Time-course analysis of nuclear events during conjugation in the marine ciliate Euplotes vannus and comparison with other ciliates (Protozoa,Ciliophora)

Ciliates represent a morphologically and genetically distinct group of single-celled eukaryotes that segregate germline and somatic functions into two types of nuclei and exhibit complex cytogenetic events during the sexual process of conjugation, which is under the control of the so-called “mating type systems”. Studying conjugation in ciliates may provide insight into our understanding of the origins and evolution of sex and fertilization. In the present work, we studied in detail the sexual process of conjugation using the model species Euplotes vannus, and compared these nuclear events with those occurring in other ciliates. Our results indicate that in E. vannus: 1) conjugation requires about 75 hours to complete: the longest step is the development of the new macronucleus (ca. 64h), followed by the nuclear division of meiosis I (5h); the mitotic divisions usually take only 2h; 2) there are three prezygotic divisions (mitosis and meiosis I and II), and two of the eight resulting nuclei become pronuclei; 3) after the exchange and fusion of the pronuclei, two postzygotic divisions occur; two of the four products differentiate into the new micronucleus and macronucleus, respectively, and the parental macronucleus degenerates completely; 4) comparison of the nuclear events during conjugation in different ciliates reveals that there are generally three prezygotic divisions while the number of postzygotic divisions is highly variable. These results can serve as reference to investigate the mating type system operating in this species and to analyze genes involved in the different steps of the sexual process.     

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: Early expression of the zygotic genotype

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1–2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macro-nuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time. © 1992 Wiley-Liss, Inc.     

DNA methylation in vegetative and conjugating cells of a protozoan ciliate: Blepharisma japonicum

We report here the presence of N⁶-methyladenine (MeAde) in the macronuclear DNA (maDNA) of Blepharisma japonicum vegetative cells. We have further investigated the relationship between DNA methylation and cell union in cells activated for conjugation. Such activation was induced by treating cells of mating type I with complementary gamone 2. We found a reduction of about 24% of MeAde content in gamone-treated cells ready for cell union. First indications of the presence and reduction of MeAde content came from electrophoresis of maDNA digested by appropriate restriction endonucleases. Chromatographic determination of the amount of methylated base by HPLC substantiated these observations. In vegetative cells, 1.576 ± 0.02% of total adenine was found to be methylated as opposed to 1.193 ± 0.04% in activated cells. The HPLC analysis of maDNA also revealed a peak with a retention time corresponding to that of 5-hydroxymethyluracil, already found in some species of dinoflagellates. In that gamone treatment is correlated with a differential gene expression (indicated by a differential RNA and protein synthesis), our results suggest that there is a relationship between macronuclear genome activation and demethylation of maDNA. This is the first report of a correlation between gene activation and adenine demethylation in a eukaryotic organism.     

Alterations in cellular gene expression without changes in nuclear matrix protein content

Cell metabolism and function are modulated in part by cell and nuclear shape. Nuclear shape is controlled by the nuclear matrix, the RNA-protein skeleton the nucleus, and its interactions with cytoskeletal systems such as intermediate filaments and actin microfilaments. The nuclear matrix plays an important role in cell function and gene expression because active genes are bound to the nuclear matrix whereas inactive genes are not. It is unknown, however, how genes move on and off the matrix, and whether these events require compositional protein changes, i.e., alterations in protein content of the nuclear matrix, or other, more subtle alterations and/or modificatins. The purpose of this investigation was to begin to determine how nuclear matrix protein composition is related to gene expression. We demonstrate that gene expression can change without apparent changes in the protein composition of the nuclear matrix in MCF10A breast epithelial cells.     

Cloning, characterization and expression of the polypeptide release factor gene, eRF1, of Blepharisma japonicum

The polypeptide release factor gene, eRF1, of Blepharisma japonicum (Bj-eRF1) was cloned and sequenced. Its coding region was 1314 base pairs and encodes a protein of 437 amino acids. The cloned gene was expressed in Escherichia coli and the recombinant Bj-eRF1 polypeptide was purified by Ni2+-nitrilotriacetic acid agarose and Superose12 chromatography. Pull-down analysis showed that the recombinant Bj-eRF1 interacts with the heterologously-expressed release factor, eRF3C, of Euplotes octocarinatus.     

