一个可介导链霉菌PKS基因 向植物转化的杂合质粒的构建

抗生素FR-008是由链霉菌FR-008所产生的一种七烯大环内酯类抗真菌抗生素。胡志浩等已克隆了长达约105kb的FR-008聚酮合酶(PKS)基因簇,对该基因簇中相邻于pabAB基因下游的3.8kbDNA进行序列分析,找到一个多功能聚酮合酶基因的起点,与数据库中蛋白质序列的比较分析揭示出一个尚未结束的大型开读框架的存在,它与抗细菌大环内酯类抗生素-红霉素生物合成所需的Ⅰ型聚酮合酶(PKS)基因中的乙酰转移酶(AT)和β-酮酰合酶(KS)的功能结构域显示出了高度的同源性,从分子水平上证实了FR-008抗生素由Ⅰ型PKS所合成。本实验将3.8kb中的编码聚酮合酶的部分开读框架通过基因工程的方法插入植物表达载体WRG2410上,从而成功构建了表达性质粒pHZ321。     

长松萝中一个聚酮合酶基因簇的克隆和鉴定

【目的】探索药用地衣长松萝(Usnea longissima Ach)聚酮化合物的生物合成基因簇,克隆聚酮合酶(PKS)基因并分析其功能。【方法】以长松萝地衣型真菌为材料,通过巢氏PCR获得聚酮合酶基因片段和原位杂交筛选基因组文库获得聚酮合酶基因及相邻基因簇。并对获得聚酮合酶进行分子系统进化分析和基因表达分析。【结果】获得药用地衣长松萝中的编码聚酮合酶基因UlPKS5的全长序列以及相邻修饰基因β-内酰胺酶和脱水酶。聚酮合酶UlPKS5含有酮体合成酶(KS),酰基转移酶(AT),产物模板(PT)以及酰基载体蛋白(ACP)结构域。分子系统进化分析显示UlPKS5属于非还原型聚酮合酶中第五组,与蒽醌类化合物生物合成相关。通过半定量RT-PCR分析表明山梨醇(10%)和蔗糖(2%和10%)能够强烈诱导UlPKS5基因表达。【结论】聚酮合酶(UlPKS5)及相邻修饰基因β-内酰胺酶和脱水酶与长松萝中蒽醌类化合物生物合成相关。     

PKSI-AT结构域及在组合生物合成研究中的应用

I型聚酮合酶（PKSI）的模块型分子结构组织方式非常适合于组合生物合成研究.结构域和模块通过二级组织方式构成了PKSI的催化单元,其它结构多肽则作为“支架”.在“支架”上对结构域和模块两个水平进行突变、替换、插入、缺失等基因操作形成重组PKS,可以理性设计并获得复杂多样的新活性或高活性的聚酮化合物.利用PKSI进行组合生物合成以期获得新聚酮化合物的研究迄今已有约25年,但是目前仍不能够对PKS进行完美的理性设计,快速合成目标活性的新聚酮化合物.PKS中的酰基转移酶结构域的研究在PKS的组合生物合成研究中一直发挥着重要作用.本文结合本课题组的研究基础,对AT结构域的结构、功能及在组合生物合成研究中的最新研究成果作以分析总结.     

草菇聚酮合酶编码基因vv-alb的克隆及其表达的初步分析

聚酮化合物（polyketides）是一类庞大的次级代谢家族,聚酮合酶（polyketide synthase,PKS）是介导聚酮化合物生物合成的关键酶。通过巢氏简并PCR与染色体步行的方法,获得了草菇中的编码PKS的基因vv-alb的全长序列,并通过荧光实时定量RT-PCR方法对vv-alb基因在草菇不同生长阶段与不同部位的表达情况进行了初步分析,为进一步研究PKS在草菇和其他食用真菌生物代谢过程中的作用奠定了一定的基础。     

牛樟芝中一个新型还原型聚酮合酶基因的克隆及表达分析

为了研究牛樟芝中PKS基因与化合物之间的关系,该研究通过对牛樟芝基因组分析获得牛樟芝聚酮合酶基因,以此序列为模板设计含有起始密码子和终止密码子的特异引物并以牛樟芝c DNA为模板克隆获得一个高度还原型PKS(HR-PKS)基因全长,命名为AcPKS2;对AcPKS2基因进行生物信息学分析,并比较该基因在不同培养基上的表达量。结果表明:AcPKS2全长7 842 bp,有24个内含子,其外显子共编码2 613个氨基酸,该蛋白的相对分子质量为293.5 kDa,理论等电点pI为5.78。用CDD分析其结构域显示,该基因属于HR-PKS,其结构域组织排列为KS-AT-DH-MT-ER-KR-ACP-TE,8个结构域其活性位点分别为β-酮基合成酶(DTACSSSL)、酰基转移酶(GHSIGETA)、脱水酶(RNDGSTSPL)、甲基转移酶(SFDIITAFDV)、烯酰还原酶(HAGVSSPAA)、酮基还原酶(GSPGQANYTAA)、酰基转移酶(YGLDSLTSVRL)、硫酯酶(KQPNGPY)。系统发育树显示AcPKS2与其他化合物未知的HR-PKS蛋白聚为一支,结构域和系统进化树分析显示该基因可能编码一种新的含TE结构域高度还原型聚酮合酶;表达分析结果显示葡萄糖和果糖能够诱导该基因的表达。     

