A simple and rapid PCR-based method to isolate complete small macronuclear minichromosomes from hypotrich ciliates: 5S rDNA and S26 ribosomal protein gene of Oxytricha (Sterkiella) nova

Hypotrich ciliates present a macronuclear genome consisting of gene-sized instead of chromosome-sized DNA molecules. Exploiting this unique eukaryotic genome feature, we introduce, for the first time in ciliates, a rapid and easy PCR method using telomeric primers to isolate small complete macronuclear DNA molecules or minichromosomes. Two presumably abundant macronuclear DNA molecules, containing ribosomal genes, were amplified from the Oxytricha (Sterkiella) nova complete genome after using this method, and then were cloned and sequenced. The 5S rDNA sequence of O. (S.) nova is the third one reported among hypotrich ciliates; its primary and secondary structure is compared with other eukaryotic 5S rRNAs. The ribosomal protein S26 gene is the first one reported among ciliates. This "End-End-PCR" method might be useful to obtain similar gene-sized macronuclear molecules from other hypotrich ciliates, and, therefore, to increase our knowledge on ribosomal genes in these eukaryotic microorganisms.     

Bryophyte 5S rDNA was inserted into 45S rDNA repeat units after the divergence from higher land plants

The 5S ribosomal RNA genes (5S rDNA) are located independently from the 45S rDNA repeats containing 18S, 5.8S and 26S ribosomal RNA genes in higher eukaryotes. Southern blot and fluorescence in situ hybridization analyses demonstrated that the 5S rDNAs are encoded in the 45S rDNA repeat unit of a liverwort, Marchantia polymorpha, in contrast to higher plants. Sequencing analyses revealed that a single-repeat unit of the M. polymorpha nuclear rDNA, which is 16103 bp in length, contained a 5S rDNA downstream of 18S, 5.8S and 26S rDNA. To our knowledge, this is the first report on co-localization of the 5S and 45S rDNAs in the rDNA repeat of land plants. Furthermore, we detected a 5S rDNA in the rDNA repeat of a moss, Funaria hygrometrica, by a homology search in a database. These findings suggest that there has been structural re-organization of the rDNAs after divergence of the bryophytes from the other plant species in the course of evolution.     

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.     

Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS^® HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.     

纤枝短月藓LEA5基因表达及耐盐性分析

该研究以纤枝短月藓为材料,利用RT-PCR和HiTail-PCR技术分别克隆得到纤枝短月藓LEA5基因的ORF和启动子序列,并进行生物信息学、基因表达及耐盐性分析,为进一步研究LEA5蛋白的保护机制奠定基础。结果显示:(1)LEA5基因包含267 bp的开放阅读框(ORF),编码88个氨基酸。(2)LEA5基因启动子序列为1 053 bp,利用PlantCARE在线工具预测顺式作用元件显示,该启动子不仅具有典型的CAAT box元件,还含有ABRE、MYB、MYC、MYB结合位点(MBS)等其他元件。(3)荧光定量分析表明,LEA5基因在纤枝短月藓不同时期和不同组织中都有表达。(4)LEA5蛋白的异源表达提高了大肠杆菌对盐胁迫的耐受性,表明LEA5蛋白可能在耐盐性中起重要作用。     

茶树CsLEA5基因的克隆及表达分析

该研究以茶树‘龙井长叶’为材料,克隆获得了茶树胚胎发育晚期丰富蛋白基因CsLEA5的cDNA序列,该序列全长515 bp,包含一个375 bp的开放阅读框,编码124个氨基酸,预测蛋白分子量为13.5 kD,理论等电点为5.92。蛋白序列分析结果显示,CsLEA5为高亲水性和稳定性蛋白,且含有一个典型的LEA_3保守结构域,属于LEA蛋白中LEA_3亚家族成员。CsLEA5基因启动子区域包含多种与逆境响应相关的顺式作用元件,如乙烯响应元件（ERE）、胁迫响应元件（STRE）、创伤响应元件（WUN motif）及MYB、MYC转录因子识别位点等。qRT PCR分析显示,CsLEA5基因表达具有明显的组织特异性,在叶片中的表达量最高,其次是嫩茎,而在其他组织器官中的表达量较低,且CsLEA5基因表达受低温和干旱胁迫的诱导。研究表明,CsLEA5基因可能在茶树响应低温和干旱胁迫过程中发挥重要作用。该研究对了解茶树抗逆分子机制,筛选抗性候选基因资源提供了重要理论依据。     

