Ribosomal transcription units integrated via T‐DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana

Wanzenböck, Eva-Maria; Schöfer, Christian; Schweizer, Dieter; Bachmair, Andreas

doi:10.1046/j.1365-313x.1997.11051007.x

Cited by 97 publications

(61 citation statements)

References 40 publications

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“…The 45S rDNA sites were localized using the R2 probe, a 6.5-kb fragment including the 18S-5.8S-25S repeat from Arabidopsis thaliana (Wanzenböck et al, 1997). The plasmid with 45S rDNA and selected BACs were labeled by nick translation using digoxigenin-11-dUTP (Roche) and Cy3 (Invitrogen), respectively.…”

Section: Methodsmentioning

confidence: 99%

Mapping the chromosomes of Poncirus trifoliata Raf. by BAC-FISH

Moraes

Mirkov

Guerra

2008

Cytogenet Genome Res

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In spite of the importance of Citrus in agriculture and recent progress in genetic mapping and cytogenetics of this group, chromosome mapping of Citrus species is still limited to rDNA probes. In order to obtain a better chromosome characterization of one species from this group, CMA/DAPI double staining followed by in situ hybridization using 45S rDNA and 24 BACs (BAC-FISH) were used on Poncirus trifoliata. The BACs used were obtained from a genomic library of this species and were selected by membrane hybridization using genomic DNA. Four of them were isolated from the Citrus tristeza virus (Ctv) resistance gene region. The P. trifoliata karyotype is composed of two chromosome pairs with one terminal and one proximal CMA⁺ band (B type chromosomes), four chromosome pairs with a single CMA⁺ band (D type) and three chromosome pairs without bands (F type). In situ hybridization with 13 of the BACs gave single copy signals on seven chromosome pairs. At least one BAC was mapped on each arm of the two B chromosome pairs. Among the four D chromosome pairs, two were identified by BACs mapped on the long arms, one has a 45S rDNA site and the other had no signal. Six BACs allowed identification of the three F chromosome pairs, with one pair hybridizing with four BACs from the Ctv resistance gene region. In summary, all nine chromosome pairs could be differentiated, seven of them by BAC-FISH, while the other two chromosomes could be recognized by the CMA⁺ band pattern and 45S rDNA sites. This first BAC-FISH map gives a general framework for comparative genome structure and evolutionary studies in Citrus and Poncirus, allowing the integration of genetic and physical maps when these BACs are included.

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Section: Methodsmentioning

confidence: 99%

Mapping the chromosomes of Poncirus trifoliata Raf. by BAC-FISH

Moraes

Mirkov

Guerra

2008

Cytogenet Genome Res

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“…For 45S rDNA FISH analyses we used the Arabidopsis thaliana R2 probe (Wanzenböck et al, 1997) which was labeled by nick translation with digoxigenin, while for locating 5S rDNA we used the Lotus japonicus D2 probe (Pedrosa et al, 2002) which was labeled by nick translation with biotin. The hybridization mixture contained 50% (v/v) formamide, 10 % (v/v) dextran sulfate, 2x SSC and 2 to 5 ng mL -1 of each probe.…”

Section: Methodsmentioning

confidence: 99%

Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae)

2006

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We investigated four orchids of the genus Maxillaria (M. discolor, M. acicularis, M. notylioglossa and M. desvauxiana) in regard to the position of heterochromatin blocks as revealed using chromomycin A 3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI) fluorochrome staining and 5S and 45S rDNA sites using fluorescence in situ hybridization (FISH). The species showed differences in chromosome number and a diversified pattern of CMA + and DAPI + bands, including heteromorphism for CMA + bands. The 5S and 45S rDNA sites also varied in number and most of them were co-localized with CMA + bands. The relationship between 5S rDNA sites and CMA + bands was more evident in M. notylioglossa, in which the brighter CMA + bands were associated with large 5S rDNA sites. However, not all 5S and 45S rDNA sites were co-localized with CMA + bands, probably due to technical constraints. We compare these results to banding data from other species and suggest that not all blocks of tandemly repetitive sequences, such as 5S rDNA sites, can be observed as heterochromatin blocks.

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“…The direction of transcription of the reporter gene was either pointing toward the left (r-constructs) or right border (f-constructs). T-DNAs containing different repetitive sequences from Arabidopsis (MartinezZapater et al, 1986;Simoens et al, 1988;Thompson et al, 1996;Wanzenbö ck et al, 1997) immediately upstream of the GUS gene ( Figure 1C), a promoterless copy of the GUS gene (DGUS, Figure 1D), or the GUS reporter gene under the control of the nopaline synthase promoter (Pro NOS ) were also introduced into Arabidopsis (NOS-GUS, Figure 1E). …”

Section: No Case Of Silencing Because Of Site Of T-dna Integrationmentioning

confidence: 99%

“…GUS 1xr 501 and GUS 1xr 502 carried one and two copies, respectively, of the 500-bp HindIII tandem repeat (Simoens et al, 1988). A fragment corresponding to the intergenic spacer of rDNA sequences (Wanzenbö ck et al, 1997) was present in T-DNA vector GUS 1xr rDNA. In constructs GUS 1xr 163 and GUS 1xr 164, the sequences cloned upstream of the CaMV 35S promoter corresponded to different dispersed repetitive sequences (Thompson et al, 1996).…”

Section: Comparable Transgene Expression Levels In Independentmentioning

confidence: 99%

Silencing in Arabidopsis T-DNA Transformants: The Predominant Role of a Gene-Specific RNA Sensing Mechanism versus Position Effects

et al. 2004

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Pronounced variability of transgene expression and transgene silencing are commonly observed among independent plant lines transformed with the same construct. Single-copy T-DNA lines harboring reporter genes of various kind and number under the control of a strong promoter were established in Arabidopsis thaliana for a comprehensive analysis of transgene expression. Characterization of 132 independent transgenic lines revealed no case of silencing as a result of site of T-DNA integration. Below a certain number of identical transgenes in the genome, gene copy number and expression were positively correlated. Expression was high, stable over all generations analyzed, and of a comparable level among independent lines harboring the same copy number of a particular transgene. Conversely, RNA silencing was triggered if the transcript level of a transgene surpassed a gene-specific threshold. Transcript level–mediated silencing effectively accounts for the pronounced transgene expression variability seen among transformants. It is proposed that the RNA sensing mechanism described is a genome surveillance system that eliminates RNA corresponding to excessively transcribed genes, including transgenes, and so plays an important role in genome defense

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Ribosomal transcription units integrated via T‐DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana

Cited by 97 publications

References 40 publications

Mapping the chromosomes of <i>Poncirus</i> <i>trifoliata</i> Raf. by BAC-FISH

Mapping the chromosomes of <i>Poncirus</i> <i>trifoliata</i> Raf. by BAC-FISH

Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae)

Silencing in Arabidopsis T-DNA Transformants: The Predominant Role of a Gene-Specific RNA Sensing Mechanism versus Position Effects

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