Comparative analysis of glue proteins in theDrosophila nasuta subgroup

The patterns of protein fractions from total salivary glands and from glue plugs were compared in seven members of the Drosophila nasuta subgroup by the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The glue protein patterns are member specific concerning the numbers and the electrophoretic mobilities of major and minor glue protein fractions. However, the major fractions of all subgroup members could be grouped into five SDS-PAGE domains according to the homologies of their electrophoretic mobilities, prominence of Coomassie blue staining, and PAS reaction. In all subgroup members, major fractions are involved in posttranslational modifications into larger protein molecules of the final glue. Quantitative estimations of the glue proteins in D. n. nasuta and D. n. albomicans reveal that they constitute between 55 and 60% of the total salivary gland proteins, whereas in D. melanogaster and in D. hydei the fraction is only 32 and 35%, respectively.     

Application of capillary electrophoresis for DNA-protein binding tests

The combination of affinity chromatography and capillary electrophoresis (CE-SDS) has been found to be a useful tool to analyse populations of proteins which specifically bind to ssor dsDNA. Proteins were extracted from tissue, cytosol or nuclei of meristems of Pisum sativum seedlings and separated on cellulose column functionalized with ss-, dsDNA (calf thymus) and ssDNA (P. sativum) at 2M concentration of sodium chloride. Electropherograms of the crude protein extracts show two fractions of proteins specific for dsDNA (calf thymus) and three fractions specific for ssDNA (calf thymus). Four and five fractions of proteins specific for ssDNA (P. sativum) were identified in the material isolated from cytosolic and nuclear extracts, respectively. Both ds- and ssDNA (calf thymus) form complexes with ca. 4.0 % of the total amount of proteins, while ssDNA (P. sativum) binds to ca. 11.0 % of cytosolic and 5.0 % of nuclear proteins.     

Grain protein variability among species of Triticum and Aegilops: quantitative SDS-PAGE studies

Summary Total proteins were extracted from degermed seeds of various species of Triticum and Aegilops with solutions containing sodium dodecyl sulfate (SDS) and mercaptoethanol. The reduced, dissociated proteins were fractionated according to molecular weight (MW) by high-resolution polyacrylamide gel electrophoresis in buffers containing SDS (SDS-PAGE). Stained SDS-PAGE patterns were measured by densitometric scanning over a suitable range of optical density. The data were normalized to equivalent total areas for each of the densitometric scans by means of a computer program that also permitted the construction of patterns of hypothetical amphiploids by averaging patterns of two or three diploid species. The grain proteins of most species examined had distinctive qualitative and quantitative aspects that were characteristic of the species even though nearly every accession or cultivar of a species exhibited at least minor differences in pattern from other accessions or cultivars. The main protein components (probably prolamins) of Triticum monococcum ssp. monococcum, T. monococcum ssp. boeoticum, T. urartu, and Aegilops squarrosa had MW's in the range 29–36 X 10³ whereas the most important components of Ae. speltoides, Ae. longissima, and Ae. searsii had MW's in the range 37–55 × 10³. Changes in the quantitative expression of particular genes, especially those coding for storage protein components, may have been associated with speciation. The strong predominance of proteins with MW's in the range 29–36 × 10³ in some accessions of AB genome tetraploids, such as T. turgidum ssp. dicoccoides, may indicate contributions to the B genome of these tetraploids by T. monococcum ssp. boeoticum, T. urartu, or Ae. squarrosa.     

Evaluation of some cereals, plants and tubers through protein composition

Wild and cultivated maize, sorghum, rice, amaranth, soybean, and cassava were screened for variability in seed storage proteins. Total seed proteins, albumin (Alb-1 and Alb-2), globulin, alcohol-soluble (A1 and A2), and glutelin (G1 and G2) fractions were investigated by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The comparison was done by the obtained protein patterns and their relative amounts. Using quantitative analysis of the protein composition and the electrophoretic patterns, the relationships between total proteins and amount of individual proteins were determined. Electrophoretic patterns of extracted proteins from investigated samples showed that the main protein subunits were concentrated between 10 and 45 kDa. Variation was found in major fractions and minor bands. Electrophoretic patterns of the protein fractions are directly related to the genetic background of the protein and can be identified and used to certify the genetic makeup of wild, cultivated, or newly derived cereals and plants.     