Phylogenetic relationships of rodent pinworms (genus Syphacia) in Japan inferred from 28S rDNA sequences

The 28S rDNA from nine species of the genus Syphacia collected in Japan was sequenced, and the phylogenetic relationship was inferred from multiple sequence alignment of 28S rDNA by the MAFFT program. Phylogenetic tree indicates that S. petrusewiczi, which was the only species belonging to the subgenus Seuratoxyuris, has diverged earlier than other rodent pinworms examined and was distantly separated from the others genetically. It was revealed that S. agraria and S. vandenbrueli, whose subgeneric status has not been specified, belonged to the subgenus Syphacia together with other 6 species. Syphacia montana from Clethrionomys, Eothenomys and Microtus was very closely related to S. obvelata from Mus, and that S. frederici from Apodemus and S. vandenbrueli from Micromys were comparatively closely related to the former two species. The phylogenetic relationship among the three species of Syphacia found in Japanese Apodemus was inconsistent with the biogeography of host rodents. The co-evolutionary relationship between pinworm species and their host rodents may not be so strict and host switching has probably occurred frequently during the course of evolution.     

Phylogenetic relationship of Alexandrium monilatum (Dinophyceae) to other Alexandrium species based on 18S ribosomal RNA gene sequences

The phylogenetic relationship of Alexandrium monilatum to other Alexandrium spp. was explored using 18S rDNA sequences. Maximum likelihood phylogenetic analysis of the combined rDNA sequences established that A. monilatum paired with Alexandrium taylori and that the pair was the first of the Alexandrium taxa to diverge, followed by Alexandrium margalefii. All three are members of the Alexandrium subgenus Gessnerium Halim nov. comb.     

Karyotype analysis and physical mapping of 45S rDNA in eight species of Sophora, Robinia , and Amorpha

The karyotype analysis and physical locations of 45S rDNA were carried out by means of fluorescence in situ hybridization in three species, and two forms of Sophora, two species of Robina, and one species of Amorpha. S. japonica L., S. japonica L. f. oligophylla Franch., S. japonica L. f. pendula Loud., and S. xanthantha C. Y. Ma. are all tetraploids with 2n = 28. There were four 45S rDNA sites in pericentromeric regions of two pairs of chromosomes in each of them. S. rubriflora Tsoong. is a triploid with 2n = 21, and three sites were located in each satellite of group 5 chromosomes. In R. pseudoacacia L. (2n = 2x = 22), we examined four intensive signals in telomeric regions of two pairs of satellite chromosomes. In R. hispida L. (2n = 2x = 30), there were four other signals in centromeric regions besides those like in R. pseudoacacia. Amorpha fruticosa L. has most chromosomes (2n = 40) among the eight materials, however, there were only six 45S rDNA loci and they laid in centromeric regions, and satellites of three pairs of chromosomes. 45S rDNA is a valuable chromosomal landmark in karyotype analysis. The distribution and genomic organization of rDNA in the three genera were also discussed. __________ Translated from Acta Botanica Yunnanica, 2005, 27(3): 261–268 [译自: 云南植物研究, 2005, 27(3): 261–268]     

Phylogenetic relationships among diploid Aegilops species inferred from 5S rDNA units

Relationships among the currently recognized 11 diploid species within the genus Aegilops have been investigated. Sequence similarity analysis, based upon 363 sequenced 5S rDNA clones from 44 accessions plus 15 sequences retrieved from GenBank, depicted two unit classes labeled the long AE1 and short AE1. Several different analytical methods were applied to infer relationships within haplomes, between haplomes and among the species, including maximum parsimony and maximum likelihood analyses of consensus sequences, “total evidence” phylogeny analysis and “matrix representation with parsimony” analysis. None were able to depict suites of markers or unit classes that could discern among the seven haplomes as is observed among established haplomes in other genera within the tribe Triticeae; however, most species could be separated when displayed on gene trees. These results suggest that the haplomes currently recognized are so refined that they may be relegated as sub-haplomes or haplome variants. Amblyopyrum shares the same 5S rDNA unit classes with the diploid Aegilops species suggesting that it belongs within the latter. Comparisons of the Aegilops sequences with those of Triticum showed that the long AE1 unit class of Ae. tauschii shared the clade with the equivalent long D1 unit class, i.e., the putative D haplome donor, but the short AE1 unit class did not. The long AE1 unit class but not the short, of Ae. speltoides and Ae. searsii both share the clade with the previously identified long {S1 and long G1 unit classes meaning that both Aegilops species can be equally considered putative B haplome donors to tetraploid Triticum species. The semiconserved nature of the nontranscribed spacer in Aegilops and in Triticeae in general is discussed in view that it may have originated by processes of incomplete gene conversion or biased gene conversion or birth-and-death evolution.     

