, in addition, three extra domains at the N-terminus. Furthermore, the serine protease domain of these CiBfs shared unique features with vertebrate complement components C1r/s and mannose-binding lectin-associated serine protease (MASP)-2/3, the absence of the disulfide bond designated histidine loop, and the usage of the AGY codon for the catalytic serine residue. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Overall deduced amino-acid identity between CiBf-1 and -2 was 88%, whereas CiBf-3 showed 49% identity to both CiBf-1 and CiBf-2. These three CiBf genes were located within an approximately 50-kb genomic region, and exons 3 and 5 of all the three Bf genes showed an extremely high degree of nucleotide identity, indicating that the CiBf genes experienced extensive reorganization, such as duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. Fluorescent in situ hybridization (FISH) to the chromosomes showed that genetic loci for the CiBfs, CiC3-1 and CiC3-2 genes are present on three different chromosomes, suggesting the possibility that the linkage among the MHC class III complement genes was established in the vertebrate lineage after its divergence from urochordates.The sequences reported in this paper have been deposited in the DDBJ database (accession nos. AB180044–AB180051).     

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in loquat (Eriobotrya japonica Lindl.) (Rosaceae, Pyrinae)

Loquat (Eriobotrya japonica Lindl.) is a fruit tree species of the Pyrinae subtribe of the Rosaceae that behaves as self incompatible. Since self-incompatibility in the Rosaceae is of the gametophytic type where a stylar ribonuclease (S-RNase) controls the female function of pollen–pistil recognition, consensus primers derived from the alignment of S-RNase sequences from other Pyrinae species were used to search for S-RNases in loquat. As a result, the first four S-RNases were sequenced for this species. The genomic sequences obtained showed the structural features of Pyrinae S-RNases. Moreover, microscopic observations of pollen tube growth in the style confirmed the inter-(in)compatibility relationships predicted from the molecular analyses. Phylogenetic analysis of the deduced amino acid sequences with other Pyrinae S-RNases confirmed that divergence of S-alleles in loquat and the Pyrinae predated speciation. This study reports for the first time the genomic characterization of S-RNases in loquat, providing a sound basis for an appropriate selection of pollinator cultivars and an adequate design of breeding programs.     

Process of invasiveness among exotic tunicates in Prince Edward Island, Canada

Over the past decade, four exotic tunicates (Styela clava, Ciona intestinalis, Botrylloides violaceus and Botryllus schlosseri) have been reported in the Brudenell estuary in Prince Edward Island (PEI), Canada. Styela clava was the first exotic tunicate to arrive in 1997, rapidly establishing, spreading, invading, and eventually becoming a nuisance in several estuaries of PEI. In the Brudenell estuary, S. clava remained the only exotic nuisance tunicate until 2003. In the fall of 2004, the vase tunicate C. intestinalis, was reported in low abundance, followed by the two colonial species, B. schlosseri and B. violaceus, reported in the spring of 2005. The abundance of C. intestinalis rapidly increased post-introduction, eventually replacing S. clava as the foremost nuisance species on mussel farms in the estuary. To date, C. intestinalis continues to colonize this estuary at epidemic proportions, resulting in the continuing drop of S. clava abundance. The current abundance of C. intestinalis is estimated at 5 cm⁻², which is similar to S. clava abundance at its height in 2003. The 2006 abundance of S. clava is estimated to have fallen to near 0 cm⁻². The dominance of C. intestinalis as a fouling organism on mussel farms is considered a serious threat to this aquaculture industry, mainly due to its unmanageable weight. The process of the detection, establishment, invasiveness, and eventual rise to nuisance level of exotic tunicates in the Brudenell River is presented.     

Phylogenetic evaluation of 5S ribosomal RNA gene and spacer in theCallistachys group (Fabaceae:Mirbelieae)

Sequences of 5S rDNA from an Australian group of papilionoid legumes were evaluated for phylogenetic informativeness. Twenty four sequences were sampled from ten species in five closely related genera:Brachysema, Callistachys, Jansonia, Nemcia andPodolobium. These sequences fell into two size classes, 200bp and 600bp, which appear to represent paralogous copies of the 5S unit at different loci. As in previous studies, the 5S rRNA gene and both ends of the inter-gene spacer are found to be conserved and almost uninformative about phylogeny at this taxonomic level. By contrast, the middle part of the spacer is highly variable, both in substitutions at individual sites and in length. The short sequences contain virtually no duplications. The long sequences contain numerous short repeats (c.20 bp) which form a regular pattern in the middle section. Phylogenies estimated from these data support monophyly of the study group, and of species. Relationships among species and genera are less clear, perhaps because divergence of the 5S spacer between species is too great. However, non-monophyly ofNemcia andBrachysema, indicated by other data, is corroborated.     

