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101.
Summary The localization of -amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts was studied using electron microscope immunocytochemistry. Antibodies were raised against total barley -amylase, i.e., -amylase containing both highisoelectric point (high-pI) and low-pI isoforms, as well as against purified high- and low-pI isoforms. All antibodies localized -amylase to the endoplasmic reticulum (ER) and Golgi apparatus (GApp) of the aleurone cell, and various controls showed that the labeling was specific for -amylase. Labeling of protein bodies and spherosomes, which are the most abundant organelles in this cell, was very low. There was no evidence that -amylase isoforms were differentially distributed within different compartments of the endomembrane system. Rather, both high- and low-pI isoforms showed the same pattern of distribution in ER and in the cis, medial, and transregions of the GApp. We conclude that in the Himalaya cultivar of barley, all isoforms of -amylase are transported to the plasma membrane via the GApp.Abbreviations ER
endoplasmic reticulum
- GA3
gibberellic acid
- GApp
Golgi apparatus
- PBS
phosphate buffered saline
- PCR
partially coated reticulum
- PM
plasma membrane
- TBS
Tris buffered saline
- TGN
trans-Golgi network 相似文献
102.
T. Kumagai Y. Umemura T. Baba M. Iwanaga 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,79(3):369-376
Summary Several sweet potato genotypes were found to lack completely or to have only traces of-amylase in their storage roots. Such genotypes do not increase in sweetness during cooking because, without a sufficient amount of-amylase, the normal hydrolysis of starch to maltose does not occur in the cooking process. In order to study the inheritance of this biochemical variant in the genotype, 41 families were generated. The following conclusions were drawn from analyzing these families. (1) This trait is controlled by one recessive allele (designated-amy) (2) It is inherited in a hexasomic or tetradisomic manner, but not disomically or tetrasomically. This conclusion supports previous cytological data that sweet potato is an autohexaploid or has two identical genomes plus one genome which is somewhat different. (3) The-amy allele appears to exist at a high frequency in cultivated germplasm. (4) Breeding sweet potato for low-amylase activity is relatively easy. New types of sweet potato without normal-amylase activity have great potential for processing and as a staple food. 相似文献
103.
We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before. 相似文献
104.
Dong-Uk Kim Seung-Kiel Park Kyung-Sook Chung Myung-Un Choi Hyang-Sook Yoo 《Molecular & general genetics : MGG》1996,252(1-2):20-32
ASchizosaccharomyces pombe homolog of mammalian genes encoding G protein subunits,gpb1
+, was cloned by the polymerase chain reaction using primer pairs that correspond to sequences conserved in several G genes of other species followed by screening of genomic and cDNA libraries. Thegpb1 gene encodes 317 amino acids that show 47% homology with human G
1 and G
2 and 40% homology withSaccharomyces cerevisiae G protein. Disruption of thegpb1 gene indicated that this gene is not required for vegetative cell growth. However,gpb1-disrupted haploid cells mated and sporulated faster than wild-type cells, both in sporulation (MEA) and in complex medium (YE): when examined 23 h after transfer to sporulation medium, 35% ofgpb1-disrupted haploid pairs had undergone conjugation and sporulation, whereas only 3–5% of wild-type haploid pairs had done so. Overexpression of thegpb1 gene suppressed this facilitated conjugation and sporulation phenotype ofgpb1-disrupted cells but did not cause any obvious effect in wild-type cells. Co-disruption of one of the twoS. pombe G-subunit genes,gpa2, in thegpb1-disrupted cells did not change the accelerated conjugation and sporulation phenotype of thegpb1
– cells. However, co-disruption of theras1 gene abolished thegpb1
– phenotype. These results suggest that Gpbl is a negative regulator of conjugation and sporulation that apparently works upstream of Ras1 function inS. pombe. The possible relationship of Gpbl to two previously identified, putative G proteins ofS. pombe is discussed.A preliminary report of this work first appeared in an abstract of the Genetic Society of America, 1993 Yeast Genetics and Molecular Biology Meeting, p. 92 and was presented at the American Association of Cancer special meeting on Cell Signalling and Cancer Treatment, 1993 相似文献
105.
