Expression profile of circular RNA in rat intimal hyperplasia and target gene prediction

Intimal hyperplasia is an important cause of stenosis or occlusion after vascular injury. Circular RNAs (circRNAs) are known to be related to various cardiovascular diseases. However, the expression profile of circRNAs in the neointima has not been reported in detail. In this study, we established a rat common carotid artery (CCA) injury model. A microarray detection showed significant differences in circRNA expression between the normal and injured CCA. Real-time quantitative polymerase chain reaction verified the differences. We used bioinformatics to predict the microRNAs that possibly interact with the differentially expressed (DE) circRNAs and linked the potential functions of circRNAs to the target genes of the microRNAs. We believe that the DE circRNA in neointima may affect the differentiation, proliferation, and migration of vascular cells through a variety of target genes. The intervention or utilization of certain circRNAs should be a new method for preventing and treating intimal hyperplasia.     

Plastid transformation of high‐biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV‐1) p24 antigen

Chloroplast transformation of the high‐biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV‐1) p24 antigen. Maryland Mammoth offers the prospect of higher yields of intact functional protein per unit floor area of contained glasshouse per unit time prior to flowering. Two different transformation constructs, pZSJH1p24 (for the insertion of a native p24 cDNA between the rbcL and accD genes) and pZF5 (for the insertion of a chloroplast‐codon‐optimized p24 gene between trnfM and trnG) were examined for the production of p24. Plants generated with construct pZSJH1p24 exhibited a normal green phenotype, but p24 protein accumulated only in the youngest leaves (up to approximately 350 µg/g fresh weight or ~2.5% total soluble protein) and was undetectable in mature leaves. In contrast, some of the plants generated with pZF5 exhibited a yellow phenotype (pZF5‐yellow) with detectable p24 accumulation (up to approximately 450 µg/g fresh weight or ~4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5‐yellow leaves was reduced by ~40%. The pZF5‐yellow phenotype was associated with recombination between native and introduced direct repeat sequences of the rbcL 3′ untransformed region in the plastid genome. Chloroplast‐expressed p24 was recognized by a conformation‐dependent monoclonal antibody to p24, and p24 protein could be purified from pZF5‐yellow leaves using a simple procedure, involving ammonium sulphate precipitation and ion‐exchange chromatography, without the use of an affinity tag. The purified p24 was shown to be full length with no modifications, such as glycosylation or phosphorylation, using N‐terminal sequencing and mass spectrometry.     

TRANSIENT TRANSFORMATION OF A CHLORARACHNIOPHYTE ALGA,LOTHARELLA AMOEBIFORMIS (CHLORARACHNIOPHYCEAE), WITH uidA AND egfp REPORTER GENES1

A transient genetic transformation system was established for a chlorarachniophyte alga, Lotharella amoebiformis K. Ishida et Y. Hara. We first isolated sequences that contain a putative promoter for a RUBISCO SSU (rbcS) gene and a terminator for another copy of rbcS gene from L. amoebiformis. With those promoter and terminator sequences, we developed two expression vectors, pLaRGus and pLaRGfp, which code uidA and egfp genes, respectively. The cells were then transformed with each vector using a microparticle bombardment system. When the cells were transformed with the pLaRGus, β‐glucuronidase (GUS) staining dyed several cells blue. Green fluorescent protein (GFP) fluorescence was observed in the cells transformed with pLaRGfp. The highest transient transformation efficiency, 35 per 2 × 10⁷ cells, was detected from the GUS staining. This study demonstrates that two reporter genes are expressed in L. amoebiformis cells when rbcS promoter and terminator are used. The conditions of transformation were also optimized. This is the first report of successful genetic transformation in chlorarachniophyte algae.     

Over-expression of the apple <Emphasis Type="Italic">spermidine synthase</Emphasis> gene in pear confers multiple abiotic stress tolerance by altering polyamine titers

An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. ‘Ballad’) plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 μM CuSO₄) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments. Xiao-Peng Wen and Xiao-Ming Pang contributed equally to this work.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

Maintaining Connexin43 Gap Junctional Communication in v-Src Cells Does Not Alter Growth Properties Associated with the Transformed Phenotype

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine “normal” cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.     

Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

γ射线诱发大鼠胚胎细胞转化与N－ras的激活

以γ射线诱发转化的大鼠胚胎细胞(REC:myc:γ33)的DNA构建粘粒基因库,用总基因库DNA转染NIH/3T3细胞,产生转化灶的DNA作二轮转染,二轮转化的NIH/3T3细胞内有大鼠REC:myc:γ33DNA中具转化活性的N-ras基因,用不对称PCR和DNA序列分析法证明,REC:myc:γ33细胞中鼠N-ras的活化是由于第61位密码子的A→G点突变.NIH/3T3转化灶中鼠N-ras也有同样点突变,但NIH/3T3细胞的内源性N-ras基因则无此突变.     

Investigation of cis-acting sequences regulating expression of the gene encoding yolk protein 3 in Drosophila melanogaster.

Summary The regulatory sequences leading to the ovarian and fat body expression of yolk proteins 1 and 2 (YP1 and 2) of Drosophila melanogaster have been characterised in some detail. These genes (yp1 and yp2) share many enhancer elements, and some important regulatory sequences lie within the coding regions. We have begun to investigate the cis-regulation of the gene encoding yolk protein 3 (yp3). We describe a system for P element transformation using the complete and unaltered yp3 gene rather than reporter genes and describe sequences conferring correct expression in the ovary and carcass.     

Partial trisomies in two spontaneously arising long-lived human keratinocyte lines

Summary During experiments concerning the introduction of oncogenes into normal human keratinocytes, we observed long-lived colonies arising spontaneously at the same low frequency in control cultures as in those transfected with Ha-rasEJ or activated c-myc or both. Two of these were karyotyped early in their life span and showed additional chromosomal material on the short arm of chromosome 9 in one case and of chromosome 18 in the other, whereas the parental cells had a normal karyotype. This indicates the presence of a partial trisomy in each line, although the origin of the extra chromosomal material is not known. A similarly long-lived human keratinocyte line containing an isochromosome of the long arm of chromosome 8 has been described elsewhere. Together these results suggest that the spontaneous occurrence of long-lived lines is more common in human keratinocytes than in fibroblasts and that a triple dose of one or more genes may be the initial event in this process. This work was supported by grant CA16754 from the National Cancer Institute, Bethesda, MD.