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The effect of total-ABL, GUS and B2M control genes on BCR-ABL monitoring by real-time RT-PCR.

We compared the effect of control genes (CG): total Abelson (total-ABL), beta-2-microglobulin (B2M) and beta-glucuronidase (GUS), recommended in the Europe Against Cancer (EAC) program, on real-time BCR-ABL monitoring in patients with chronic myeloid leukemia (CML). We focused on the stability of CG expressions during therapy and the effect of the CGs on BCR-ABL ability to characterize the disease status and disease prognosis, issues that have not been addressed yet. The results showed B2M as a very convenient CG for BCR-ABL monitoring. On the contrary, the widely used total-ABL was not confirmed as appropriate for normalization of gene expression in CML.

Adult↗

Introduction of a NIa proteinase cleavage site between the reporter gene and HC-Pro only partially restores the biological properties of GUS- or GFP-tagged LMV.

Lettuce mosaic virus (LMV) isolates LMV-E and LMV-0 differ in their virulence on lettuce varieties carrying the mo1(2) resistance gene, which reduces viral accumulation and blocks the expression of symptoms after infection with avirulent isolates such as LMV-0. Previous work had indicated that reporter genes such as GUS or GFP affect the biological properties of recombinant LMV isolates in both susceptible and resistant lettuce varieties when fused to the N-terminus of the viral protein HC-Pro. The impact of the addition of a cleavage site for the NIa proteinase between the reporter gene and HC-Pro was evaluated, in an effort to recover the full spectrum of the biological properties of parental isolates. Symptoms, accumulation, cell-to-cell and long distance movement of the recombinant viruses containing the NIa cleavage site were studied in susceptible and mo1(2) lettuce varieties. Both LMV-0 and LMV-E recombinant viruses recovered the behaviour of their wild-type parent in susceptible plants upon addition of the NIa cleavage site. While the recombinant LMV-E modified in this way recovered the breaking properties of its wild-type counterpart in mo1(2) plants, similar modification of the LMV-0 derived recombinants failed to rescue a severe inhibition in systemic accumulation in mo1(2) plants, despite the fact that neither cell-to-cell movement nor phloem loading or unloading seemed to be severely affected.

Cysteine Endopeptidases↗

The GUS reporter gene system.

The GUS reporter gene system is already a powerful tool for the assessment of gene activity in transgenic plants. Further developments may lead to routine in vivo analysis and fusion genetics.

Animals↗

Characterization and mapping of Ds-GUS-T-DNA lines for targeted insertional mutagenesis.

The transposition patterns of the Ds-GUS transposon T-DNA in 23 independent single-copy lines have been characterized and the map positions of 10 of them on three of the five Arabidopsis chromosomes are reported. Using overexpressed Activator (Ac) elements as a transposase source, it was found that the primary determinant of transposition frequency is the insertion site of the Ac-T-DNA. Neither the structure of the transposon T-DNA nor, in most cases, its insertion site have a significant effect on transposition frequency. Both the frequency and timing of transposition are influenced by the parent through which the transposon and transposase T-DNAs are transmitted. Overall, nearly 75% of plants in which excision has occurred bear a reinserted element and very short-range transpositions predominate, underlining the advantage of using mapped transposons for insertional mutagenesis.

Arabidopsis↗

Fluorescent imaging of GUS activity and RT-PCR analysis of gene expression in the shoot apical meristem.

The use of promoter-reporter gene constructs in transgenic plants is a powerful tool in the analysis of gene expression which can, however, be limited in the resolution of small structures, such as the apical meristem. This paper reports on a fluorescent imaging technique for the analysis of GUS reporter gene expression to cellular resolution in the apical meristem of tomato. Using this technique in combination with an RT-PCR analysis of RBS gene-specific transcript levels, it is shown that: 5' upstream sequences of RBCS genes are sufficient to mimic the pattern of transcripts revealed by in situ hybridisation (no expression in the apical meristem, high expression in the initiated leaf primordia); the genes RBCS2, RBCS3A and RBCS3B are transcriptionally activated upon primordium initiation with transcripts for RBCS1 and RBCS3C accumulating later in leaf development; and that RBCS promoter activity cannot be induced in the apical meristem by light, an environmental signal which elevates RBCS transcript level in other aerial parts of the plant. These data provide a detailed picture of the complex transcriptional events occurring on leaf initiation and the establishment of the photosynthetic machinery; they describe two complementary techniques which allow the analysis of such complex events at the tissue and cellular level; and they characterize an in vivo assay system which can be used to analyse the factors involved in the initiation and maintenance of gene expression patterns in the apical meristem.

Gene Expression Regulation, Plant↗

Multiple photoreceptors mediate the light-induced reduction of GUS-COP1 from Arabidopsis hypocotyl nuclei.

The subcellular localization of COP1, a key photomorphogenic repressor, is regulated by light in Arabidopsis seedlings. Photoreceptor loss-of-function mutants and dominant gain-of-function overexpression transgenes were both used to analyze the influences of the three photoreceptors, phyA, phyB, and CRY1, on the light-regulated subcellular localization of COP1. Through a semiquantitative analysis of the nuclear abundance of GUS-COP1 in the various genetic backgrounds, the specific roles of the individual photoreceptors have been established. The data suggest that multiple photoreceptors influence the light-regulated subcellular localization of COP1 in white light. Under specific wavelengths of light, phyA, phyB, and CRY1 each play critical roles in mediating far-red, red, and blue light signals, respectively. Our data also support an interdependency between CRY1 and the phytochromes in mediating the light-regulated subcellular localization of COP1 and thus seedling development.

