Novel cDNA sequences of aryl hydrocarbon receptors and gene expression in turtles (Chrysemys picta and Pseudemys scripta) exposed to different environments

Reproductive changes have been observed in painted turtles from a site with known contamination located on Cape Cod, MA, USA. We hypothesize that these changes are caused by exposure to endocrine-disrupting compounds and that genes involved in reproduction are affected. The aryl hydrocarbon receptor (AHR) is an orphan receptor that is activated by environmental contaminants. AHR mRNA was measured in turtles exposed to soil collected from a contaminated site. Adult turtles were trapped from the study site (Moody Pond, MP) or a reference site and exposed to laboratory environments containing soil from either site. The red-eared slider was used to assess neonatal exposure to soil and water from the sites. The environmental exposures occurred over a 13-month period. Juveniles showed an age-dependent increase in brain AHR1. Juvenile turtles exposed to the MP environment had elevated gonadal AHR1. Adult turtles exposed to the MP environment showed significantly decreased brain AHR2. The painted turtle AHR is the first complete reptile AHR cDNA sequence. Phylogenetic analysis of the painted turtle AHR showed that it clusters with other AHR2s. Partial AHR1 and partial AHR2 cDNA sequences were cloned from the red-eared slider. MEME analysis identified 18 motifs in the turtle AHRs, showing high conservation between motifs that overlapped functional regions in both AHR isoforms.     

纤枝短月藓BeLEA2基因的克隆及表达分析

为探究纤枝短月藓LEA2基因的结构和表达特征,该研究以纤枝短月藓为材料,首次利用PCR克隆技术得到纤枝短月藓BeLEA2基因序列,并对该基因进行分析。结果表明:(1)该基因序列中含有2个外显子和1个内含子,其开放阅读框(ORF)为456 bp,编码151个氨基酸,预测其相对分子质量为16515.96 Da。(2)将纤枝短月藓与其他植物LEA2基因氨基酸序列进行比对,构建系统进化树,结果显示纤枝短月藓与小立碗藓的亲缘关系最近。(3)利用HiTail-PCR技术克隆获得1072 bp的BeLEA2启动子序列,用PlantCARE在线工具对该启动子的顺式作用元件进行预测,结果表明该启动子除了含有核心启动子元件TATA-box和CAAT-box外,还含有ABRE、MYB、MYC、MYB结合位点(MBS)等其他顺式元件。(4)实时荧光定量PCR分析表明,BeLEA2基因在纤枝短月藓不同发育时期和不同组织中都有表达,且对脱水胁迫有响应。以上结果为进一步探究LEA2基因在苔藓植物中的功能及作用机制奠定了基础。     

柠条锦鸡儿F3H基因克隆及功能分析

柠条锦鸡儿为豆科灌木,对各种环境胁迫具有较强的适应能力,类黄酮是天然的抗氧化剂,花青素属类黄酮化合物,逆境胁迫会影响植物体内花青素的合成,而黄烷酮3-羟化酶(F3H)是花青素生物合成所必需的一种关键酶。该研究成功分离克隆了柠条锦鸡儿的F3H基因,命名为CkF3H。CkF3H基因的开放阅读框(ORF)为1095 bp,编码364个氨基酸,推测的蛋白质分子量为41.3 kDa,理论等电点为5.9。生物信息学分析发现,CkF3H基因序列与其它植物F3H有较高的一致性,推测CkF3H蛋白与其它植物F3H蛋白具有相似的功能。利用染色体步移法克隆得到CkF3H起始密码子ATG上游468 bp的启动子序列,PlantCARE软件分析表明,该序列具有启动子的基本元件CAAT-box和TATA-box以及多种与逆境胁迫相关的顺式调控元件。实时荧光定量PCR分析表明,CkF3H在柠条的根、茎和叶中均有表达,没有组织特异性;CkF3H的表达受低温、高盐、干旱和高温胁迫的诱导,并且在低温胁迫下,CkF3H的表达还受到光周期的影响。综上所述,研究结果表明CkF3H基因在柠条锦鸡儿适应低温、高盐、干旱和高温胁迫的过程中发挥作用。     