Evolutionary patterns in the antR-Cor gene in the dwarf dogwood complex (Cornus, Cornaceae)

The evolutionary pattern of the myc-like anthocyanin regulatory gene antR-Cor was examined in the dwarf dogwood species complex (Cornus Subgenus Arctocrania) that contains two diploid species (C. canadensis and C. suecica), their putative hybrids with intermediate phenotypes, and a tetraploid derivative (C. unalaschkensis). Full-length sequences of this gene (∼4 kb) were sequenced and characterized for 47 dwarf dogwood samples representing all taxa categories from 43 sites in the Pacific Northwest. Analysis of nucleotide diversity indicated departures from neutral evolution, due most likely to local population structure. Neighbor-joining and haplotype network analyses show that sequences from the tetraploid and diploid intermediates are much more strongly diverged from C. suecica than from C. canadensis, and that the intermediate phenotypes may represent an ancestral group to C. canadensis rather than interspecific hybrids. Seven amino acid mutations that are potentially linked to myc-like anthocyanin regulatory gene function correlate with petal colors differences that characterize the divergence between two diploid species and the tetraploid species in this complex. The evidence provides a working hypothesis for testing the role of the gene in speciation and its link to the petal coloration. Sequencing and analysis of additional nuclear genes will be necessary to resolve questions about the evolution of the dwarf dogwood complex.     

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protistTrichomonas vaginalis

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones fromTrichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which containedT. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of theT. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists likeEntamoeba histolytica, Trypanosoma cruzi, andLeishmania infantum.     

The sex-lethal gene homologue in Chrysomya rufifacies is highly conserved in sequence and exon-intron organization

A great variety of sex determination mechanisms exists in insect species. In Drosophila melanogaster sex is determined by the ratio between X chromosomes and autosomes, while in the blowfly Chrysomya rufifacies it is maternally determined. A cascade of genes which are involved in sex determination has been identified in D. melanogaster with the Sex-lethal gene (Sxl) as the key gene. We screened genomic libraries of C. rufifacies with a probe of the Sxl gene from D. melanogaster and isolated a genomic region that included most of the homologous gene. DNA- and protein-sequence comparison showed a high percent identity between the Chrysomya and the Drosophila gene. Up to 90% identity of the amino acid sequences was found in the region that contained the RNA-binding domains. The degree of identity is much lower outside of this functionally important region (18% identity). cDNA analysis showed a highly conserved exon-intron structure between the two species, although sex-specific splicing as used in D. melanogaster for the regulation of Sxl activity, could not be detected in C. rufifacies.     

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wildtype cells under phototrophic conditions.     

Isolation and characterization of microsatellite DNA primers for <Emphasis Type="Italic">Sinadoxa corydalifolia</Emphasis> (Adoxaceae)

Sinadoxa corydalifolia is the only species of Sinadoxa (Adoxaceae) with the aberrant morphology. This species has become extremely endangered in the Qinghai-Tibetan Plateau. To provide a population-level genetic profile for investigation and conservation of genetic diversity of this species, we developed 10 new microsatellite loci for this species by the combining biotin capture method. About 31 microsatellites were screened from the library, 10 of the screened microsatellites are polymorphic. The number of alleles per locus in 18 individuals ranged from 3 to 11, expected heterozygosity and observed heterozygosity ranged from 0.3071 to 0.6243 and from 0.1675 to 0.4357, respectively. We further performed cross-priming tests of these primers in another species of the Adoxaceae: Adoxa moschatellina and found 9 of 10 successfully amplified the targeted sequences. These newly developed loci provide a useful tool to investigate the genetic diversity and design the conversation measures of S. corydalifolia and study the genetic divergence and the initial speciation pattern between it and the related species in the Adoxaceae.     

Historical biogeography and phylogenetic relationships of the genus Chamaecyparis (Cupressaceae) inferred from chloroplast DNA polymorphism

The genus Chamaecyparis comprises five species and one variety native to Taiwan, Japan, Canada, and USA, which demonstrates a classical eastern Asian, western North American, and eastern North American disjunct distributional pattern. The phylogenetic relationships of the species of Chamaecyparis were inferred by comparing 1130 bp of the combined data set of chloroplast trnV intron and petG-trnP intergenic spacer. The phylogenetic tree shows that Chamaecyparis nootkatensis (Cupressus nootkatensis or Xanthocyparis nootkatensis) is clearly diverged from other Chamaecyparis species. For Chamaecyparis species, C. thyoides is sister to C. formosensis and C. pisifera and these together form a monophyletic group. C. lawsoniana is sister to C. obtusa and C. taiwanensis; and these form another monophyletic group. Homogeneity in evolutionary rates was found among species in these two monophyletic groups. Results indicate the divergent evolution of C. taiwanensis and C. formosensis and molecular evidence in this investigation supports C. taiwanensis as a variety of C. obtusa. Utility of cpDNA intergenic spacer petG-trnP in Chamaecyparis is also discussed. Several biogeographical implications were inferred: (1) at least two divergence events have produced the eastern Asian, and both western and eastern North American disjunct distribution in Chamaecyparis; (2) intercontinental sister species pairs are found in Chamaecyparis; (3) cpDNA divergence between two intercontinental sister pairs of C. thyoides and C. pisifera, and C. lawsoniana and C. obtusa is 2.8% and 1.1%, which suggest an estimated divergence time of 14 and 5.5 million years ago during middle and late Miocene, respectively; (4) cpDNA divergence of two Asian Chamaecyparis groups between C. obtusa and C. taiwanensis, and between C. pisifera and C. formosensis is 0.25% and 0.57%, which suggest an estimated divergence time of 1.3 and 2.9 million years ago during Pleistocene and late Pliocene, respectively; these estimated divergence times suggest a relatively recent migration of Chamaecyparis to Taiwan from the Japanese Archipelago; (5) that climatic deterioration caused the disappearance of Chamaecyparis in continental Asia is probable.     