Arabidopsis↗

Phytochrome-Mediated Light Regulation of PHYA- and PHYB-GUS Transgenes in Arabidopsis thaliana Seedlings.

Phytochrome wild-type gene-[beta]-glucuronidase (PHY-GUS) gene fusions were used in transgenic Arabidopsis to compare the activity levels and light regulation of the PHYA and PHYB promoters and to identify the photoreceptors mediating this regulation. In dark-grown seedlings, both promoters are 4-fold more active in shoots than in roots,but the PHYA promoter is nearly 20-fold more active than that of PHYB in both organs. In shoots, white light represses the activities of the PHYA and PHYB promoters 10- and 2-fold, respectively, whereas in roots light has no effect on the PHYA promoter but increases PHYB promoter activity 2-fold. Consequently, PHYA promoter activity remains higher than that of PHYB in light in both shoots (5-fold) and roots (11-fold). Experiments with narrow-waveband light and photomorphogenic mutants suggest that no single photoreceptor is necessary for full white-light-directed PHYA repression in shoots, but that multiple, independent photoreceptor pathways are sufficient alone or in combination. In contrast, phytochrome B appears both necessary and sufficient for a light-mediated decrease in PHYB activity in shoots, and phytochrome A mediates a far-red-light-stimulated increase in PHYB promoter activity. Together, the data indicate that the PHYA and PHYB genes are regulated in divergent fashion at the transcriptional level, both developmentally and by the spectral distribution of the prevailing light, and that this regulation may be important to the photosensory function of the two photoreceptors.

Journal Article↗

Localization of mouse melanoma growth stimulatory activity gene (Mgsa) between Afp and Gus on chromosome 5 using interspecific backcross mice.

Melanoma growth stimulatory activity (Mgsa) is a polypeptide mitogen and a possible autocrine growth factor for melanoma and other tumor cells. A restriction fragment length variant of Mgsa was followed in interspecific backcross mic Mgsa was localized between Afp and Gus on mouse chromoson 5, distal to known loci affecting skin pigmentation.

Animals↗

[Establishment of a method for GUS gene transferring into wheat (Triticum astivum L.) embryos by low energy ion beam implantation].

Physical parameters influencing transformation of wheat mediated by low energy ion beam, including type of ion, parameters of ion energy, dose and dose rate, were studied. Ar+ was regarded as suitable ions implanted in transformation. 20-25 keV of energy, 4.68 x 10(16) ions/cm2 of dose, 2.6 x 10(15) ions/cm2 of dose rate were chosen as appropriate implantation parameters. The suitable culture conditions for induction and growth of callus and the optimal selection scheme were established, After implantation and selection, resistant calli and hygromycin-resistant plantlets were obtained from three varieties. Molecular analysis data proved that GUS gene had integrated into the wheat genome. The mature embryo transformation efficiency of wherat variety Yangmai 5, Yangmai 158, Wanmai 32 reached 9.5%, 10.8%, 11.2% measured in produced hygromycin-resistant callus and 1.4%, 3.4%, 1.7% measured in regenerated plants, respectively, This experiment provides a basis for further investigation of wheat transformation system. Low energy ion beam mediated transformation can be extended to other plant recalcitrant to Agrobacterium tumefaciens as soon as methodological parameters are optimized.

Gene Transfer Techniques↗

Activation of genes presumed in cytokinin signal pathway of two-component system in response to the increased cytokinin contents in ipt-GUS transgenic Arabidopsis.

The ipt-GUS activated transgenic Arabidopsis had 20-25 fold higher cytokinin contents than the wild type. Changes in cytokinin content in vivo triggering gene expressions involved in signal pathway of two-component system have been analyzed on the day of 6 d, 12 d, 20 d and 30 d after seed cultivation on MS medium in light conditions. The results showed that the two cytokinin receptors, His-kinase CRE1 was more sensitive to the increased cytokinin contents than CKI1. Arabidopsis response regulators, ARR4 and ARR5, were induced by the increased cytokinin contents at different time after seed germination. ARR4 responded to cytokinins at early time of seed germination, especially on the 6 d when seedlings were around true leaf initiation, while cytokinins induced ARR5 activation after 6 d of seed cultivation in light conditions, an obvious increase was on the 20 d when seedlings were around inflorescent shoot initiation. Hpt-type transmitter kinase AHP4 increased its activation by cytokinin induction only between the 20 d and 30 d after seed cultivation in light conditions, and an obvious increase was on the 20 d.

Arabidopsis↗

Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures.

Genetic transformation of cereals by direct DNA delivery via microprojectile bombardment has become an established procedure in recent years. But the derivation of functional transgenic plants, especially in wheat, is still problematic, mainly due to low efficiency of DNA delivery and the reduced regeneration capability of microprojectile-bombarded tissue. We focussed on these two aspects and found that the regeneration of scutellar calli of wheat can be rendered highly efficient and considerably accelerated by a liquid culture phase in screen rafts. We also found that the expression of a reporter gene following DNA delivery by microprojectile can be improved by maintaining the scutellar calli in 0.25 M mannitol before and after bombardment, by bombardment in the presence of silver thiosulfate and Ca(NO3)2 (rather than CaCl2) and by the elimination of spermidine from the DNA/microprojectile mixture. A protocol that includes all these features leads to several-fold higher transient expression of the reporter gene than have previously published procedures.

Calcium↗

Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene.

We obtained carrot (Daucus carota) cells possessing the 5'-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial beta-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in beta-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of beta-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of beta-glucuronidase activities, in concord with embryogenesis, were also noted.

Journal Article↗