小麦胚胎发育过程中蛋白质、核酸的动态变化

小麦每胚总核酸、RNA 和每细胞 RNA 含量均随胚的发育而增加,直到开花后21天达到高峰。每胚蛋白质和每细胞蛋白质含量也相应增加,但每胚蛋白质含量的增加持续到开花后24天。每毫克胚干重为单位的 RNA 含量在胚发育早期有下降,在后期有增加,每毫克胚干重的蛋白质含量也相应变化。每胚 DNA 含量在胚发育早期有增加,但之后即相对稳定。作者对 RNA 含量的变化与细胞蛋白质的合成和胚胎发育的关系进行了讨论。     

水稻胚胎形成期间蛋白质和RNA合成活力的变化

用~3H-亮氨酸和~3H-尿苷标记不同发育时期的稻胚,发现蛋白质合成活力呈四阶段变化;各种RNA的合成与胚胎各发育阶段密切有关。α-鹅膏蕈碱(1.0μg/ml)对稻胚RNA合成的抑制作用在开花后7、15和18天较强,13天时很弱。在水稻胚胎形成期间,胚细胞蛋白质合成活力高峰先后出现于胚分化后期(开花后11天)和成熟中期(18天);mRNA的合成在分化初期(7天)和成熟中期(15～18天)较强;而rRNA和/或tRNA的合成高峰则出现在胚胎器官原基分化已经完成时(13天)。     

小麦返白系返白期间Poly(A)RNA与蛋白质水平的变化

比较了小麦返白系与其原始品种矮变1号在返白期间Poly(A)RNA与蛋白质水平的变化。在心叶变白过程中。返白系的蛋白质含量下降,随着复绿又回升;而RNA含量变化与此大致呈平行关系。在全白阶段,返白系的Poly(A)RNA含量大幅度下降。体外合成的蛋白质也少得多,但其翻译活性/Poly(A)RNA及各期的蛋白质/Poly(A)RNA比例并不比对照低。可能返白系在返白期间蛋白质水平的变化与转录水平的基因调控有关。     

莲胚发育与核酸,蛋白质含量的变化

莲胚发育达到最大鲜重(开花后21d)前,胚轴和子叶的DNA,RNA都持续增长。开花13d后,蛋白、淀粉等贮藏物质显著积累,核酸增长速度加快。成熟胚轴的DNA和RNA含量很高,而子叶中积累大量的淀粉、可溶性糖和蛋白质。发育前期胚乳的生长速度较快,开花后16d左右鲜重和物质积累达到高峰。胚生长后期胚乳逐渐败育,贮藏物质和结构物质都减少,膨大的子叶逐步取代了胚乳的地位。 莲胚生物大分子物质含量的模式属于双子叶植物类型。讨论了莲胚细胞多倍化的问题。     

高等植物胚胎的发育生物学研究——Ⅱ.水稻胚胎发育过程中的生化变化

应用微量生化分析的方法测定了开花后6～30天水稻幼胚分化过程中的DNA、RNA,蛋白质、类脂和干重的动态变化。在人工气候室条件下(25℃,10小时光照)培养的水稻,在开花后第6～15天,即从第一叶原基形成至胚分化完成期间,每胚总核酸含量不断增加。尤其是在胚发育的早期阶段,DNA、RNA含量迅速增加。此后,在开花后18～30天,核酸含量的增加逐趋减缓。在此过程中,每胚蛋白质、类脂含量和干重也相应增加。然而,以胚干重为单位的DNA、RNA含量却随胚的发育而减少。同时,蛋白质含量仍不断增加。所以,DNA、RNA相对含量的减少,可能是由于细胞内含物和结构物质的增加而引起的。至于平均每个细胞的DNA含量,在整个胚发育过程中几乎稳定;而RNA含量,在胚发育的早期阶段有所增加,以后则基本保持同样水平。在胚发育早期RNA的强烈合成可能与细胞的分化和器官形成有关。     