植物类型Ⅲ聚酮合酶超家族基因结构、功能及代谢产物

植物聚酮类化合物主要包括酚类、芪类及类黄酮化合物等,在植物花色、防止紫外线伤害、预防病原菌、昆虫危害以及作为植物与环境互作信号分子方面行使着重要的生物学功能。该类化合物具有显著多样的生物学活性,对人体保健及疾病治疗有显著意义。植物类型Ⅲ聚酮化合物合酶(PKS)在该类化合物生物合成起始反应中行使着关键作用,决定该类化合物基本分子骨架建成和代谢途径碳硫走向,为合成途径关键酶和限速酶。以查尔酮合酶为原型酶的植物类型Ⅲ PKS超家族是研究系统进化和蛋白结构与功能关系的模式分子家族,目前已经分离得到14种植物类型Ⅲ PKS基因,这些同祖同源基因及其表达产物既有共性,也表现出许多独特个性,这些个性赋予此类次生代谢产物结构上的多样性。以下综述了植物类型Ⅲ PKS超家族基因结构、功能及代谢产物研究进展。     

植物类型III聚酮合酶超家族基因结构、功能及代谢产物

植物聚酮类化合物主要包括酚类、芪类及类黄酮化合物等,在植物花色、防止紫外线伤害、预防病原菌、昆虫危害以及作为植物与环境互作信号分子方面行使着重要的生物学功能。该类化合物具有显著多样的生物学活性,对人体保健及疾病治疗有显著意义。植物类型III 聚酮化合物合酶 (PKS) 在该类化合物生物合成起始反应中行使着关键作用,决定该类化合物基本分子骨架建成和代谢途径碳硫走向,为合成途径关键酶和限速酶。以查尔酮合酶为原型酶的植物类型III PKS超家族是研究系统进化和蛋白结构与功能关系的模式分子家族,目前已经分离得到14种植物类型III PKS基因,这些同祖同源基因及其表达产物既有共性,也表现出许多独特个性,这些个性赋予此类次生代谢产物结构上的多样性。以下综述了植物类型III PKS超家族基因结构、功能及代谢产物研究进展。     

利玛原甲藻中聚酮合酶基因克隆与分析

为探讨聚酮合酶 (polyketide synthase, PKS)基因与藻毒素合成的关系,揭示PKS基因在赤潮毒素合成中的作用,采用兼并引物,通过PCR技术获得利玛原甲藻(Prorocentrum lima)可能存在的I型PKS基因;并对所获得PKS基因的同源性进行了分析,构建了基于PKS氨基酸序列的系统进化树;采用RT-PCR技术分析了PKS基因在利玛原甲藻中的表达状况;并通过多聚腺苷酸RNA的扩增、细菌的分离鉴定、限制性内切酶酶切、Southern blotting等技术对PKS基因进行了分析.结果表明,利玛原甲藻中PKS基因与海洋原甲藻聚为一支,在利玛原甲藻中有显著表达;以Oligo(T)引物进行RT-PCR扩增时,可出现18S rRNA和PKS基因相应条带;限制性内切酶酶切和Southern blotting结果显示,该基因中存在明显的甲基化;16S rRNA基因序列分析显示,从利玛原甲藻培养液中分离到的细菌与海洋放线菌假诺卡氏菌属(Pseudonocardia)基因序列同源性达到99%,该菌株中并不存在PKS基因.结果显示,所获得的PKS基因是利玛原甲藻聚酮合酶基因,基因序列已提交GenBank (EF521601);PKS可能在腹泻性贝毒合成中起着关键作用.     

硫酯酶结构域对酮内酯类化合物合成的影响

在I型聚酮合成酶(polyketide synthase,PKS)中,硫酯酶结构域(Thioesterase,TE)负责将聚酮长链从PKS上水解下来,并协助环化为大环内酯环.分别将红霉素TE、ACP6-TE和苦霉素模块6(PikM6)基因转入糖多孢红霉菌KR6突变体中表达,仅PikM6显著促进酮内酯类化合物的合成,表明TE只有融合于模块中才能充分发挥硫酯酶的功能.     