Peripheral nervous system myelin assembly in vitro: perturbation by the ionophore monensin

Abstract: Sciatic nerves from 13-day-old rats were incubated in vitro with [³⁵S]methionine in the presence or absence of 0.22 μM monensin and total paniculate and myelin fractions prepared. The total particulate was further subfractionated by continuous density gradient centrifugation, after which the maximal specific activities of three marker enzymes, 2′,3′-cyclic nucleotide phospho-diesterase (myelin), 5′-nucleotidase (plasma membrane), and cerebroside sulphotransferase were recovered at 0.72, 0.82, and 0.92 M sucrose, respectively. The radiolabelled proteins present in the gradient subtractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, and bands corresponding to the P₀ and myelin basic proteins were identified by co-migration with unlabelled myelin marker proteins on both one-dimensional SDS-PAGE and two-dimensional nonequilibrium isoelectric focussing/SDS-PAGE systems. Following a 90-min incubation with [³⁵S]methionine, newly synthesized myelin basic proteins were recovered in fractions between 0.5 and 0.7 M sucrose; this distribution was unaltered by monensin. In contrast, the distribution of newly synthesized P₀ protein across the gradients was influenced by monensin: a bimodal distribution across the control gradients with peaks of recovery of 0.60 and 0.82 M sucrose was altered to give a single peak at an intermediate density of 0.72 M sucrose. The total proportions of newly synthesized P₀ and myelin basic proteins (MBP) present across the entire gradients were calculated from the fluorograms, and the ratio was found to be 2.8 P₀: (LBP + SBP), in both the presence and absence of the ionophore. However, only 70% and 50% of the control levels of MBP and P₀ were recovered with a purified myelin fraction after incubation with monensin. The results are discussed with reference to different intracellular transport processes for the P₀ glycoprotein and the MBP within the Schwann cell, and also to the differential compartmentation of the sites of synthesis and membrane export within the Golgi body.     

Chemical analysis of major outer membrane proteins of Neisseria meningitidis: comparison of serotypes 2 and 11

Most of the 15 protein serotypes found in group B Neisseria meningitidis have distinct major outer membrane protein patterns when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the Weber-Osborn system. Both serotypes 2 and 11 contain major outer membrane proteins with apparent molecular weights of 41,000 and 28,000 (41K and 28K). The 41K and 28K proteins were purified from the prototype strains of these two serotypes (M986 type 2 and M136 type 11) by preparative slab SDS-PAGE and were chemically characterized. No hexosamine was found in the purified 41K and 28K proteins. Although the two 41K proteins had similar amino acid compositions, their mobilities in Laemmli SDS-PAGE and their fragmentation patterns on SDS-PAGE after cyanogen bromide cleavage were different. The two 28K proteins differed in their amino acid composition, mobilities in Laemmli SDS-PAGE, and cyanogen bromide cleavage products. Peptide maps following chymotrypsin digestion of radioiodinated 41K and 28K proteins revealed distinct peptide maps for all four proteins. Comparison of the peptide maps of two 41K or two 28K proteins indicated that most of the unique peptides were hydrophilic, whereas most of the common peptides were hydrophobic. These results indicated that both of the 41K proteins and the 28K proteins from serotypes 2 and 11 were chemically different, although the proteins having the same molecular weights appeared to share common peptides.     

Variation of starch granule proteins and chromosome mapping of their coding genes in common wheat