The 18S rRNA gene sequences of four commercially important seaweeds

18S rRNA gene sequences are presented forAhnfeltia plicata, Chondrus crispus, Furcellaria lumbricalis andPalmaria palmata, commercially important marine algae of the North Atlantic. The sequences range from 1765 to 1777 nucleotides in length, with guanine + cytosine content of 50.1% to 52.4%. Sequence divergence between species in different orders was 11.3–12.3%, whereas the variation betweenC. crispus andF. lumbricalis, both from the Gigartinales, was only 3.6%. Based on limited experience with other groups of Rhodophyta, these sequences obtained from single populations are likely to be representative of the species as a whole, with little variation expected among conspecifics regardless of morphological aberration or apparent genetic isolation.NRCC 34824.     

基于nad5的西施舌漳州群体遗传分化水平分析——以蛤蜊属2个物种差异水平为参照

扩增了西施舌日照、连云港、北海、漳州4个野生群体、四角蛤蜊和中国蛤蜊各1个群体共73个样本的NAD5基因片段,测序获得了480bp核苷酸序列,分析核苷酸的多态性,旨在评估福建漳州西施舌与日照、连云港、北海西施舌之间的分化水平。结果:从73个序列中共检测到44种单倍型(Hap),其中西施舌4个群体有29种Haps,四角蛤蜊和中国蛤蜊分别有10种和5种Haps,漳州群体与北海、日照、连云港群体单倍型有明显差异;将西施舌分为北海、日照、连云港组(GP1)和漳州组(GP2)2个组,分析核苷酸差异,GP1与GP2间的T、A、G含量差异极显著(P0.01)。GP1与GP2间的遗传距离与组内(GP1、GP2)遗传距离之比为25.1—41.8,四角蛤蜊与中国蛤蜊之间的遗传距离与种内个体间遗传距离之比为24.4—36.7,GP1、GP2间的差异达到了四角蛤蜊和中国蛤蜊种间差异水平,而日照、北海群体间的遗传距离只有0.009,北海与日照群体地理位置虽远,但遗传差异则很小;AMOVA分析显示漳州西施舌发生了极显著遗传分化(FST=0.966—0.978,P0.01)。     

Cloning and comparative analysis of the gene encoding diacylglycerol acyltransferase from wild type and cultivated soybean

Diacylglycerol acyltransferase (DGAT), as an important enzyme in triacylglycerol synthesis, catalyzes the final acylation of the Kennedy pathway. In the present study, the GmDGAT gene was cloned from Glycine max by using AtDGAT as a query to search against the soybean EST database and the rapid amplification of cDNA ends (RACE) method. Allelic genes were also isolated from 13 soybean accessions and the divergence of the deduced amino acid sequences were compared. The comparison reveals that although GmDGAT is a highly conserved protein, several differences of insertion/deletion were identified in the N-terminal region of the GmDGATs from various soybean accessions. In the C-terminal regions, a single amino acid mutation specific to both G. max and G. soja was also found. The GmDGAT genomic sequences were further cloned and the number and size of exons in the DGAT genomic sequence were very similar among different plant species, whereas the introns were more diverged. These results may have significance in elucidating the genetic diversity of the GmDGAT among the soybean subgenus.     

Enzyme polymorphism inCitrullus lanatus andC. colocynthis in Israel and Sinai

Electrophoretic and morphological variation was studied in 13 cultivars ofC. lanatus and 31 accessions ofC. colocynthis from Israel. Twelve enzyme systems were assayed, representing 19 loci. We found 12 commercially grown cultivars to be monomorphic at all loci. OneC. lanatus accession collected from Israel is highly polymorphic and carries alleles ofC. colocynthis; this accession is probably a representative of a locally cultivated land race grown by Bedouins for animal feed. Over a range of 500 km two forms ofC. colocynthis were identified: one which grows along the coastal plains of the Mediterranean and the other in the arid Negev and Sinal deserts. A high level of electrophoretic and morphological divergence was found between plants of the two regions, whereas within the ecotypes little variation was observed.