parB: an auxin-regulated gene encoding glutathione S-transferase.

We have isolated an auxin-regulated cDNA, parB, from the early stage of cultured tobacco mesophyll protoplasts. The expression of parB was observed during transition from G0 to the S phase of tobacco mesophyll protoplasts cultured in vitro. The predicted amino acid sequence of parB cDNA has 213 amino acid residues with a relative molecular weight of 23,965. Nucleotide sequence analysis revealed that parB cDNA has homology to glutathione S-transferase (GST; RX:glutathione R-transferase, EC 2.5.1.18) from several sources including plant and animal cells. When we introduced expression vector pKK233-2, which retains parB cDNA, into Escherichia coli, we could detect GST activity in the parB gene product. Accordingly a significant increase of GST activity was detected in the tobacco mesophyll protoplasts cultured in the presence of 2,4-dichlorophenoxyacetic acid. This is an example in which the function of auxin-regulated gene product is shown to be ascribed to a specific enzymatic activity. As GST, and its substrate glutathione, are shown to be related to cell proliferation as well as detoxification of xenobiotics in plant and animal cells, the role of parB is discussed in relation to the induction of proliferative activity in differentiated and nondividing mesophyll protoplasts of tobacco.     

Isolation of cDNA of an auxin-regulated gene encoding a G protein beta subunit-like protein from tobacco BY-2 cells.

The addition of 2,4-dichlorophenoxyacetic acid to tobacco BY-2 cells that had been cultured in modified Linsmaier and Skoog medium deprived of auxin for 3 days induced cell division, whereas without 2,4-dichlorophenoxy-acetic acid application, no such induction of cell division was seen. When differential cDNA screening for auxin was done at 4 hr after the addition of 2,4-dichlorophenoxyacetic acid, the cDNA of an auxin-responsive gene designated arcA was isolated. The predicted gene product of arcA is a polypeptide with a M(r) of 35,825. arcA, thus, is a plant hormone-regulated gene that encodes a protein structurally related to the beta subunit of a guanine nucleotide-binding regulatory protein, which is composed of seven repetitive segments of Trp-Asp 40-aa repeats. The possibility that arcA gene products induce cell division is discussed.     

Expression of an auxin- and cytokinin-regulated gene in cambial region in Zinnia

The expression patterns of a cDNA clone, p48h-10, of an auxin-induced gene were examined in isolated mesophyll cells of Zinnia and in the organs of Zinnia plants. In the isolated mesophyll cells, the mRNA accumulates in 48 hr of culture with 1-naphthaleneacetic acid alone. Because the first cell division occurs before 36 hr of culture, the gene probably is not involved in cell division. Benzyladenine does not induce expression of this gene, but the combination of 1-naphthaleneacetic acid and benzyladenine induces the mRNA accumulation about 24 hr earlier than does 1-naphthaleneacetic acid alone. Tissue print hybridization shows that the mRNA is present predominantly in the cambial region in stems, leaves, and roots and in the vascular bundles in flower buds but does not occur in the apical regions of shoot or root. The characteristics of the gene expression, including auxin- and cytokinin-regulated induction and cambial region localization, encourage us to suggest that the gene is involved in the early process of vascular differentiation.     

HIF-1α质粒转染NIH3T3细胞的体外实验

目的探讨低氧诱导因子1(HIF-1)在NIH3T3细胞的体外表达.方法首先构建真核表达质粒,以脂质体为介导,体外转染细胞,经RT-PCR,Western blot,ELISA检测基因在mRNA、蛋白质等方面的表达.结果半定量RT-PCR证实在NIH3T3细胞表达外源基因,Western blot检测到外源HIF-1蛋白质表达,ELISA试验证明表达的基因产物具有生物活性.结论PcDNA3-HIF1真核表达质粒能够有效地在体外表达目的基因,为进一步的动物实验奠定了基础.     

Cloning of the cocaine-sensitive bovine dopamine transporter.