独一味苯丙氨酸解氨酶基因的克隆与表达分析

以独一味叶片为材料,采用RT-PCR和RACE方法克隆了独一味苯丙氨酸解氨酶基因（PAL）的全长cDNA,命名为LrPAL基因。测序结果表明,LrPA L基因全长2 298 bp,含有1个2 145 bp的完整开放阅读框（ORF）,编码714个氨基酸。蛋白序列分析表明,其包含典型的PAL活性中心序列（GTITASGDLVPLSYIA）,与其他植物的PAL蛋白有很高的同源性。系统进化树分析表明,独一味LrPAL与唇形科植物的PAL蛋白聚为一类,说明两者的亲缘关系较近。用 Real-Time PCR方法检测发现,LrPAL基因在独一味的叶中表达量最高,茎中表达量最少。研究结果推测,从独一味中克隆获得的苯丙氨酸解氨酶基因（LrPAL）是典型的PAL家族成员,在独一味各组织发育过程中具有重要功能。     

Conversion of α-methyltropate to optically active α-phenylpropionate by tropate-degrading Rhodococcus sp. KU1314

Microorganisms which can assimilate tropate were screened from soil. Among them, we found a microorganism which has an ability to convert α-methyltropate to optically active α-phenylpropionate, and it was identified as Rhodococcus sp. KU1314. Substrate specificity of the microorganism has been studied. When the aryl group was phenyl, 4-methoxyphenyl and 2-naphthyl, the substrate gave optically active α-propionate in good yields. To estimate the reaction mechanism, some compounds considered to be the intermediates were subjected to the reaction. Both enantiomers of α-methyltropate were converted to (R)-α-phenylpropionate with almost the same enantiomeric excess (68 and 72% from R-and S-enantiomers, respectively) and yield (605 and 48% from R-and S-enantiomers, respectively).     

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.     

麻竹miR172a靶基因DlAP2的克隆及其表达

为了解开花麻竹(Dendrocalamus latiflorus)的Dl AP2基因功能,采用RT-PCR和RACE技术克隆了mi R172a靶基因AP2同源序列c DNA全长,命名为Dl AP2。结果表明,Dl AP2基因c DNA全长为1729 bp,包含5′端非编码区81 bp、开放阅读框1464 bp、3′端非编码区160 bp和24个碱基的Poly A尾巴,在编码框靠近3′端130 bp处有1个高度匹配mi R172a的结合位点(CTGCAGCATCATCAGGATTCT)。Dl AP2编码487个氨基酸的蛋白,具有两个AP2结构域,属于AP2/ERF家族AP2亚家族的AP2组,与来自其它单子叶植物的AP2蛋白均有较高同源性。RLM-5′RACE分析表明,mi R172a主要在靶序列的第11~12个碱基之间剪切靶基因Dl AP2的mi RNA。q RT-PCR结果表明,麻竹花芽中Dl AP2基因的表达规律与mi R172a表达变化正好相反,证明mi R172a对Dl AP2基因的表达具有调控作用。     

Genetic instability at the cut locus of Drosophila melanogaster induced by the MR-h12 chromosome

Summary Unstable mutations were generated at the cut locus by the MR-h12 factor which induces male recombination. The unstable allele ct ^MR2, containing the MR-transposon in the cut locus is a very powerful mutator producing a number of different viable and lethal mutations both in the cut locus and outside it.I describe several types of mutations: stable reversion to wild type, which were sometimes associated with the appearance of unstable mutations in other loci; of stable deficiencies at the cut locus (lethals); new unstable mutations at different loci with the ct ^MR2 allele conserved; new unstable cut alleles with a phenotype other than that of ct ^MR2. The possible mechanisms of these mutational events are discussed. The genetic system constructed in the present work affords an opportunity for molecular studies of the cut locus and the MR-transposon, as a sequence from the cut locus has recently been cloned (Tchurikov et al. 1981).     

Doc and copia instability in an isogenic Drosophila melanogaster stock

A high degree of heterogeneity and an overall increase in number of insertion sites of the mobile elements Doc and copia were revealed in one substock of an isogenic Drosophila melanogaster stock, while in two other substocks the distribution of copia sites was highly homogenous, but that of Doc sites was again heterogenous. We therefore concluded that copia was unstable in one of the substocks and Doc was unstable in all. Doc instability presumably arose earlier than copia instability. Doc and copia transpositions were directly observed in experiments with one substock. An abundance of copia insertions was revealed in the X chromosome where insertions with deleterious effects are exposed to selection in hemizygous condition. The locations of many other mobile elements (mdg1, mdg2, mdg3, mdg4, 297, B104, H.M.S. Beagle, I, P, BS, FB) were found to be conserved in each substock and did not differ between them, indicating that these mobile elements were stable. This homogeneity is a strong argument against any possibility of inadvertent contamination.     

