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851.
Imerio Capone Maura Cardarelli Maurizio Trovato Paolo Costantino 《Molecular & general genetics : MGG》1989,216(2-3):239-244
Summary Root differentiation could be elicited on carrot discs by transformation with the agropine Ri plasmid rolB gene cloned in the binary vector Bin19, provided two conditions were met. Firstly, an adequate auxin supply had to be provided. This was achieved by co-inoculation with a strain carrying only the auxin synthetic genes of the TR-DNA. Most of the resulting roots were then shown to harbour only rolB and no aux genes. Secondly, an extended non-coding region (1200 bp) at the 5 end of rolB had to be included in the construction. A shorter (300 bp) 5 region, including TATA and CCAAT boxes, was not sufficient to trigger root differentiation. Both the extended (B1185) and reduced (B310) 5 regions of rolB were then cloned upstream of the -glucuronidase (GUS) reporter gene and infections carried out both on the apical and on the basal side of carrot discs. Strong expression of GUS, visualized histochemically as an intense blue colouring of transformed cells was observed with B1185-GUS constructions on the apical side of the discs. Only occasionally could coloured cells be observed on the basal side of the discs with B1185-GUS and on both apical and basal sides with B310-GUS constructions. Strong GUS expression was, on the contrary, achieved on cells of both auxin-rich (apical) and auxin-depleted (basal) sides of the discs with the strong constitutive viral promoter, CaMV35S. These results indicate the presence of an upstream regulatory region which confers polar expression to the rolB gene and suggest a role for auxin in its activation. 相似文献
852.
Integration of HBV-DNA into liver and hepatocellular carcinoma cells during persistent HBV infection 总被引:1,自引:0,他引:1
D A Shafritz 《Journal of cellular biochemistry》1982,20(3):303-316
The hepatitis B virus carrier state (persistent HBV infection) is characterized by the presence of viral surface antigen (HBsAg) and virion particles (Dane particles) in the blood. From 1% to 10% of carriers develop chronic liver disease and/or hepatocellular carcinoma. Recent studies have demonstrated integrated HBV-DNA in hepatocellular carcinomas and in several human hepatoma cell lines. In hepatoma patients, integrated HBV-DNA has been found in all HBsAg carriers. Nontumorous liver also revealed integrated HBV-DNA with the same or a different hybridization pattern from that observed in the tumor. To explore when integration occurs, carriers of short-term (less than 2 years) or long-term (greater than 8-10 years) were evaluated. DNA extracts from percutaneous (needle) liver biopsies showed free viral DNA with no specific integration bands in short-term carriers. In long-term carriers, HBV-DNA was integrated into the host genome with either a diffuse or a unique hybridization pattern. HBV-DNA integration correlated with the duration of the carrier state and absence of virions in the serum but did not correlate with histologic evidence of chronic hepatitis. These studies suggest that integration of HBV-DNA occurs during persistent HBV infection irrespective of liver disease and precedes development of hepatocellular carcinoma. 相似文献
853.
An 8 bp sequence repeated 6 times is present to the right of the mannopine type pRi8196 T-DNA righ-border sequence. Experiments were designed to test whether these repeats have a role in T-DNA transfer. Several constructs in which different lengths of pRi8196 right-border region were linked to the cucumopine synthesis gene on anAgrobacterium-Escherichia coli shuttle vector were made. The recombinant plasmids were tested for their efficiency to act as a source of T-DNA in a binary system in which a wild-type Ri plasmid provided virulence and root-inducing functions. The T-DNA transfer efficiency of the constructs was assessed by computing the relative frequency of roots containing cucumopine. Depending on the Ri plasmid used as source of virulence functions, a high level of T-DNA transfer was observed only if 6 (pRi8196) or 5 (pRiA4) repeats were present. These results were confirmed by looking for single-stranded T-DNA molecules (T-strands) in bacteria induced for virulence. The repetition of the 8 bp unit was named T-DNA transfer stimulator sequence (TSS). 相似文献
854.
Linda Oskam Dirk J. Hillenga Gerard Venema Sierd Bron 《Molecular & general genetics : MGG》1992,233(3):462-468
Summary pTB19, a 27 kb plasmid originating from a thermophilic Bacillus species, contains integrated copies of two rolling-circle type plasmids on a 10.6 kb DNA fragment. In the present study we analysed the part of pTB19 that contains the rolling-circle plasmid pTB913 and the region in between the two rolling-circle plasmids. We show that, in the integrated state, pTB913 was flanked by a 55 by direct repeat that duplicated part of the replication initiation gene repB. Since repB was interrupted, the integrated pTB913 could not initiate rolling-circle replication. Autonomously replicating pTB913 was produced from pTB19, probably through recombination between the 55 by direct repeats; this was a rare event. Since the second integrated rolling-circle type plasmid also contained a non-functional replication initiation gene, replication of pT1319 must be controlled by the RepA determinant. Theta-type replication, controlled by RepA is likely to account for the high stability of pTB19. In between the two integrated rolling-circle plasmids was present an open reading frame (447 codons) which could encode a protein of unknown function. 相似文献
855.