Starch granule proteins (SGPs) of common wheat (Triticum aestivum L.) were analyzed by two electrophoretic techniques: sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2D-PAGE). These analyses identified three kinds of SGPs which were tentatively designated SGP-1, SGP-2 and SGP-3. SDS-PAGE resolved the products of three homoeologous genes for SGP-1 into three protein fractions, SGP-A1, -B1 and -D1. While SDS-PAGE resolved SGP-3 into one fraction, 2D-PAGE separated it into three protein fractions encoded by homoeologous genes Sgp-A3, B3 and -D3. SGP-2 was detected as one protein by SDS-PAGE and was present as one protein on 2D-PAGE. Aneuploid (nullisomic-tetrasomic and ditelosomic) analyses in the cultivar Chinese Spring showed that the genes for two SGPs (SGP-1 and -3) were located on the short arms of group-7 chromosomes. The results obtained from deletion lines for chromosome arms 7AS, 7BS and 7DS suggested that the gene order along the arms is centromere-Sgp-1-Sgp-3-Wx. An electrophoretic survey of wheat germ plasm identified a few cultivars lacking one of the proteins SGP-A1, -B1, -D1, SGP-A3 and -B3. The null alleles Sgp-A1b, Sgp-B1b and Sgp-D1b will be useful for the production of a variant wheat lacking SGP-1.     

小麦小花高纯度线粒体的分离及其蛋白质双向电泳体系建立

应用差速离心和Percoll不连续密度梯度法分离纯化小麦三核期小花线粒体. 在裂解液选择、IPG胶条pH值范围、SDS-PAGE胶浓度及蛋白质上样量等方面对线粒体蛋白质双向电泳体系进行探索和优化,确立了一套适用于小麦小花高纯度完整线粒体的分离方法及其蛋白质双向电泳的技术体系. 结果表明,采用20%、24%和40% Percoll密度梯度和28% Percoll自形成密度高速离心体系,获得了有活性、高纯度且较完整的线粒体;经TCA-丙酮法提取蛋白,以7 mol/L尿素,2 mol/L硫脲,4% CHAPS(W/V),65 mmol/L DTT,0.5% IPG缓冲液(V/V),0.001% 溴酚蓝(W/V)裂解液溶解蛋白,采用17 cm,pH 4～7 IPG胶条和11% SDS-PAGE分离胶,上样量为160 μg,硝酸银染色法,更适合小麦小花线粒体蛋白质组双向电泳分离. 经PDQuest 2DE 8.0.1软件包统计分析,在2-DE图谱上分辨出约150个蛋白点,蛋白点清晰呈圆形,无横条纹干扰,这为利用双向电泳技术在亚细胞水平对线粒体进行蛋白质组学研究与分析奠定了基础,更为进一步分析研究线粒体与雄性不育的关系提供了理论与技术支撑.     

雌核发育银鲫和两性生殖彩鲫精子蛋白组份的比较研究

对雌核发育银鲫和两性融合彩鲫精子蛋白组份进行了比较分析。通过分级抽提得到精子的不同组份精浆、精头的膜、鞭毛和脱膜精头等,然后经不同的凝胶电泳系统,比较分析了银鲫精子和其两性亲缘种彩鲫精子相应组份可溶性蛋白成份的差异。研究表明,经分级抽提的银鲫精子和彩鲫精子的各个组份都含有其特定的蛋白谱带。精浆蛋白在两种鱼之间和两种鱼的不同个体之间都存在一定差异。精头膜、鞭毛和脱膜精头的可溶性蛋白在同种鱼不同个体间高度一致,但在两种鱼之间表现出差异。两种鱼精头膜的可溶性蛋白在SDS－PAGE电泳图谱上基本一致,而在非变性聚丙烯酰胺凝胶电泳图谱上则具有各自的特征性谱带。鞭毛可溶性蛋白的SDS－PAGE分析在雌核发育银鲫中揭示出一条特异的蛋白带。脱膜精头的可溶性蛋白在SDS－PAGE电泳图谱上差异明显,存在几条特征性蛋白带,并经Acid-Urea PAGE系统分析,证实这些特征性蛋白为碱性蛋白。这些发现为进一步鉴定雌核发育银鲫雄鱼精子的特异性蛋白和揭示其分子机制打下了基础。     

Glue proteins inDrosophila nasuta

Larval glue protein fractions ofDrosophila nasuta nasuta were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Seven major and at least four minor glue protein fractions were recognized. Six of the major fractions are glycosylated. They migrate as three prominent doublets (>100, 43, and 30/28 kd). The synthesis of traceable amounts of these major fractions begins already during the second as well as during the early stages of the third larval instar. The 43-kd and the 30/28-kd fractions are coded by X-chromosomal genes. They are probably clustered within the huge puff of division 10, which is the most prominent X-chromosomal puff in the polytene chromosomes of the third larval instar. Complex posttranslational modification of all but one major glue protein fraction (14 kd) leads to the formation of about 15 different protein fractions in the final glue product. The amount of glue protein produced byD. n. nasuta larvae (in relation to the total saliva proteins) is nearly twice the amount produced byD. melanogaster larvae (ca. 55 and 32%, respectively). This work was supported by the University Grants Commission, New Delhi, India, the Deutscher Akademischer Austauschdienst, FR Germany (to S.R.R.), and the Deutsche Forschungsgemeinschaft (Ka 309/9-1).     