一个源于皮革肾岛衣地衣型真菌中的HR-PKS基因克隆与鉴定

本研究通过对皮革肾岛衣(Nephrmopsis pallescens)地衣型真菌转录组数据的分析,首次克隆得到一种高度还原型聚酮合酶(Highly reducing PKS)基因全长,并对该基因进行生物信息学分析,并检测该基因在不同培养基上的表达量。该基因cDNA全长7 491 bp (命名为NpPKS1),可编码2 496个氨基酸,是一种稳定存在于细胞质基质中的非分泌蛋白;结构域与其他真菌的洛伐他汀九酮合成酶(LNKS)结构域极为相似,且聚类分析与其他真菌的洛伐他汀九酮合成酶(LNKS)聚为一类;在以葡萄糖和麦芽糖作为碳源的情况下,番茄浸粉、牛肉浸粉、酪蛋白胨等作为氮源可强烈刺激NpPKS1基因的表达,而胰蛋白胨对该基因的表达存在一定程度的抑制效应。本研究为皮革肾岛衣地衣型真菌中的基因资源利用、聚酮化合物异源表达和其合成机制的研究奠定了基础。     

Purification and molecular properties of ferredoxin-glutamate synthase from Chlamydomonas reinhardii

Ferredoxin-glutamate synthase (EC 1.4.7.1) from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity, with a specific activity of 10.4 units mg^-1 protein, by a method which included chromatography on diethylaminoethyl sephacel and hydroxylapatite, and ferredoxin-sepharose affinity treatment. The enzyme is a single polypeptide chain of M _r 146000 dalton which shows an absorption spectrum with maxima at 278, 377 and 437 nm, and an A₂₇₆/A₄₃₇ absorptivity ratio of 7.0. The anaerobic addition of dithionite results in the loss of the absorption peak at 437 nm, which is restored upon reoxidation of the enzyme with an excess of 2-oxoglutarate, alone or in the presence of glutamine. This indicates the presence in the enzyme of a flavin prosthetic group, which is functional during the catalysis. The ferredoxin-glutamate synthase can be assayed with methyl viologen, chemically reduced with dithionite, but it is unable to use reduced pyridine nucleotide. Azaserine, 6-diazo-5-oxo-norleucine, bromocresol green and p-hydroxymercuribenzoate are potent inhibitors of this activity, which, on the other hand, is stable upon heating at 45°C for 10 min.Abbreviations DEAE-sephacel diethylaminoethyl sephacel - Fd ferredoxin - GOGAT glutaniate synthase (glutamine: -ketoglutarate aminotransferase) - SDS sodium dodecyl sulfate     

The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos

Soluble starch synthase was purified 10000-fold from developing embryos of pea (Pisum sativum L.). The activity was resolved into two forms which together account for most if not all of the soluble starchsynthase activity in the embryo. The two isoforms differ in their molecular weights but are similar in many other respects. Their kinetic properties are similar, neither isoform is active in the absence of primer, and both are unstable at high temperatures, the activity being abolished by a 20-min incubation at 45° C. Both isoforms are recognised by antibodies raised to the granule-bound starch synthase of pea. Isoform II, which has the same molecular weight (77 kDa) as the granulebound enzyme, is recognised more strongly than Isoform I.     

Glucomannan synthesis in pea epicotyls: The mannose and glucose transferases

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a -1,4-[su14C]mannan from GDP-d-[U-¹⁴C]-mannose, a mixed -1,3- and -1,4-[¹⁴C]glucan from GDP-d-[U-¹⁴C]-glucose and a -1,4-[¹⁴C]-glucomannan from both GDP-d-[U-¹⁴C]mannose and GDP-d-[U-¹⁴C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The -glucan synthase had different properties from other preparations which bring about the synthesis of -1,3-glucans (callose) and mixed -1,3- and -1,4-glucans and which use UDP-d-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-d-xylose in addition to GDP-d-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-d-glucose acted competitively in the presence of GDP-d-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-d-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-d-glucose and GDP-d-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-d-mannose and GDP-d-glucose to bring about the synthesis of the heteropolysaccharide.Abbreviations CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate - CHAPSO 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate - CHD 1,2-cyclohexanedione - CDP cytidine 5-diphosphate - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - GDP guanosine 5-diphosphate - NAI N-acetyl-imidazole - NEM N-ethylmaleimide - PGO phenylglyoxal This work has been made possible by grants of M.A.F. and M.U.R.S.T. 40% of Italy. Dr. A. Zuppa wishes to thank the C.N.R. of Italy for his research scolarship.     

Biphenyl synthase,a novel type III polyketide synthase

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.     