Phylogenetic relationships in the harmful dinoflagellate Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) inferred from LSU rDNA sequences

Phylogenetic relationships among chain-forming Cochlodinium species, including the harmful red tide forming dinoflagellate Cochlodinium polykrikoides, were investigated using specimens collected from coastal waters of Canada, Hong Kong, Japan, Korea, Malaysia, México, Philippines, Puerto Rico, and USA. The phylogenetic tree inferred from partial (D1–D6 regions) large subunit ribosomal RNA gene (LSU rDNA) sequences clearly differentiated between C. polykrikoides and a recently described species, Cochlodinium fulvescens. Two samples collected from the Pacific coasts of North America (British Columbia, Canada and California, USA) having typical morphological characters of C. fulvescens such as the sulcus located in the intermediate region of the cingulum, were closely related to C. fulvescens from western Japan in the phylogenetic tree. Cochlodinium polykrikoides formed a monophyletic group positioned as a sister group of the C. fulvescens clade with three well-supported sub-clades. These three clades were composed of (1) East Asian, including specimens collected from Hong Kong, western Japan, and southern Korea, (2) Philippines, from Manila Bay, Philippines and Omura Bay, Japan, and (3) American/Malaysian, from the Atlantic coasts of USA, the Pacific coast of México, Puerto Rico, and Borneo Island, Malaysia. Each of these clades is considered to be a so-called “ribotype” representing the population inhabiting each region, which is distinguished based on ribosomal RNA gene sequences in the species despite similarities in their morphological characters.     

A Phylogenetic Study of the Anopheles punctulatus Group of Malaria Vectors Comparing rDNA Sequence Alignments Derived from the Mitochondrial and Nuclear Small Ribosomal Subunits

A phylogenetic study of the members of the Anopheles punctulatus group was performed using structural and similarity-based DNA sequence alignments of the small ribosomal subunit (SSU) from both the nuclear and the mitochondrial genomes. The mitochondrial SSU gene (12S, 650 bp) proved to be highly restricted by its secondary structure and displayed little informative sequence variation. Consequently, it was considered unsuitable for a phylogenetic study of these closely related mosquito species. A structural alignment of the nuclear ribosomal DNA SSU (18S, 2000 bp) proved to be more informative than similarity-based alignments. Analyses showed the A. punctulatus group to be monophyletic with two major clades; a Farauti clade containing members displaying an all-black-scaled proboscis (A. farauti 1–3 and 5–7) and the Punctulatus clade containing members displaying extensive white scaling on the apical half of the proboscis (A. farauti 4, A. punctulatus, and An. sp. near punctulatus). Anopheles koliensis was positioned basal to the Farauti clade.     

新疆无苞芥锌指蛋白基因的克隆及表达分析

利用同源克隆法从新疆无苞芥中克隆获得1个锌指蛋白基因(OpZFP)。序列分析表明,OpZFP基因的开放阅读框为684bp,推测编码含227个氨基酸的蛋白质。生物信息学分析显示,OpZFP蛋白含有1个典型的C2H2型锌指结构,在C端含有一个可能具有转录抑制功能的EAR结构域。系统进化树分析表明OpZFP编码产物与拟南芥AtZFP1、琴叶拟南芥AlZFP1的进化关系较近。分离了OpZFP基因2 095bp的启动子序列,发现该启动子与拟南芥AtZFP1基因的启动子序列只有84.4%的相似性,启动子分析表明二者存在多处不同的顺式作用元件。半定量RT-PCR分析表明,OpZFP在根、茎、叶、花和果荚中均有表达,在根中的表达量最高。OpZFP基因受高盐、干旱和低温等胁迫的诱导表达,表明该蛋白涉及多种胁迫相关的信号传导途径。     