高等植物胚胎的发育生物学研究——Ⅵ.粳稻胚分化发育期间一些大分子物质的动态

鉴测了粳稻胚的发育进程及不同分化发育时期中DNA、RNA、蛋白质、淀粉含量和鲜重、干重、细胞数的变化。 开花后6～13天(胚分化第一到第四叶原基期间)胚细胞数增加时,DNA、RNA含量迅速上升。此后细胞数和DNA、RNA含量都趋于稳定,但RNA在18～25天再次增长。每胚蛋白质和干重基本上随RNA含量相应地变化。 每毫克胚于重的核酸和蛋白质含量在13天出现明显的转折。平均每细胞的DNA含量在整个发育期保持稳定,RNA第一阶段的增长只持续到分化完成的前夕,而蛋白质在25天以前一直增加。 胚内淀粉的累积在整个胚形成期呈现三段斜率不同的直线。以单位干重表示时则在8天左右和21天出现两个高峰,先于RNA和蛋白质两次积累的高峰。 在大分子物质的变化与胚胎发育进程相互关系并与籼稻比较的基础上,将稻胚发育划分为原胚期、分化期、成熟期和休止期。     

长豇豆胚和胚乳的发育及营养物质积累

长豇豆(Vigna sesquipedalis (L.)Fruwirth)开花前7—10小时传粉,开花后8—10小时完成双受精。合子期珠孔端及合点部位胚囊的周界壁有壁内突。胚发育属柳叶菜型。胚柄的基部细胞及基部区域外层细胞的外切向壁发生壁内突。成熟胚中胚柄宿存。开花后9—16天为子叶细胞中淀粉积累期,开花后12—18天为蛋白质积累期。胚乳发育为核型,珠孔端胚乳细胞化,合点端保持游离核状态。胚乳外层细胞为传递型细胞,珠孔端的胚乳细胞形成折叠细胞群,亦有壁内突。心形胚期胚乳开始退化解体,成熟胚期胚乳完全消失。     

水稻胚胎发育与萌发过程中凝集素活性、含量与免疫学性质的变化

稻胚凝集素(RGL)存在胚中,胚乳中没有测得凝集素活性。水稻开花后7～21天胚中RGL括性与含量迅速增加、积累,基本达到成熟胚的最高水平。在开花后7与13天胚中除了有RGL存在外还发现有与RGL免疫学性质无关的凝集素存在。在萌发早期RGL活性与含量迅速下降,在浸种萌发后1～4天之间则又保持相对恒定。水稻胚胎发育与萌发过程中没有观察到与RGL免疫学性质相关但分子性质不同的凝集素存在。RGL是稻胚发育过程中形成的专一蛋白质,它的表达与积累有严格的时空专一性,它的活性与含量变化与细胞分裂、分化等胚胎发育过程是相关联的。     

水稻胚和胚乳内源ABA含量的变化及其与发育和萌发的关系

用放射免疫方法测定了种子发育和萌发过程中胚和胚乳的游离态(f-)和结合态(c-)内源 ABA 水平的变化。发育中稻胚 ABA 含量的双峰曲线与胚的两阶段发育模式一致。胚分化期和成熟期各有一个 ABA 含量的高峰。分化期以 f-ABA 为主,可能主要来自母体组织,与同化物迅速输入种胚有关;成熟期以 c-ABA 为主,可能主要是原位合成的,更直接地涉及胚基因表达的调节。胚乳的 ABA 含量占整个种子的90％左右,但 ABA 浓度(按 ABAng/mg鲜重表示)仅为胚的一半左右。除在线性充实期有一个 ABA 浓度的高峰外,整个发育期间胚乳的 ABA 浓度非常稳定。萌发期间胚的 ABA 含量呈“V”字形曲线变化,萌发开始时 ABA含量迅速下降,胚芽伸长生长开始以后再逐渐回升。讨论了内源 ABA 与种胚发育和萌发的可能关系。     

小麦醇溶蛋白mRNA的分离及体外翻译

本文中采用受粉后发育25天的小偃麦种子制备总RNA。然后用高效Oligo(dT)—Celluiose分离Poly(A)—mRNA。根据它在麦胚无细胞蛋白质合成体系中的翻译活性和对体外翻译产物进行聚丙烯酰胺凝胶电泳放射自显影分析说明,我们分离的Poly(A)—mRNA     

