Altered Patterns of Protein Phosphorylation and Synthesis Caused by Methyl Mercury in Cerebellar Granule Cell Culture

In the preceding report we demonstrated a dose-dependent increase in 32P-phosphoprotein labeling after 24-h exposure of cultured cerebellar granule neurons to methyl mercury (MeHg), a response that was not observed in glial cultures. In the present study we have examined 32P-labeled phosphoproteins by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. At concentrations of 0.5 and 1 microM, which were not extensively cytotoxic, MeHg enhanced phosphorylation of numerous acidic proteins, particularly a cluster of proteins with Mr approximately 28,000 and pI approximately 5.7-5.9 (pp 28/5.7-5.9) and a protein with Mr approximately 58,000 and pI approximately 5.6. The pp28 cluster displayed considerable two-dimensional pattern variability from one experiment to the next, suggesting susceptibility to subtle structural modifications. Time course studies revealed that increased 32P phospholabeling of pp28/5.7-5.9 was detectable after 12-h exposure to 3 microM MeHg and reached values of 300-500% of control by 24 h. These studies also showed that among the 21 proteins analyzed by two-dimensional densitometry, 32P phospholabeling of four proteins increased by 20-50% and of two proteins decreased by 20-50% after 24-h treatment. However, exposure to 10 microM MeHg produced stimulation of pp28/5.7-5.9 32P phospholabeling within 2 h. Under these conditions a relatively high stimulation (sevenfold) of pp28/5.7 phospholabeling occurred, while pp28/5.9 32P phospholabeling was only moderately (5-20%) enhanced. 35S and 32P double-label analysis of cells treated with 0, 0.5, and 1 microM MeHg indicated specific stimulation of 32P phospholabeling of these proteins without increased polypeptide synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

An electrophoretic karyotype of Aspergillus niger

Summary An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomesized DNA was separated into four bands. Seven of the eight linkage groups could be correlated with specific chromosomal bands. For this purpose DNA preparations from seven transformant strains of A. niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed. Some of the assignments were confirmed with linkage groupspecific A. niger probes. The estimated sizes of the A. niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A. nidulans. The total genome size of A. niger significantly exceeds that of A. nidulans and is estimated to be about 35.5–38.5 Mb. Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.     

Filtration stress-induced variations of peroxidase activity in cell suspension cultures of sycamore (Acer pseudoplatanus) cells

Filtration stress, consisting in the rapid filtration of Acer pseudoplatanus L. cell suspension cultures, resulted in significant differences between the peroxidases (EC 1.11.1.7) released during cell growth and those released after filtered cells were resuspended in fresh medium (recovery medium). These differences concerned mainly modifications of (i) the pH optimum of peroxidase activity (guaiacol as electron donor), (ii) the number and the pI values of the peroxidase isoenzymes as shown by isoelectric focusing, and (iii) the molecular weights of the different peroxidase fractions determined by gel filtration chromatography. The presence of 1 m M Li⁺ in the recovery medium inhibited the release of peroxidase and this effect was partially reversed by K⁺. The release of peroxidase by stressed cells was also strongly inhibited by Na₂CO₃ in the recovery medium. The results presented are consistent with the proposal that the characteristic isoperoxidase patterns induced by filtration stress might be used as a model to study the response of plant cells to stress.     

Denaturing gradient gel electrophoresis identifies genomic DNA polymorphism with high frequency in maize

Summary We have used denaturing gradient gel electrophoresis (DGGE) to identify genomic DNA polymorphism in maize (Zea mays L.). DGGE probes detect polymorphism in maize at a frequency comparable to the incidence of restriction fragment length polymorphism (RFLP). Probes identifying polymorphism were mapped to maize chromosome arms by utilizing DGGE and maize lines carrying B-A chromosomal translocations. The methods for library construction, probe screening, and genome analysis, described here for maize, can also be applied to the genomic analysis of other organisms.     

Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance

Summary We have begun to apply techniques for the preparation and anaylsis of large DNA segments from sugar beet (Beta vulgaris) addition lines carrying a mitotically stable chromosome fragment from B. procumbens that confers monogenic resistance to the nematode Heterodera schachtii, with a view towards isolating the resistance gene. DNA probes specific for this chromosome fragment were selected, and various methods for cloning genome-specific fragments, including probes from megabase DNA separated in pulsed-field slab gels, are compared. Probes that display high homology to B. procumbens have been used for hybridization of a representative genomic library and for initial step in mapping the chromosome fragment via pulsed-field gel electrophoresis after restriction with infrequently cutting enzymes. Our data indicate that DNA molecules from the entire chomosome fragment can be separated from protoplast DNA lysates.     

Methylated and undermethylated rDNA repeats are interspersed at random in two higher plant species

The organization of methylated rDNA repeats of radish and pea is described and it is shown that methylated repeats and non-methylated repeats are interspersed one with another. Methylated arrays are not much longer than 100 kb, or about 10 repeat units in length.     

Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

凝胶电泳与共头霉分类

对于共头霉的唯一已知种总状共头霉Syncephalastrum racemosum Cohn ex Schroet.(S 1)和新发现的具单孢孢子囊的新种单孢共头霉下的三个变种,原变种S.monosporum Zheng et al. var. monosporum( S7, S8, S9),冠囊变种var. cristatum Zheng et al. (S10, S11)及多重生变种var. pluriproliferum Zheng et al.(S12) 6株菌的蛋白和酯酶进行了聚丙烯酰胺凝胶电泳的研究.这4个分类群的蛋白图谱和酯酶酶谱互不相同,而同一分类群内各个菌株之间则有很高的相似性.从计算出的相似性百分数来看,3个新变种相互之间有较为密切的关系,而和S. racemosum虽然较疏远,但也有相当程度的亲缘关系.这些结果被认为支持了根据形态鉴定而定这三个新变种属于同一新种内并将其和S. racemosum一并归属于一个属内.由此可见,聚丙烯酰胺凝胶电泳是共头霉分类的有用的辅助手段.     

Comparison of the specific activity of ribulose-1,5-bis-phosphate carboxylase-oxygenase from some C3 and C4 plants

The specific activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39) was measured from the crude extracts of five C₃ plants consisting of wheat ( Triticum aestivum L. cv. Maris Mink), spinach ( Spinacia oleracea L.), pea ( Pisum sativum L. cv. Greenfeast), pumpkin ( Cucurbita pepo L. cv. Jättiläismeloni) and Ceratodon purpureus (Hedw.) Brid., and two C₄ plants, maize ( Zea mays L. ETA F₁) and sugar sorghum [ Sorghum saccharatum (L. emend, L.) Moench]. The amount of Rubisco in the crude extracts was estimated by polyacrylamide gel electro-phoresis with the Coomassie Brilliant Blue staining procedure. The amounts of the dye bound to the purified Rubisco of different higher plants were similar. The method gave a linear response for both purified enzyme and crude extracts, and the results agreed with those observed by immunochemical methods. The addition of positive effectors such as inorganic phosphate was necessary to obtain maximal activity in the crude extracts of all the studied plants except in that of maize. No significant differences in the specific carboxylase activity at 25°C were found between the C₃ and C₄ plants.