Developmental regulation of expression of the malate synthase gene in transgenic plants

The cucumber malate synthase (MS) gene, including 1856 bp of 5 non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5 sequence was linked to the -glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of -glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.     

Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development

In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family. This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development. The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported. One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangements in its 5 region and intron splicing sites. The remaining genes (Bp4A and Bp4C), as well as the cDNA clones, appear to code for small proteins of unique structure. Three different types of proteins can be predicted as a result of the deletion of carboxy or amino terminal portions of a conserved core protein. These proteins all share a common alternation of hydrophobic and hydrophilic domains. A fragment of the genomic clone containing the gene Bp4A, as well as the non-functional gene Bp4B, was introduced into tobacco plants via Agrobacterium-mediated transformation. The functional gene Bp4A is expressed in transgenic tobacco plants and shows spatial and temporal regulation consistent with the expression patterns seen in Brassica napus.     

Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium

A chimaeric gene has been constructed that expresses -D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3–4 days and then declined; if selection was imposed expression increased again after 10–14 days. The extent of transient expression after 4 days correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3–4 days after inoculation. The most surprising result was that the blue staining was concentrated in localized zones on the circumference of the disc; within these zones, essentially all the cells appeared to be expressing GUS. We suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of leaf explants, but that the frequency of stable integration is several orders of magnitude lower.     

A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

白细胞介素2受体α链基因转录起始点和5'调控区结合蛋白的初步研究

 借助于5'和3'末端删切后重建的IL-2R a链基因调控区次级克隆,在体外合成有放射性同位素参入的反意义RNA探针与总RNA进行液相杂交,结果表明TPA或PHA分别活化的T细胞在IL-2R a链表达过程中都在不同程度上有选择地利用了调控区内分别为-58(5')和+1(3')位两个转录起始点中3'转录起始点。热休克使PHA活化细胞更明显地利用+1位点。PHA诱导Jurkat细胞表达IL-2RamRNA斑点杂交证实,Jurkat细胞在活化16小时表达IL-2Ra基本达到高峰,至24小时已明显下降。根据这一规律提取PHA诱导活化15小时的Jurkat细胞S100和NE,进行有关结合蛋白的研究,初步结果显示磷酸纤维素柱的KCI洗脱组分中存在着DNA结合蛋白,有关结合蛋白性质的研究正在进行中。     

用人DNA介导的基因转移修复中国仓鼠细胞的HPRT缺陷

 中国仓鼠卵巢细胞(CHO-K1)经N-甲基-N'-硝基-N-亚硝基胍(MNNG)诱变和6-巯基鸟嘌呤(6-TG)选择,得到稳定的次黄嘌呤磷酸核糖转移酶(HPRT)缺陷细胞株,酶活性仅为野生型的6.5％。用磷酸钙共沉淀法和电脉冲法向HPRT-细胞转移人宫颈癌细胞(HeLaS_3)基因组DNA,纠正了CHO细胞的HPRT缺陷。酶活性提高了6.9倍,达到野生型的45％。用Alu序列探针进行分子杂交,证实经过基因转移并连续传代15次以上的受体细胞中含人DNA序列。表明人的有关基因已稳定地整合到CHO细胞的染色体中。     

Embryogenic cells in plant tissue cultures: Occurrence and behavior

Summary Plants develop from meristems where cells proliferate and are partitioned into layers that eventually differentiate to form the various tissues and organs of the plant. A phenomenon unique to certain plant tissues is the ability to inducede nova a range of developmental patterns, including embryogenesis. The temporal and spacial distribution of these developmental competencies suggests that regulatory proteins, rather than a lack of signals or signal receptors, shield specific developmental genes from signals that otherwise would confuse development. Studies involving embryogenic cells demonstrate that they are not strongly shielded from developmental signals, thus they are not determined. Furthermore, the normal development of zygotic and somatic embryos is readily perturbed by abnormal physicochemical environments. This suggests that embryogeny remains developmentally plastic until differentiation is largely completed. The ability to induce somatic embryogenesis from specific tissues by specific signals is providing opportunities to further the molecular characterization of the menagerie of genetic regulation involved in development. Much of this review was presented at a Moet-Hennessy Louis Vuitton sponsored conference, Control of Morphogenesis in Plants, Aix Les Bains, France, September, 1989. Studies in our laboratory were supported in part by grants from the National Aeronautics and Space Administration (Cooperative Agreement No. NCC2-139), the Utah State University Biotechnology Center, and by the Utah Agricultural Experiment Station, Utah State University, Logan, UT 84322-4845 (approved as journal paper no. 3928).