Summary Translocation of 14C-labelled carbohydrates between the parent stolon and branches, and among branches, of Trifolium repens plants was investigated in two glasshouse experiments to determine patterns of physiological organisation in this clonal species. Differential defoliation treatments were applied to the parent stolon and/or branches to test the sensitivity of translocation to the short-term carbon needs of defoliated sinks. Strong reciprocal exchange of carbohydrate between the parent stolon and branches was observed, with 18 41% of the 14C exported from leaves on the parent stolon moving to branches, while branches simulta-neously exported 25% (for old source branches) to 54% (for young source branches) of the 14C they assimilated to the parent plant, including translocation to other branches. Branch-to-branch translocation occurred both acropetally and basipetally. Parent-to-branch, branch-to-parent and branch-to-branch carbon fluxes all increased in response to defoliation of the sink, at the expense of carbon supply to stolon tissue or roots of the source module. Reduced export to stolon tissue of the parent axis played a major role in facilitating C reallocation from leaves on the parent stolon to defoliated branches. The observed patterns of C allocation and translocation could be adequately explained by accepted source-sink theory, and are consistent with a high degree of intra-plant physiological integration in resource supply and utilisation. This information provides mechanistic explanations for aspects of the growth dynamics and ecological interactions of T. repens in the patchy environment of a grazed pasture. 相似文献
856.
Klaus Hensler 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1992,171(2):273-284
Summary In Locusta migratoria, the major pathway from descending deviation detectors (DDNs; preceding paper, Hensler 1992) to wing motoneurons involves a population of thoracic interneurons (TINs). Nine TINs are characterized which receive input from cervical proprioreceptors. Responses to the combination of exteroreceptive input (signalling course deviation) and proprioreceptive input (monitoring movement and position of the head) are described and compared to those of DDNs to the same stimuli.Abbreviations
AP
action potential
-
DDN
descending deviation detector neuron
-
TCG
tritocerebral giant neuron
-
TIN
thoracic interneuron 相似文献
857.
Kazuhito Akama Hideaki Shiraishi Shozo Ohta Kenzo Nakamura Kiyotaka Okada Yoshiro Shimura 《Plant cell reports》1992,12(1):7-11
Summary The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.Abbreviations AIM
Agrobacterium infection medium
- CIM
callus-inducing medium
- CTAB
cetyltrimethylammonium bromide
- 2,4-D
2,4-dichlorophenoxy-acetic acid
- GUS
ß-glucuronidase
- hph
hygromycin phosphotransferase
- IAA
indole-3-acetic acid
- IBA
indole-3-butyric acid
- 2ip
N -(2-isopentenyl) adenine
- NPTII
neomycin phosphotransferase II
- RIM
root-inducing medium
- 35S
cauliflower mosaic virus 35S promoter
- SIM
shoot-inducing medium 相似文献
858.
Irradiation of protoplasts with X-rays or ultraviolet light does not seem to influence the level of transient expression of
foreign DNA inPetunia protoplasts, whereas the number of stably transformed colonies is significantly raised. This may indicate that irradiation
influences integration and/or the expression of marker genes and does not result in enhanced uptake rates of plasmids into
protoplasts and cell nuclei. Co-transformation with plasmids carrying a gene for kanamycin resistance (neomycin phosphotransferase
II) and a gene for hygromycin resistance (hygromycin phosphotransferase) revealed that the cotransformation rates were not
stimulated by irradiation when measuring expression. Twenty-five kanamycin resistant but hygromycin sensitive colonies were
examined with Southern or slot blotting and all were found to contain the coding sequence for the hygromycinphosphotransferase
gene in their genomes. No obvious differences regarding copy number of integrated genes were observed when comparing transformed
colonies derived from irradiated and non-irradiated protoplasts. 相似文献
859.
The T-region of Ti plasmids expresses two genes (No. 1 and 2) in crown-gall cells which are essential for auxin effects. It has been shown that gene 2 (=IaaH) codes for an amidohydrolase which converts indole-3-acetamide into indole-3-acetic acid and which is functional in bacteria and in crown-gall cells (Schröder et al. (1984), Eur. J. Biochem. 138, 387–391). In this report we describe a quantitative assay for the enzyme and its application to analyze the properties of the enzyme as expressed in plant cells and in Escherichia coli. The enzyme requires no cofactors, and the temperature optimum (30–37°C), pH optimum (8.5–9.5), and Km (about 1 M) were very similar in both systems. Besides indole-3-acetamide, the enzyme also hydrolyzed indole-3-acetonitrile, esters of indole-3-acetic acid with glucose and myo-inositol, a-naphthaleneacetamide, and phenylacetamide, indicating that it may have a general function in converting substances of low auxin activity into those with high auxin activity. The results are discussed in relation to the possible function of T-DNA gene 1 which cooperates with gene 2 in evoking auxin effects in crown-gall cells.Abbreviations HPLC
high-pressure liquid chromatography
- T-DNA
transferred DNA 相似文献
860.
Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of Nicotiana plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical
isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine
dehydrogenase activity (encoded by the T-region of Agrobacterium tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All eight cell lines selected
on the screening medium were identified as nuclear hybrids, while only three of the eight evaluated clones obtained by mechanical
isolation and cloning were found to be nuclear hydrids; the rest of them were nuclear segregants of tobacco [1] or N. plumbaginifolia [4] type. These data give independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products,
that can be revealed only by using nonselective methods for hybrid screening. In this paper we demonstrate the value of microisolation
for the recovery of cytoplasmic hybrids. 相似文献