Role of the RRM domain in the activity, structure and stability of poly(A)-specific ribonuclease

Poly(A) specific ribonuclease (PARN), which contains a catalytic domain and two RNA-binding domains (R3H and RRM), acts as a key enzyme in eukaryotic organisms to regulate the stability of mRNA by degrading the 3' poly-(A) tail. In this research, the activity, structure and stability were compared between the full-length 74kDa PARN, the proteolytic 54kDa fragment with half of the RRM, and a truncated 46kDa form completely missing the RRM. The results indicated that the 46kDa one had the lowest activity and substrate binding affinity, the most hydrophobic exposure in the native state and the least stability upon denaturation. The dissimilarity in the activity, structure and stability of the three PARNs revealed that the entire RRM domain not only contributed to the substrate binding and efficient catalysis of PARN, but also stabilized the overall structures of the protein. Spectroscopic experiments suggested that the RRM domain might be structurally adjacent to the R3H domain, and thus provide a basis for the cooperative binding of poly(A) by the two RNA-binding domains as well as the catalytic domain.     

Effect of 2,4-dichlorophenoxyacetic acid on polysomal profiles and polyadenylated RNA in excised tuber tissue of Jerusalem artichoke (Helianthus tuberosus)

Dormant tuber tissue of Jerusalem artichoke ( Helianthus tuberosus L.) can be stimulated by wounding to initiate RNA and protein synthesis. No DNA synthesis or cell divisions occur unless an auxin is provided. Changes in polysomal profiles and levels of Poly(A)⁺-RNA in response to wounding and auxin treatment were studied. Polysomes were isolated at various times after excision and incubation of tissue in the presence or absence of 10⁻⁵ M 2,4-dichlorophenoxyacetic acid. Polysomal profiles were studied by sucrose density gradient centrifugation. Dormant tissue contained ribosomes mainly in monosome form. Within 4 h of excision, a significant increase in the polysomal fraction was observed both in control and auxin-treated tissue. Increases in polysomes continued during the next 20 h. Poly(A)⁺-RNA was isolated from total polysomal RNA by oligo(dT)-cellulose column chromatography. There was a large increase in the amount of poly(A)⁺-RNA within 4 h of excision. During the first 43 h of incubation, levels of total polysomal RNA as well as poly(A)⁺-RNA in tissue treated with 2,4-dichlorophenoxyacetic acid were significantly higher than those in controls.     

Regulation of poly(A) polymerase activity and poly(A)+ RNA in germinated wheat embryos under conditions of pathogenesis

The induction of poly(A) polymerase was accompanied by a rise in the level of poly(A)⁺ RNA during early germination of excised wheat embryos (48 h). Fractionation of this RNA-processing enzyme by acrylamide gel electrophoresis and also by molecular sieving on Sephadex G-200 revealed a single molecular form of poly(A) polymerase with a molecular weight of 125 000. Wheat poly(A) polymerase specifically catalyzed the incorporation of [³H]AMP from [³H]ATP into the polyadenylate product only in the presence of primer RNA. Substitution of [³H]ATP by other labelled nucleoside triphosphates, such as [³H]GTP, [³H]UTP or [α-³²P]CTP in the assay mixture did not yield any labelled polynucleotide reaction product. The ³H-labelled reaction product was retained on poly(U)-cellulose affinity column and was not degraded by RNAase A and RNAase T₁ treatment. In addition, the nearest-neighbour frequency analysis of the ³²P-labelled reaction product predominantly yielded [³²P]AMP. Thus, characterization of the reaction product clearly indicated its polyadenylate nature. The average chain length of the [³H]poly(A) product was 26 nucleotides. Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) brought about a severe inhibition (62–79%) of poly(A) polymerase activity. Concurrently, there was a parallel decrease (73%) in the level of poly(A)⁺ RNA. Inhibition of poly(A) polymerase activity in infected embryos could be due to enzyme inactivation, which in turn brought about a downward shift in the level of poly(A)⁺ RNA. The crude extract of the cultured pathogen contains a non-dialysable, heat-labile factor, which, along with a ligand, inactivates (65–74%) poly(A) polymerase in vitro. The fungal extracts also contained a dialysable, heat-stable stimulatory effector which activated wheat poly(A) polymerase (3.6–4.0-fold stimulation) in vitro. However, the stimulatory fungal effector was not expressed in vivo, but was detectable after the inhibitory fungal factor had been destroyed by heat-treatment in our in vitro experiments.     