Molecular phylogeny and evolution of the genus Neoerysiphe (Erysiphaceae, Ascomycota)

The genus Neoerysiphe belongs to the tribe Golovinomyceteae of the Erysiphaceae together with the genera Arthrocladiella and Golovinomyces. This is a relatively small genus, comprising only six species, and having ca 300 species from six plant families as hosts. To investigate the molecular phylogeny and evolution of the genus, we determined the nucleotide sequences of the rDNA ITS regions and the divergent domains D1 and D2 of the 28S rDNA. The 30 ITS sequences from Neoerysiphe are divided into three monophyletic groups that are represented by their host families. Groups 1 and 3 consist of N. galeopsidis from Lamiaceae and N. galii from Rubiaceae, respectively, and the genetic diversity within each group is extremely low. Group 2 is represented by N. cumminsiana from Asteraceae. This group also includes Oidium baccharidis, O. maquii, and Oidium spp. from Galinsoga (Asteraceae) and Aloysia (Verbenaceae), and is further divided into four subgroups. N. galeopsidis is distributed worldwide, but is especially common in western Eurasia from Central Asia to Europe. N. galii is also common in western Eurasia. In contrast, the specimens of group 2 were all collected in the New World, except for one specimen that was collected in Japan; this may indicate a close relationship of group 2 with the New World. Molecular clock calibration demonstrated that Neoerysiphe split from other genera of the Erysiphaceae ca 35–45 M years ago (Mya), and that the three groups of Neoerysiphe diverged between 10 and 15 Mya, in the Miocene. Aloysia citriodora is a new host for the Erysiphaceae and the fungus on this plant is described as O. aloysiae sp. nov.     

Comprehensive molecular phylogenetic analysis and evolution of the genus Phyllactinia (Ascomycota: Erysiphales) and its allied genera

Phyllactinia is a unique genus within the Erysiphales (Ascomycota) having a partly endo-parasitic nature of the mycelium within the host plant tissues. We constructed phylogenetic trees for the genus Phyllactinia and its allied genera based on a total of 120 nucleotide sequences of the 28S rDNA and ITS regions to discuss their phylogenetic relationships with special references to host plants, biogeography, evolutionary dating, and taxonomy. The analysis of the Erysiphales confirmed the monophyly of the endo-parasitic genera, i.e. Leveillula, Phyllactinia, and Pleochaeta. Phyllactinia specimens used in this study were divided into six distinctive groups and three subgroups. Interestingly, Leveillula, an obligately endo-parasitic genus of the Erysiphales, grouped together with Phyllactinia, although this was not significantly supported by the Kishino–Hasegawa and Shimodaira–Hasegawa tests. This suggests that the evolution within this group of fungi occurred from partial endo-parasitism to obligate endo-parasitism. The host range of Phyllactinia is mostly confined to woody plants, especially deciduous trees. Betulaceae, Fagaceae, Ulmaceae, Moraceae, and Rosaceae may have close connections to the divergence of the groups and subgroups of Phyllactinia concerned. Most of these plant families are known as major members of the boreotropical flora of the Tertiary, which suggests an early Tertiary origin of this genus. A comparison of the phylogenies of hosts and parasites revealed that host range expansion at higher taxonomic levels (higher than family level) is independent of the phylogeny of plants. Conversely, host range expansions in lower taxonomic levels (infrafamilial or infrageneric) tend to occur within a single family or genus. An estimation of the evolutionary timing using a molecular clock approach suggested that Phyllactinia split from Pleochaeta about 60 M years ago (Ma) in the early Tertiary and divergence of the six major clades of Phyllactinia occurred between 5 and 40 Ma during the Oligocene and Miocene. Divergence within the major clades and within Leveillula occurred maybe from more than 5 Ma onwards during the Pliocene and Quaternary. This is the first comprehensive phylogenetic study of Phyllactinia and other endo-parasitic genera of the Erysiphales.     