Purification and characterization of pregastric esterase from calf

Calf pregastric esterase (PGE) was purified from calf gullet tissues. The tissue was excised and lyophilized, and lipid materials were extracted with acetone and n-butanol at -20 degrees C. Proteins were extracted from the delipidated tissue with a buffer containing a chaotropic salt (NaSCN) to solubilize hydrophobically bound protein aggregates. Calf PGE precipitated from the crude extract at pH 5.0. The precipitated, solubilized proteins were subjected to anion-exchange chromatography on DEAE-Sephacel, and the enzymatic activity was eluted using a linear gradient from 0.10 to 0.50 M NaCl at pH 8.0. Fractions with high specific activities were then chromatographed twice using gel filtration on Sephadex G-100. The resultant enzyme was shown to be pure upon discontinuous electrophoresis in 12% polyacrylamide containing 0.1% sodium dodecyl sulfate (SDS-PAGE). From SDS-PAGE gel patterns, a molecular weight of 49,000 was determined. The amino acid composition of the enzyme allowed calculation of an "average hydrophobicity" (Bigelow index) of 1150 cal/residue. This indicates that calf PGE is relatively hydrophobic, being similar to proteins such as alpha-lactalbumin and bovine serum albumin in average hydrophobicity.     

Plastid changes during the conversion of chloroplasts to chromoplasts in ripening tomatoes

Methods were developed for the isolation of plastids from mature green and ripening tomatoes (Lycopersicon esculentum Mill.) and purification by sucrose or Percoll density-gradient centrifugation. Assessment of the purity of preparations involved phase-contrast and electron microscopy, assays for marker enzymes and RNA extraction and analysis. Proteins were extracted from isolated plastids at different ripening stages and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The profiles obtained from chloroplasts and chromoplasts showed many qualitative and quantitative differences. Labelling of proteins with [³⁵S]methionine in vivo showed that there was active protein synthesis throughout ripening, but there was a change in the plastid proteins made as ripening proceeded. The cellular location of synthesis of specific proteins has yet to be established.Abbreviations CS citrate synthase - EDTA ethylenediaminetetraacetic acid,-acetate - GAPDH NADP⁺-glyceraldehyde-3-phosphate dehydrogenase - rRNA ribosomal RNA - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - Tris 2-amino-2(hydroxymethyl)-1,3-propanediol     

Protein synthesis within dendrites: Ionic and neurotransmitter modulation of synthesis of particular polypeptides characterized by gel electrophoresis

This study evaluates whether physiological variables differentially affect the local synthesis of protein constituents of synapses in subcellular fractions containing pinched-off dendrites (synaptodendrosomes). Synaptodendrosomes were pulse-labeled in a medium containing³⁵S-methionine with 3 or 25 mM KCl and in the presence or absence of 0.5 mM EGTA or 10 μM glutamate. Synaptodendrosomes were then subfractionated to prepare synaptic plasma membranes and synaptic junctional complexes. The protein constituents of the synaptic plasma membrane and synaptic junctional complex fractions that were locally synthesized were identified using SDS-PAGE and two-dimensional gel electrophoresis and the extent of labeling of individual bands was analyzed using a Phosphorimager. Analysis of incorporation into individual bands resolved by SDS-PAGE revealed that depolarizing conditions (25 mM KCl) increased the extent of labeling of different bands to a different extent (ranging from 10–70% increases in labeling). Addition of 0.5 mM EGTA decreased the extent of labeling of the same group of bands in both 3 mM KCl and 25 mM KCl conditions. Addition of 10 μM glutamate reduced incorporation especially in the synaptodendrosomes incubated in 25 mM KCl. Two-dimensional gel electrophoresis analyses revealed that the labeled spots that showed differential labeling under the different conditions did not correspond to the most prominent Coomassie-stained spots. These results indicate that the proteins that are synthesized in synaptodendrosomes and regulated by physiological variables are not amongst the more abundant protein constituents of the fractions. Taken together, these results are consistent with the idea that protein synthesis within dendrites may be regulated by synaptic activity.     