A cDNA encoding the dopamine transporter from bovine brain substantia nigra was identified on the basis of its structural homology to other, recently cloned, neurotransmitter transporters. The sequence of the 693-amino acid protein is quite similar to those of the rat gamma-aminobutyric acid, human norepinephrine, and rat serotonin transporters. Dopamine transporter mRNA was detected by in situ hybridization in the substantia nigra but not in the locus coeruleus, raphe, caudate, or other brain areas. [3H]Dopamine accumulation in tissue culture cells transfected with the cDNA was inhibited by amphetamine, cocaine, and specific inhibitors of dopamine transport, including GBR12909.     

Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco.

Little is known about the metabolic origin of petroselinic acid (18:1 delta 6cis), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the delta 9-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriander endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and delta 4-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase.     

Transfer of resistance traits from carrot into tobacco by asymmetric somatic hybridization: Regeneration of fertile plants

Transfer of methotrexate and 5-methyltryptophan resistance from carrot (Daucus carota) to tobacco (Nicotiana tabacum) was achieved by fusion between leaf mesophyll protoplasts of tobacco and irradiated cell culture protoplasts of carrot. Some of the regenerated somatic hybrids exhibited normal tobacco morphology with coexpression and independent segregation of the transferred resistance markers. Chromosomal instability resulted in aneuploid somatic hybrids with significantly lower chromosome number than predicted by simple addition of parental chromosome number. The methotrexate resistance phenotype was correlated with the expression of carrot-specific dihydrofolate reductase as judged by isozyme and immunological characteristics of the enzyme. The genomic construct of these somatic hybrids made the transmission of the resistance character into the next sexual generation possible.     

Transferrin messenger ribonucleic acid: molecular cloning and hormonal regulation in rat Sertoli cells

Transferrin-specific cDNA clones were isolated from a rat liver cDNA library prepared from transferrin-enriched mRNA. Hybrid selection and sequence analysis confirmed that the selected clone contained the carboxy-terminal coding region of the transferrin mRNA. Northern blot analysis was used to demonstrate the presence of transferrin mRNA in liver and Sertoli cells. Transferrin mRNA levels were measured in total RNA isolated from cultured rat Sertoli cells after treatment with FSH, insulin, retinol, and testosterone. The results showed a 2- to 4-fold increase in the level of transferrin mRNA, which peaked on the fourth day of culture after initiation of treatment, with FSH, insulin, retinol, and testosterone. This induction is gene specific, since no change in the mRNA levels for either the catalytic or regulatory subunits of cAMP-dependent protein kinase was observed. The effects of hormones, vitamin A (retinol), and Bu2 cAMP on transferrin mRNA and transferrin secretion (measured by RIA) in cultured Sertoli cells were compared. In general, a direct relationship between the amount of transferrin mRNA present in the cells and the amount of transferrin secreted into the culture medium was observed. These results demonstrate the important role that vitamin A, testosterone, and peptide hormones play in modulating transferrin gene expression in Sertoli cells.     

INFECTION OF TOBACCO MESOPHYLL PROTOPLASTS BY TOBACCO MOSAIC VIRUS

It was proved that the protoplasts prepared from mesophyll of Nicotiana tabacum are infected by tobacco mosaic virus. The infection occurred when purified tobacco mosaic virus particles were added to a protoplast suspension in the presence of poly-L-ornithine. The virus multiplied in these protoplasts to a level of 10(6) virus particles per infected protoplast during 24 hours of incubation. The efficiency of infection was remarkably high, exceeding that by mechanical inoculation of tobacco leaves.     

Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain.

Subtractive library construction and differential screening were used to identify a cDNA for a cell type-specific immediate early gene induced in rat PC12 pheochromocytoma cells. Sequencing identified the protein product of this gene as rat synaptotagmin IV (SytIV). Synaptotagmins are synaptic vesicle proteins thought to play a role in depolarization-induced, calcium-mediated exocytosis and neurotransmitter release. SytIV mRNA accumulation is transiently induced in PC12 cells by potassium depolarization, calcium ionophore, ATP, and forskolin. In contrast, growth factors and phorbol 12-myristate 13-acetate induce little or no SytIV mRNA accumulation. Kainic acid-induced seizures in rats are followed by accumulation of SytIV message in the hippocampus and piriform cortex. The SytIV gene may provide a direct link between depolarization-induced neuronal gene expression and subsequent modulation of synaptic structure and function.     

Corticotropin-releasing factor mRNA in rat thymus and spleen.

Corticotropin-releasing factor (CRF) initiates stress-induced immunosuppression via the hypothalamic-pituitary-adrenal axis. CRF has also been shown to have direct stimulatory and suppressive effects on immune cells. We have previously detected immunoreactive and bioactive CRF in the rat spleen and thymus. To determine if CRF is synthesized in these tissues, we analyzed rat spleen and thymus for the presence of CRF mRNA. RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction with CRF gene-specific oligonucleotide primers. After Southern blotting and hybridization with an internal CRF gene probe, a product of the expected size was detected in the spleen, thymus, and hypothalamus (positive control) but not in liver or kidney (negative controls), indicating that CRF is synthesized in the spleen and thymus. Furthermore, CRF could be secreted from splenic and thymic adherent cells in culture. Secretion increased severalfold in response to nordihydroguaiaretic acid (NDGA), a lipoxygenase pathway inhibitor, whereas interleukin 1 had no effect, suggesting that regulation of CRF secretion may differ from that in the hypothalamus. CRF mRNA was detected in NDGA-stimulated thymic adherent cells and in both control and NDGA-stimulated splenic nonadherent cells. The finding that CRF is synthesized in the spleen and thymus suggests that locally synthesized "immune" CRF, acting as an autocrine or paracrine cytokine, may have direct regulatory effects on immune function.     

有机阴离子转运蛋白3在人血管平滑肌细胞的表达

目的检测有机阴离子转运蛋白3（OAT3）及其mRNA在人血管平滑肌细胞的表达,并观察尿酸刺激对其表达的影响。方法分离培养人脐动脉血管平滑肌细胞,随机分为正常对照组及尿酸处理组,提取细胞总RNA,RT-PCR方法扩增特异性OAT3 cDNA片段,制备探针,用Northern blot方法检测各组OAT3 mRNA的表达。提取细胞膜蛋白,用Westernblot方法检测各组OAT3的蛋白表达。结果在正常对照组人血管平滑肌细胞可检测到OAT3mRNA和蛋白的表达。与对照组相比,尿酸处理组血管平滑肌细胞OAT3的表达在mRNA及蛋白水平均显著上调（P<0.05,n=3）。结论人血管平滑肌细胞表达OAT3,尿酸处理可以使其表达上调。提示OAT3可能部分介导血管平滑肌细胞对尿酸的摄取过程。     

Functional evidence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts

Tobacco mesophyll protoplasts were previously shown to respond to naphthaleneacetic acid by modifying their transmembrane potential difference. In the present work, evacuolated protoplasts were used to show that this response resides only at the plasmalemma. This electrical response was investigated by using polyclonal antibodies directed against plasma membrane antigens presumably involved in the reception and transduction of the auxin signal. An IgG fraction from an antiserum directed against the membrane auxin-binding protein from maize coleoptile completely inhibited the naphthaleneacetic acid-induced response of tobacco protoplasts. The suppression of the auxin-induced variation in the transmembrane potential difference by an IgG preparation directed against the plasmalemma ATPase from yeast demonstrated the involvement of the ATPase in the electrical response. Variation induced by fusicoccin in the transmembrane potential difference of tobacco protoplasts was unaffected by the anti-auxin-binding protein IgG fraction but was completely suppressed by the anti-ATPase IgG preparation. These results demonstrate the presence of a membrane receptor for auxin at the plasmalemma, the binding of the hormone to this receptor leading to the activation of the proton-pumping ATPase. They also show that at least the primary steps of activation by naphthaleneacetic acid are distinct from those of the fusicoccin-induced response.