Molecular cloning and expression of retinoic-acid synthesizing enzyme raldh2 from Takifugu rubripes

Retinaldehyde dehydrogenases (raldhs) synthesize retinoic acid (RA), which is required for pattern formation and organogenesis during embryogenesis. To elucidate the common role of RA on vertebrate embryos, we first sought to clone a homologous gene to human raldh2 from fugu, Takifugu rubripes. We cloned a 1837 bp cDNA that encodes fugu raldh. The deduced amino acid sequence of the fugu raldh comprises 502 amino acids. The fugu Raldh showed highest sequence identity to zebrafish, Danio rerio, Raldh2 (79.9%). The fugu Raldh also showed high sequence identity to other vertebrate Raldh2: Xenopus laevis (77.2%), human (77.4%), mouse (74.3%) and chick (73.9%). Comparative genomic analysis showed that the gene arrangement around fugu raldh agreed with that of human raldh2. Fugu raldh mRNA was expressed through embryogenesis similarly to raldh2 in other vertebrates. These results and phylogenetic analyses suggest that pufferfish raldh is a fugu orthologue of other species' raldh2.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

A polysaccharide from Lichina pygmaea and L. confinis supports the recognition of Lichinomycetes

The lichen-forming order Lichinales, generally characterized by prototunicate asci and the development of thalli with cyanobacteria, has recently been recognized as a separate class of ascomycetes, Lichinomycetes, as a result of molecular phylogenetic studies. As alkali and water-soluble (F1SS) polysaccharides reflect phylogeny in other ascomycetes, a polysaccharide from Lichina pygmaea and L. confinis was purified and characterized to investigate whether these F1SS compounds in the Lichinomycetes were distinctive. Nuclear magnetic resonance (NMR) spectroscopy and chemical analyses revealed this as a galactomannan comprising a repeating unit consisting of an α-(1→6)-mannan backbone, mainly substituted by single α-galactofuranose residues at the O-2- or the O-2,4- positions linked to a small mannan core. With the exception of the trisubstituted mannopyranose residues previously described in polysaccharides from other lichens belonging to orders now placed in Lecanoromycetes, the structure of this galactomannan most closely resembles those found in several members of the Onygenales in Eurotiomycetes. Our polysaccharide data support molecular studies showing that Lichina species are remote from Lecanoromycetes as the galactofuranose residues are in the α-configuration. That the Lichinomycetes were part of an ancestral lichenized group can not be established from the present data because the extracted polysaccharide does not have the galactofuranose residue in the β configuration; however, the data does suggest that an ancestor of the Lichinomycetes contained a mannan and was part of an early radiation in the ascomycetes.     

拟南芥抵抗丁香假单胞杆菌过程中AZI1基因功能研究

AZI1属于脂转移蛋白家族,它在拟南芥抵抗病原菌侵染过程中可能起着传递信号物质的作用。该实验以过表达和T-DNA插入突变体及野生型拟南芥植株为材料,通过RNA印迹、蛋白质免疫印迹和原位免疫组织化学方法,研究了拟南芥壬二酸诱导基因AZI1对丁香假单胞杆菌的抗性功能。结果表明:(1)AZI1基因可以被丁香假单胞杆菌、H2O2和乙烯利诱导,它可能参与水杨酸和乙烯介导的抗菌途径。(2)蛋白质免疫印迹实验结果显示,丁香假单胞杆菌侵染叶片的叶柄渗出液中存在AZI1蛋白及其同源物EARLI1,并能够与其他蛋白质形成复合体,说明AZI1有可能通过维管组织移动到个体的其他部位,与信号分子的转移有关。(3)AZI1及其同源物EARLI1主要在花序茎的木质化部位表达,过表达AZI1基因能够促进木质素的合成,提高拟南芥对丁香假单胞杆菌的抗性。     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Conditionallethality of Escherichia coli strains carrying dnaE anddnaQ mutations

Summary A double mutant of Escherichia coli K12 which carries a conditional lethal mutator mutation, dnaQ49 (Horiuchi et al. 1978), and a DNA polymerase III-deficient mutation, dnaE486 (Wechsler and Gross 1971), was found to be more thermolabile than was either of the dnaQ49 or dnaE486 single mutants. The double mutant is able to grow at28° C but not at 30° C. Under the restrictive conditions DNA synthesis, but not protein synthesis, of the double mutant was suppressed. All the other combinations of dnaQ and dnaE mutation alleles tested so far rendered the cells thermolabile. a dnaZ mutation exerted a similar effect on the dnaQ strain. However, when non-specific temperaturesensitive graowth mutations were conbined with the dnaQ49 mutation, no such increase in thermosensitivity was observed. There is a possibility that the product of the dnaQ gene interacts directly with the DNA replicating enzyme complex.     