Detection of mRNA degradation intermediates in tissues using the 3'-end poly(A)-tailing polymerase chain reaction method

It has become increasingly clear that mRNA stability is an important determinant of mRNA abundance in virtually all organisms. Although our understanding of prokaryotic lower eukaryotic mRNA stability mechanisms has progressed considerably, little is known about mammalian mRNA stability mechanisms, particularly at the tissue and animal levels. This is due largely to the lack of suitable methods to approach the problem. In this study, we have developed and refined the 3'-end poly(A)-tailing polymerase chain reaction (PCR) method to detect degradation intermediates in vivo. Using an in vitro transcribed RNA as a template, we found that the method could be used to detect a homogeneous pool of RNA down to 0.1 ng. The addition of 10 microg of total RNA from tissues decreased the sensitivity limit to 4 ng. Detection limits of the technique were determined precisely by varying the concentrations of in vitro transcribed RNA in a constant amount of total RNA and varying the concentration of total RNA while maintaining a constant amount of in vitro transcribed RNA. Our overall results showed that the poly(A)-tailing PCR method could be used to detect specific RNA species of approximately 1000 nt in a pool of heterogeneous RNA in the range of 1 in 2500 to 1 in 10,000. To our knowledge, this is the most sensitive method to date for identifying mRNA degradation intermediates. Employing sense strand gene-specific primers in this method, we have discovered the class II and class III P-glycoprotein (Pgp) mRNA degradation intermediates in normal rat tissues. This method should serve as an additional tool to help us understand mRNA decay mechanisms in tissues and at animal levels.     

Electroosmotic properties of microfluidic channels composed of poly(dimethylsiloxane)

Microfluidic devices fabricated from polymers exhibit great potential in biological analyses. Poly(dimethylsiloxane) (PDMS) has shown promise as a substrate for rapid prototyping of devices. Despite this, disagreement exists in the literature as to the ability of PDMS to support electroosmotic (EO) flow and the stability of that flow over time. We demonstrate that in low ionic strength solutions near neutral in pH, oxidized PDMS had a four-fold greater EO mobility (μ_eo) compared to native PDMS. The greater μ_eo was maintained irrespective of whether glass or PDMS was used as a support forming one side of the channel. This enhanced μ_eo was preserved as long as the channels were filled with an aqueous solution. Upon exposure of the channels to air, the mobility decreased by a factor of two with a half-life of 9 h. The EO properties of the air-exposed, oxidized PDMS were regenerated by exposure to strong base. High ionic strength, neutral in pH buffers compatible with living eukaryotic cells diminished the EO flow in the oxidized PDMS devices to a much greater extent than in the native PDMS devices. For analyses utilizing intact and living cells, oxidation of PDMS may not be an effective strategy to substantially increase the μ_eo.     

Solvatochromic studies in polyethylene glycol–salt aqueous biphasic systems

The polarities of the co-existing phases of a polyethylene glycol (PEG)-2000–K₃PO₄ aqueous biphasic system (ABS) have been examined using Reichardt’s carboxylated pyridinium-N-phenoxybetaine dye as a probe. Using this probe, the polarities of these phases have been compared to those of conventional solvent extraction systems and micellar systems using values obtained from the literature. In general, these extraction systems are comparable in polarity to rather polar solvents. Data on the free energy of transfer of solvents suggests that this may be due to the failure of the probe to account for the real polarity of the salt-rich phase compared to the polymer-rich phase. Examination of the monophasic region of these systems suggests that the reason for this is that the probe is partitioned to a discreet solvent domain dominated by PEG, even though phase separation of the solution is not observed. The use of linear free energy relationships for the characterization of ABS is briefly discussed.