Protein phosphorylation patterns during aestivation in the land snailOtala lactea

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of³²P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)Abbreviations CY cytosol - dpm radioactive disintegrations per minute - IEF isoelectrofocusing - GP glycogen phosphorylase - MC microsomes - MT mitochondria - PAGE polyacrylamide gel electrophoresis - PKF phosphofructokinase - PK pyruvate kinase - PM plasma membrane - SDS sodium dodecyl sulphate     

The procuticle of Drosophila: heterogeneity of urea-soluble proteins.

Proteins, soluble in 7 M urea, 4 M guanidine hydrochloride, or 2% sodium dodecyl sulfate, have been extracted from untanned larval cuticles of Drosophila melanogaster. A major protein fraction, apparent molecular weight 8000 - 10 000, is resolved into eight different components (five major, three minor) by gradient gel electrophoresis under nondenaturing conditions. Proteins extracted in 7 M urea have been resolved by diethylaminoethylcellulose chromatography into five fractions, three of which are greatly enriched for electrophoretically homogeneous proteins. The five fractions have different amino acid compositions. Electrophoretic variants involving four of the five major proteins have been obtained. Preliminary genetic analysis indicates that at least three of the five proteins are specified by separate structural genes.     

Composition of cell wall preparations of rice bran and germ

Cell walls of petrol-defatted non-waxy IR32 rice bran and germ were prepared by protein removal with 0.5% SDS—0.6% β-mercaptoethanol, heating the residue to 80°, and destarching with Bacillus licheniformis α-amylase. A waxy rice, IR29, had a similar cell wall composition as IR32. Principal wall sugars were arabinose, xylose, and glucose. The 0.5 M sodium or potassium hydroxide and 8 M urea preferentially extracted arabinose-, xylose- and uronic acid-rich polysaccharides but 6 M sodium hydroxide—0.81 M boric acid extracted mannose-rich polysaccharides. DEAE-cellulose BO₃^3? chromatography of the 0.5 M sodium hydroxide extracts gave fractions of similar arabinose— xylose ratios. Proteins in the cell wall preparations had only 0.4–1.6% hydroxyproline, and were bound mainly to polysaccharides, based on disc gel electrophoresis. The preparations were autofluorescent in UV and rich in phenols, mainly ferulic acid. The cell wall preparations and their 8 M urea fractions had a softening effect on defatted waxy starch aqueous gel at 0.2–2% of the starch.     

Electron Microscopy of the Major Outer Membrane Protein of Campylobacter jejuni

The surfaces of the disrupted-cell surfaces of the Campylobacter jejuni strains FUM158432 and M1 were examined using the negative-staining technique and electron microscopy. The surfaces of the whole cells and the outer membranes were covered with small dark dots which, in some areas, were arranged in hexagonal patterns. The hexagonal arrangement was more clearly seen in extracted outer membrane. The size of each structure was measured based on a center-to-center distance with the adjacent structure, and was determined to be 9.9±0.9 nm. A profile of the proteins in the outer membrane by SDS-PAGE, performed in 0.1% SDS and at 100 C, showed 42 kDa proteins to comprise the major outer membrane protein of this bacterium. Digestion of the outer membrane materials with proteinase reduced this protein band in the SDS-PAGE, and the amount of dark dots on the electron micrograph indicated the structure to be the major outer membrane protein (porin) of this bacterium. The power spectrogram of a computer-assisted Fourier transformation of the hexagonally arranged porin proteins suggests that the porin has a trimeric structure rather than a monomeric one.     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

In-vitro translation of different mRNA-containing fractions of Chlamydomonas chloroplasts

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.Abbreviation SDS sodium dodecyl sulfate     

Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.