Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes: Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee α-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin “signal” seque?ce is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.     

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.     

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.     

接触二氧化硫后小麦叶片中逆境乙烯的生物合成

AVG和AOA强烈抑制二氧化硫处理小麦叶片中乙烯产生和ACC合成,对MACC的形成也有一定的抑制作用。CoGl_2明显抑制乙烯产生,而ACC大量积累,MACC含量则未因ACC增加而相应增加。DNP和CCCP也抑制乙烯产生,但前者引起ACC大量积累,后者引起ACC含量下降。CHI对乙烯产生和ACC形成均显示强烈的抑制作用,同时也明显抑制MACC形成。这表明小麦叶片接触SO_2引起的逆境乙烯也是循蛋氨酸→SAM→ACC→乙烯途径。     

Patterns of protein synthesis during the germination of pea axes,and the effects of an interrupting desiccation period

As germination of axes of Pisum sativum L. seeds progressed, profound quantitative and qualitative changes occurred in the patterns of protein synthesis. This was shown by fluorography of gels following two-dimensional polyacrylamide gel electrophoresis separation of [³⁵S]methioninelabelled proteins. The effects of desiccation during germination on these in-vivo protein-synthesis patterns were followed. Desiccation differentially affected the synthesis of proteins. Usually, however, upon rehydration following desiccation the types of proteins being synthesized were recognizable as those synthesized earlier during imbibition of control, once-imbibed axes: seeds imbibed for 8 h, and then dried, did not recommence synthesis of proteins typical of 8-h-imbibed control seeds, but rather of 4-h-imbibed control seeds. Seeds imbibed for 12 h, and then dried and rehydrated, synthesized proteins typical of 4-h-and 8-h-control seeds. Thus drying of germinating pea axes caused the proteinsynthesizing mechanism to revert to producing proteins typical of earlier stages of imbibition. Drying during germination never caused the seed to revert to the metabolic status of the initial mature dry state, however.Abbreviation DR dried and rehydrated     

Monoclonal antibodies to plant plasma-membrane antigens

Murine monoclonal antibodies to membrane antigens were generated by immunization with a crude cellular membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. From a panel of thirteen monoclonal antibodies, seven were found to be directed against antigens present on the plasma-membrane by immunofluorescence visualization of antibody binding to the surface of isolated protoplasts. The corresponding set of plasma-membrane antigen(s) were present in root, shoot and leaf tissue and some but not all of these antigens were of wide species distribution, being found in Nicotiana tabacum L., N. plumbaginifolia L., Glycine max L., Phaseolus vulgaris L. and Triticum aestivum L. Topologically specific labeling of intact protoplasts with a monoclonal antibody reactive with an epitope present on the plasma-membrane specifically labeled a membrane fraction which equilibrated at a density of 1.14 kg/l following centrifugation in a sucrose gradient. In addition to use as biochemical markers for fractionation and molecular characterization of plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations.     

The relationship between carbon-dioxide-limited photosynthetic rate and ribulose-1,5-bisphosphate-carboxylase content in two nuclear-cytoplasm substitution lines of wheat,and the coordination of ribulose-bisphosphate-carboxylation and electron-transport capacities

Photosynthesis in two cultivars of Triticum aestivum was compared with photosynthesis in two lines having the same nuclear genomes but with cytoplasms derived from T. boeoticum. The in-vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) isolated from lines with T. boeoticum cytoplasm was only 71% of that of normal T. aestivum. By contrast, the RuBPCase activities calculated from the CO₂-assimilation rate at low partial pressures of CO₂, p(CO₂), were the same for all lines for a given RuBPCase content. This indicates that both types of RuBPCase have the same turnover numbers in-vivo of 27.5 mol CO₂·(mol enzyme)^–1·s^–1 (23°). The rate of CO₂ assimilation measured at normal p(CO₂), p _a =340 bar, and high irradiance could be quantitatively predicted from the amount of RuBPCase protein. The maximum rate of RuBP regeneration could also predict the rate of CO₂ assimilation at normal ambient conditions. Therefore, the maximum capacities for RuBP carboxylation and RuBP regeneration appear to be well-balanced for normal ambient conditions. As photosynthetic capacity declined with increasing leaf age, the capacities for RuBP carboxylation and RuBP regeneration declined in parallel.Abbreviations PAR photosynthetically active radiation - RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