弯枝藻属rbcL基因的适应性进化分析

为探讨淡水红藻的叶绿体基因及其适应性进化特征,选取弯枝藻属(Compsopogon)及相近外类群的rbc L基因共17条,利用PAML 4.9软件,对弯枝藻属rbc L基因编码蛋白进行生物信息学分析,并分别采用分支模型、位点模型以及分支-位点模型对基因的选择位点进行检测。结果表明,弯枝藻属rbc L基因编码蛋白的二级结构主要由α螺旋和β折叠构成,结构稳定。采用最大似然法构建的系统发育树表明,内类群为单一物种,分为3个小分支,具有一定地理分布规律。在3种进化模型中均未检测到统计上显著的正选择位点,表明绝大多数位点处于负选择压力下。因此,弯枝藻属rbc L基因未发生适应性进化。     

新疆无苞芥类钙调素蛋白基因OpCML13的克隆与分析

通过对无苞芥幼苗cDNA文库的随机克隆测序分析,获得1条与拟南芥编码类钙调素13(calmodulinlike13,CML13)高度相似的EST(GenBank登录号为JZ152285)。利用RT-PCR技术从无苞芥cDNA中克隆了该基因,命名为OpCML13,该基因开放阅读框(ORF)为447bp,编码148个氨基酸。生物信息学分析表明,OpCML13蛋白的二级结构含有4个Ca2+结合基序(EF-hands)结构,是一个亲水蛋白,无信号肽,无跨膜区域,为非跨膜蛋白;同源建模发现该蛋白包含8个α-螺旋结构和10个β-转角。系统进化树分析表明,OpCML13编码产物与拟南芥AtCML13和AtCML14进化关系较近,属于同一进化分支。半定量RT-PCR结果显示,OpCML13基因在无苞芥的不同组织中都有表达,在根中表达量最高;高盐胁迫处理6~24h,OpCML13基因的表达明显增强,随后逐渐恢复正常表达水平;低温、干旱和ABA处理6h后基因表达明显上调,并且一直保持较高的表达水平。研究表明,OpCML13基因在无苞芥逆境胁迫中可能起着重要作用。     

5S rDNA variation and its phylogenetic inference in the genus <Emphasis Type="Italic">Leporinus</Emphasis> (Characiformes: Anostomidae)

5S rDNA sequences have proven to be valuable as genetic markers to distinguish closely related species and also in the understanding of the dynamic of repetitive sequences in the genomes. In the aim to contribute to the knowledge of the evolutionary history of Leporinus (Anostomidae) and also to contribute to the understanding of the 5S rDNA sequences organization in the fish genome, analyses of 5S rDNA sequences were conducted in seven species of this genus. The 5S rRNA gene sequence was highly conserved among Leporinus species, whereas NTS exhibit high levels of variations related to insertions, deletions, microrepeats, and base substitutions. The phylogenetic analysis of the 5S rDNA sequences clustered the species into two clades that are in agreement with cytogenetic and morphological data.     

盐穗木HcPEAMT基因的克隆及表达分析

依据盐穗木编码PEAMT的EST序列设计引物,通过快速扩增cDNA末端技术,获得盐穗木磷酸乙醇胺甲基转移酶(phosphoethanolamine N-methyltransferase,PEAMT)全长cDNA,命名为HcPEAMT。序列分析表明HcPEAMT基因开放阅读框为1 482bp,编码494个氨基酸,推测分子量为56.3kD,理论等电点为5.51。保守结构域分析表明,HcPEAMT含有2个独立的S-腺苷甲硫氨酸依赖性甲基转移酶的保守结构域,每个结构域含有4个基序。系统进化树分析确认HcPEAMT与盐生植物盐角草的亲缘关系较近。实时荧光定量PCR分析表明,盐胁迫3h时,盐穗木同化枝和根中HcPEAMT基因的表达迅速上调并达到最大值,分别为对照的4.3倍和6.7倍。脱落酸(ABA)胁迫3h时,同化枝中HcPEAMT的表达量达到最高,而根中HcPEAMT的表达在12h才达到最高,表达量分别为对照的2.6和2.5倍。研究结果表明,HcPEAMT基因表达受盐胁迫的强烈诱导,也受ABA胁迫的诱导。该研究结果有助于阐明HcPEAMT基因表达与植物抗逆性的相关性。