Resolution of two molecular forms of sucrose-phosphate synthase from maize,soybean and spinach leaves

Two forms of sucrose-phosphate synthase (EC 2.4.1.14) were resolved from leaves of three species, maize (Zea mays L. cv. Pioneer 3184), soybean (Glycine max (L.) Merr., cv. Ransom) and spinach (Spinacia oleracea L. cv. Resistoflay) by hydroxyapatite Ultrogel chromatography, using a 75-mM (designated peak 1) and 250-mM (peak 2) K-phosphate discontinuous-gradient elution. Rechromatography of the two forms showed that they were not readily interconvertible. The distribution of activity between the two forms differed among species and changed during purification of the enzyme. Recovery of peak-1 activity was specifically lowered when maize leaf extracts were prepared in the absence of magnesium, indicating that the two forms may differ in stability. In addition, the forms of the enzyme from maize differed in the extent of glucose-6-phosphate activation. These results provide evidence for the existence of multiple forms of sucrose-phosphate synthase in leaves of different species and that the forms differ in regulatory properties.Abbreviations Fru6P fructose 6-phosphate - Glc6P glucose 6-phosphate - HAU hydroxyapatite Ultrogel - Pi inorganic phosphate - SPS sucrose-phosphate synthase - UDP uridine 5-diphosphate - UDPG uridinediphosphate glucose Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10511 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. Supported in part by USDA Competitive Research Grant No. 85-CRCR-1-1568     

Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants

Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS¹ type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.     

Action of light,nitrate and ammonium on the levels of NADH- and ferredoxin-dependent glutamate synthases in the cotyledons of mustard seedlings

In mustard (Sinapis alba L.) cotyledons, NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) is only detectable during early seedling development with a peak of enzyme activity occurring between 2 and 2.5 d after sowing. With the beginning of plastidogenesis at approximately 2 d after sowing, ferredoxindependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) appears while NADH-GOGAT drops to a very low level. The enzymes were separated by anion exchange chromatography. Both enzymes are stimulated by light operating through phytochrome. However, the extent of induction is much higher in the case of Fd-GOGAT than in the case of NADH-GOGAT. Moreover, NADH-GOGAT is inducible predominantly by red light pulses, while the light induction of Fd-GOGAT operates predominantly via the high irradiance response of phytochrome. The NADH-GOGAT level is strongly increased if mustard seedlings are grown in the presence of nitrate (15 mM KNO₃,15 mM NH₄NO₃) while the Fd-GOGAT level is only slightly affected by these treatments. No effect on NADH-GOGAT level was observed by growing the seedlings in the presence of ammonium (15 mM NH₄Cl) instead of water, whereas the level of Fd-GOGAT was considerably reduced when seedlings were grown in the presence of NH₄Cl. Inducibility of NADH-GOGAT by treatment with red light pulses or by transferring water-grown seedlings to NO ₃ ^- -containing medium follows a temporal pattern of competence. The very low Fd-GOGAT level in mustard seedlings grown under red light in the presence of the herbicide Norflurazon, which leads to photooxidative destruction of the plastids, indicates that the enzyme is located in the plastids. The NADH-GOGAT level is, in contrast, completely independent of plastid integrity which indicates that its location is cytosolic. It is concluded that NADH-GOGAT in the early seedling development is mainly concerned with metabolizing stored glutamine whereas Fd-GOGAT is involved in ammonium assimilation.Abbreviations and symbols c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light (3.5 W·m^-2) - NADH-GOGAT NADH-dependent glutamate synthase (EC 1.4.1.14) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W· m^-2) - RG9-light long wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Metabolic engineering in strawberry fruit uncovers a dormant biosynthetic pathway

Wild strawberry (Fragaria vesca) fruit contains several important phenylpropene aroma compounds such as eugenol, but cultivated varieties are mostly devoid of them. We have redirected the carbon flux in cultivated strawberry (Fragaria×ananassa) fruit from anthocyanin pigment biosynthesis to the production of acetates of hydroxycinnamyl alcohols, which serve as the precursors of the phenylpropenes, by downregulating the strawberry chalcone synthase (CHS) via RNAi-mediated gene silencing and, alternatively, by an antisense CHS construct. Simultaneous heterologous overexpression of a eugenol (EGS) and isoeugenol synthase (IGS) gene in the same cultivated strawberry fruits boosted the formation of eugenol, isoeugenol, and the related phenylpropenes chavicol and anol to concentrations orders of magnitude greater than their odor thresholds. The results show that Fragaria×ananassa still bears a phenylpropene biosynthetic pathway but the carbon flux is primarily directed to the formation of pigments. Thus, partial restoration of wild strawberry flavor in cultivated varieties is feasible by diverting the flavonoid pathway to phenylpropene synthesis through metabolic engineering.     

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k _i=2.6 mmol · l^-1 for gCS, k _i=0.33 mmol · l^-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)⁺RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)⁺RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)⁺RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - gCS glyoxysomal citrate synthase - gMDH glyoxysomal malate dehydrogenase - k _i inhibitor constant - mCS mitochondrial citrate synthase - OAA oxaloacetate - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis