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31.
Maize chloroplast RNA polymerase: the 78-kilodalton polypeptide is encoded by the plastid rpoC1 gene. 总被引:5,自引:0,他引:5 下载免费PDF全文
The 180-, 120- and 38-kDa polypeptides found in highly purified maize plastid RNA polymerase preparations are encoded by the maize plastid genes rpoC2, rpoB, and rpoA, respectively [Hu, J. and Bogorad, L. (1990) Proc. Natl. Acad. Sci. USA. 87, pp. 1531-1535]. These genes have segments that specify amino acid sequences homologous to those of E. coli RNA polymerase subunits. The plastid gene products are designated b", b and a, respectively. We report here that the amino-terminal amino acid sequence of a 78-kDa polypeptide also found in highly purified maize plastid RNA polymerase preparations matches precisely the sequence deduced from the maize plastid rpoC1 gene which has segments homologous to the 5' end of the E. coli rpoC gene. Thus, the 78-kDa polypeptide is likely to be a functional component of maize plastid DNA-dependent RNA polymerase. This polypeptide is designated subunit b'. Three polypeptides unrelated to RNA polymerase have also been identified in this preparation. 相似文献
32.
A novel translation initiation region from Mycoplasma genitalium that functions in Escherichia coli. 总被引:6,自引:1,他引:5 下载免费PDF全文
The tuf gene of Mycoplasma genitalium uses a signal other than a Shine-Dalgarno sequence to promote translation initiation. We have inserted the translation initiation region of this gene in front of the Escherichia coli lacZ gene and shown that it is recognized by the translational machinery of E. coli; the signal operates in vivo at roughly the same efficiency as a synthetic Shine-Dalgarno sequence. The M. genitalium sequence was also used to replace the native translation initiation region of the cat gene. When assayed in E. coli, the M. genitalium sequence is equivalent to a Shine-Dalgarno sequence in stimulating translation of this mRNA also. Site-directed mutagenesis enabled us to identify some of the bases that comprise the functional sequence. We propose that the sequence UUAACAACAU functions as a ribosome binding site by annealing to nucleotides 1082-1093 of the E. coli 16S rRNA. The activity of this sequence is enhanced when it is present in the loop of a stem-and-loop structure. Additional sequences both upstream and downstream of the initiation codon are also involved, but their role has not been elucidated. 相似文献
33.
Characterization of epitopes on the cationic peanut peroxidase by four monoclonal antibodies 总被引:1,自引:0,他引:1
The epitope sites on the cationic peanut peroxidase were characterized by four monoclonal antibodies raised against this isozyme. Evidence is presented showing that the epitope for monoclonal antibody 1B is located on the polypeptide. Sensitivity of the epitopes recognized by 1M and 2F to 0.1M HCl, boiling, 10 mM periodate and trifluoromethane sulfonic acid treatment indicate that they occur at regions where oligosaccharides are linked to the polypeptide backbone. The antigenic specificity of 2A is, in addition, dependent on the conformation of the epitope site which is destroyed after partial proteolysis of the peroxidase. 相似文献
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35.
Regulation of differentiation of the BC3H1 muscle cell line through cAMP-dependent and -independent pathways 总被引:7,自引:0,他引:7
Serum mitogens, fibroblast growth factor (FGF), and type beta transforming growth factor (TGF-beta) suppress differentiation of the mouse muscle cell line BC3H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC3H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC3H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E1, and cholera toxin, prevented up-regulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC3H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-beta. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC3H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-beta inhibit differentiation through a mechanism independent of cAMP or protein kinase C. 相似文献
36.
C W Hu T A Griffin K S Lau R P Cox D T Chuang 《The Journal of biological chemistry》1986,261(1):343-349
To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue. 相似文献
37.
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39.
Five kinds of artificial sea water with similar salinity, pH and osmolarity were prepared with different concentrations of calcium and magnesium ions. They were complete sea water, calcium-free sea water, half calcium-free sea water, magnesium-free sea water and half magnesium-free sea water. The fertilized eggs from induced spawnings of grey mullet Mugil cephalus , were transferred to these sea waters at two development stages, i.e. before the formation of 2-blastomere stage and at the gastrula stage. Survivals of fertilized egg in different sea waters were checked after several hours incubation, the percentage survival was used as an index of the importance of calcium and magnesium to the embryonic development. The results showed that calcium is indispensable to the embryonic development of mullet egg. Magnesium, however, is not the necessary element in the incubation medium. 相似文献
40.
Alston-Mills Brenda Li Qi Chang Ottinger Mary Ann 《In vitro cellular & developmental biology. Plant》1989,25(10):934-938
Summary Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large
quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo
and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing
hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were
collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm2 tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with
SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin
and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were
expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive
expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with
LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed
a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse.
This work was funded by the Maryland Agricultural Experiment Station artical no. A4975, contribution no. 8019. 相似文献