5种相思树和尾巨桉人工林土壤养分和酶活性特征

为揭示固氮树种土壤养分转化的酶学机制,对固氮树种[厚荚相思(Acacia crassicarpa)、黑木相思(A. melanoxylon)、卷荚相思(A.cincinnata)、大叶相思(A.auriculiformis)和马占相思(A.mangium)]及非固氮树种尾巨桉(Eucalyptusurophylla×E.grandis)人工林的土壤养分含量、酶活性及其相关性进行研究。结果表明,相思林40～60cm土层的pH均高于尾巨桉林;5种相思林土壤各土层的TP、TK含量均低于尾巨桉林,而20～40 cm土层的TC、TN含量均高于尾巨桉林,黑木相思林和马占相思林各土层的有效养分均显著高于尾巨桉林(P0.05)。0～10 cm土层中,相思林的土壤酸性磷酸酶和纤维素酶活性均高于尾巨桉林,大叶相思林的土壤脲酶、蔗糖酶、纤维素酶和芳基硫酸酯酶活性显著高于尾巨桉林(P0.05),卷荚相思林的土壤脲酶、纤维素酶、几丁质酶和淀粉酶活性显著高于尾巨桉林(P0.05)。相关分析结果表明,土壤脲酶、蔗糖酶和几丁质酶活性与AP显著负相关(P0.05),蔗糖酶和纤维素酶活性与NH4+-N显著负相关(P 0.05),脲酶、纤维素酶、芳基硫酸酯酶与土壤TK显著负相关(P0.05),几丁质酶活性与TN含量呈显著正相关(P0.05),土壤淀粉酶活性与NH4+-N呈显著正相关(P 0.05),过氧化氢酶活性与土壤TK含量呈显著正相关。可见,与尾巨桉人工林相比,在我国南方退化山地引种相思树可提高土壤关键酶的活性,对土壤有效养分具有明显改良作用,有利于退化地土壤的生态修复及人工林长期生产力的维持。     

Complementation studies with the repair-deficient uvrD3, uvrE156, and recL152 mutations in Escherichia coli

Summary Previous work showed that the mutations uvrD3, uvrE156, and recL152 were closely linked and increased UV-sensitivity. They were phenotypically distinguishable in that only the uvrD3 mutation significantly decreases host cell reactivation of UV-irradiated phage (Hcr^-) and repair of methylmethane sulfonate (MMS)-induced damage, and only the uvrE156 mutation increased mutation rates (Mut^-). MMS-resistant revertants of a uvrD3 mutant were still UV-sensitive and fell into two phenotypic classes, Hcr^- Mut⁺ (non-mutator) and Hcr⁺ Mut^-. In this work complementation tests were done by examining UV-and MMS-sensitivity and host cell reactivation in heterogenotes containing combinations of uvrD3, uvrE156, recL152, and the MMS-resistant mutations derived from uvrD3. The mutations could not complement each other in the repair of UV-damage, the one trait all had in common, indicating that they were in one gene. For the most part, the different mutations were able to complement each other in respect to traits in which one was deficient and the other had wild type activity.     

Cloning and expression analyses of AtMRP4 , a novel MRP -like gene from Arabidopsis thaliana

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55–63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes – safeners – in monocotyledonous species) revealed that the members of this gene family are differentially regulated. Received: 20 February 1998 / Accepted: 9 March 1998     

Flamingomyces and Parvulago, new genera of marine smut fungi (Ustilaginomycotina)

Teliospore walls, teliospore germinations, hyphal septations, cellular interactions, and nucleotide sequences from the D1/D2 region of the nuLSU rRNA gene of the marine smut fungi Melanotaenium ruppiae and Ustilago marina were examined and compared with findings in other Ustilaginomycotina. The data show that Melanotaenium ruppiae belongs to the Urocystaceae and Ustilago marina to the Ustilaginaceae. Within the Urocystaceae, Melanotaenium ruppiae is morphologically similar to Melanustilospora and Vankya. However, according to the molecular results Melanotaenium ruppiae can neither be ascribed to Melanustilospora nor to Vankya. Therefore, the new genus Flamingomyces is proposed for Melanotaenium ruppiae. Ustilago marina differs from the other Ustilaginaceae in the mode of sporulation, which exclusively occurs at the base of the host plant culms. Accordingly, the new genus Parvulago is proposed for Ustilago marina.