J. Daniel 《Molecular & general genetics : MGG》1996,253(3):393-396
A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of
high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the
ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination
in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible
implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general
approaches to gene selection, such as the gene-gene interference method, are discussed.
Received: 18 March 1996 / Accepted: 23 August 1996 相似文献
106.
The grass flora of Namibia (374 species in 110 genera) shows surprisingly little variation in 13C values along a rainfall gradient (50–600 mm) and in different habitat conditions. However, there are significant differences in the 13C values between the metabolic types of the C4 photosynthetic pathway. NADP-ME-type C4 species exhibit the highest 13C values (–11.7 ) and occur mainly in regions with high rainfall. NAD-ME-type C4 species have significantly lower 13C values (–13.4 ) and dominate in the most arid part of the precipitation regime. PCK-type C4 species play an intermediate role (–12.5 ) and reach a maximum abundance in areas of intermediate precipitation. This pattern is also evident in genera containing species of different metabolic types. Within the same genus NAD species reach more negative 13C values than PCK species and 13C values decreased with rainfall. Also in Aristida, with NADP-ME-type photosynthesis, 13C values decreased from –11 in the inland region (600 mm precipitation) to –15 near the coast (150 mm precipitation), which is a change in discrimination which is otherwise associated by a change in metabolism. The exceptional C3 species Eragrostis walteri and Panicum heterostachyum are coastal species experiencing 50 mm precipitation only. Many of the rare species and monotypic genera grow in moist habitats rather than in the desert, and they are not different in their carbon isotope ratios from the more common flora. The role of species diversity with respect to habitat occupation and carbon metabolism is discussed. 相似文献
107.
大肠癌中p53基因突变的研究 总被引:2,自引:0,他引:2
应用聚合酶链反应(PCR)──单链构型多态性(SSCP)结合银染法对14例大肠癌p53基因的第4、第5─6和第7外显子进行了点突变的研究,结果共检测出6例点突变,而且发现各外显子的突变频率存在差异。另外,利用购自ATCC的两个探针 (p53cDNA探针和pYNZ22探针)对大肠癌中p53基因的杂合性失去进行了研究,在14例大肠癌中共检出6例杂合性丢失。将点突变检测结果同杂合性丢失结果进行比较分析, 并着重探讨了大肠癌中p53基因失活导致肿瘤的作用方式。
Abstract:The exons 4-7 of p53 gene were examined in 14 colorectal Cancer patients by using PCR-SSCP-silver staining method.The results showed 6 cases of point mutation and the mutation frequencies of exons were different from each other.p53 cDNA and pYNZ22 VNTR were used as probes to examine LOH(Loss of heterozygosity)of 14 colorectal cancers.6 cases with LOH were found.The results of present research suggest that mutation and LOH of p53 gene are critical events in the progress and development of Cancer.There were different kinds of inactivation model of p53 gene in the process of development of cancer and transformation of cells. 相似文献
108.
109.
Robert N. Fontaine Ruanna E. Gossett Friedhelm Schroeder Barbara A. O'Toole Thomas Doetschman Ann B. Kier 《Molecular and cellular biochemistry》1996,159(2):149-153
The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP
liver fatty acid binding protein
- I-FABP
intestinal fatty acid binding protein
- TGF1
transforming growth factor beta-1
- TNF-
tumor necrosis factor-
- MIP-
macrophage inflammatory protein-
- PMSF
phenylmethyl sulfonyl fluoride
- PBS
phosphate buffered saline 相似文献
110.
Summary During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not -galactosidase activity, were detected iroculture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl--D-galactopyranoside (ONPG), indicating that -galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that =galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of -galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed. 相似文献