The specific activity of ribulose-1,5-bisphosphate carboxylase in relation to genotype in wheat

The specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) in crude extracts of leaves from euploid, amphiploid and alloplasmic lines of wheat fell into high or low categories (3.75 or 2.70 mol·mg^–1·min^–1, 30°C). For the alloplasmic lines, where the same hexaploid nuclear genome was substituted into different cytoplasms, the specific activity of RuBPCase was consistent with the type of cytoplasm (high for the B and S cytoplasms and low for the A and D cytoplasms). There was no evidence from the euploid and amphiploid lines that small subunits encoded in different nuclear genomes influenced the specific activity. High specific activity was conferred by possession of the chloroplast genome of the B-type cytoplasm which encodes the large subunit of RuBPCase. All lines with a cytoplasm derived from the Sitopsis section of wheat, with the exception of Aegilops longissima and A. speltoides 18940, had RuBPCase with high specific activity. In contrast with the euploid lines of A. longissima, the alloplasmic line containing A. longissima cytoplasm from a different source had RuBPCase with high specific activity. The difference in specific activity found here in-vitro was not apparent in-vivo when leaf gas exchange was measured.Abbreviation RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

Evidence for a uridine-5′-diphosphate-glucose-protectedp-chloromercuribenzene sulfonic acid-binding site in sugarcane vacuoles

The uptake of uridine-5-diphosphate (UDP) glucose into vacuoles isolated fromSaccharum sp. cells was fully inhibited by pretreatment with 50 Mp-chloromercuribenzenesulfonic acid (PCMBS) and was not affected by N-ethylmaleimide up to a concentration of 5 mM. The addition of 10 mM UDP-glucose during the pretreatment partially protected the uptake mechanism from PCMBS inhibition, while the presence of adenosine-5-diphosphate (ADP) glucose or of various hexose-phosphates had no protective effect. Parallel experiments on the binding of [²⁰³Hg]PCMBS to the vacuoles showed that UDP-glucose and UDP added at 10 mM concentrations caused a 40% decrease in the binding of PCMBS while ADP-glucose did not inhibit the binding. The results indicate the presence in a previously proposed group translocator of at least one site that can bind UDP-glucose. This site, which is blocked by PCMBS, interacts with the nucleotide moiety of UDP-glucose.Abbreviations ADP-glucose adenosine-5-diphosphate glucose - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzenesulfonic acid - UDP uridine-5-diphosphate - UDP-glucose uridine-5-diphosphate glucose     

Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures

Out of the eight enzymes involved in the biosynthesis of the isoquinoline alkaloid berberine, at least, two enzymes, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, are exclusively located in a vesicle with a specific gravity of =1.14 g·cm^–3 as shown by direct enzymatic assay as well as immunoelectrophoresis. Electronmicroscopic examination of the enzyme-containing particulate preparation from Berberis wilsoniae var. subcaulialata cultured cells demonstrated that it is composed mainly of membranous vesicles. The protein composition of this preparation reveals the presence of only about 20 separable proteins, of which two major ones are berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase. Incubation of these vesicles with the substrate (S)-reticuline in the presence and absence of S-adenosyl-l-methionine leads to the formation of a red product which was identified as dehydroscoulerine. If the cytoplasmic enzyme S-adenosyl-l-methionine:(S)-scoulerine-9-O-methyltransferase is added to the vesicle preparation in the presence of (S)-reticuline and S-adenosyl-l-methionine, not dehydroscoulerine but columbamine, the immediate precursor of berberine is formed. Some of the quaternary alkaloids are located inside the vesicles; fusion of these vesicles leads to vacuoles containing the quaternary alkaloids. These vesicles are the first highly specific and unique compartment serving only alkaloid biosynthesis; they are found in members of four different plant families and in cell cultures as well as in differentiated tissue.Abbreviations BBE berberine bridge enzyme - STOX (S)-tetrahydroprotoberberine oxidase Dedicated to Professor Karl Decker, Freiburg, on the occasion of his 60^th birthday