基因芯片技术与基因表达谱研究

基因芯片技术是近年来出现的分子生物学与微电子技术相结合的最新DNA分析检测技术,该技术将成为信息科学与生命科学之间的联系纽带,为后基因组时代基因功能的分析提供一种最重要的技术手段,目前基因芯片技术已在基因表达谱等研究中得到广泛应用。     

Progesterone-dependent and -independent expression of the multidrug resistance type I gene in porcine granulosa cells

A primary role of plasma membrane P-glycoprotein (P-gp), encoded by multidrug resistance type I (MDR1), is to protect against naturally occurring xenotoxics. Progesterone (P₄) profoundly influences MDR1 expression in granulosa cells and luteal cells. Here, P₄ regulation of MDR1 expression was investigated in porcine granulosa cells using the P₄-mediated promoter activity assay and a P4 receptor (PR) antagonist (RU-486). The promoter activity was measured chronologically for 48 h in cells transfected with the PR response element-containing pGL3. LH could stimulate the promoter activity through endogenous P4, with a maximum activity at 5 h. MDR1 mRNA level was highly maintained at 24–36 h. Conversely, exogenous P4 prolonged the promoter activity to further 10 h, and the high level of MDR1 mRNA was maintained even at 48 h. RU-486 completely inhibited the promoter activity, but the level of MDR1 mRNA rapidly increased in the presence of RU-486. The granulosa cells may become susceptible to RU-486 as a xenotoxic to rapidly express MDR1 for protection against it. These results indicate that MDR1 is expressed in porcine granulosa cells through P4-dependent and -independent regulations.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

We have isolated and characterized the genomic clone CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5 upstream region fused to the coding sequence of -glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5 deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.     

Overexpression of Cas-interacting zinc finger protein (CIZ) suppresses proliferation and enhances expression of type I collagen gene in osteoblast-like MC3T3E1 cells

Osteoblasts are the cells which form bone under the regulation not only by hormones and cytokines but also by ECM molecules via their attachment. To obtain insights into the role of intracellular signaling molecules operating to mediate the attachment-related regulation of osteoblastic functions, we investigated in osteoblast-like MC3T3E1 cells the effects of the overexpression of CIZ, a novel signaling protein which interacts with p130Cas. In MC3T3E1 cells, CIZ mRNA is expressed constitutively. Endogenous CIZ was localized in the MC3T3E1 cells with relatively high levels of accumulation at the attachment sites when the cells were cultured on fibronectin, collagen, or BSA. CIZ overexpression increased the number of adhesion plaques and reduced proliferation of the cells compared to that of control cells transfected with an empty vector. Furthermore, CIZ overexpression enhanced type I collagen mRNA expression, the most abundant constituent of bone matrix and a major product of osteoblasts. Analysis of the promoter region of type I collagen gene identified the presence of a consensus CIZ-binding sequence, which indeed conferred responsiveness to CIZ overexpression to a heterologous promoter. These data indicate that CIZ acts as a novel regulatory molecule in controlling osteoblastic function.     

日本血吸虫32kDa蛋白质基因在真核表达载体中的亚克隆

设计和合成特定寡核苷酸引物,TRIZOL提取日本血吸虫成虫RNA,RT-PCR法扩增日本血吸虫32kDa蛋白质(Sj32)基因编码序列,将扩增产物连接pGEM-T克隆载体,再亚克隆到真核表达载体pBKCMV中.结果RT-PCR法特异性扩增出Sj32编码基因片段,其大小约为1270bp,经双酶切、PCR鉴定表明所构建的质粒pGEM-Sj32和pBK-Sj32中含有目的基因.pBK-Sj32重组质粒的成功构建,为进一步表达Sj32及其在血吸虫病免疫诊断、免疫预防中的作用研究提供了条件.