Luciferase-based transgenic recombination assay is more sensitive than β-glucoronidase-based

Ilnytskyy, Yaroslav; Boyko, Alexander; Kovalchuk, Igor

doi:10.1016/j.mrgentox.2004.02.001

Cited by 30 publications

(33 citation statements)

References 46 publications

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“…Strand breaks in one of the overlapping regions can be repaired via homologous recombination, thus restoring transgene structure. The luciferase activity is then visualized in vivo using a long exposure CCD camera (Ilnytskyy et al 2004). …”

Section: Plants Used For Experimentsmentioning

confidence: 99%

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

Rahavi

Kovalchuk

2013

Physiol Mol Biol Plants

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In the past, we showed that exposure to abiotic and biotic stresses changes the homologous recombination frequency (HRF) in somatic tissue and in the progeny. In current work we planned to answer the following question: do stress intensity/duration and time during exposure influence changes in somatic HRF and transgenerational changes in HRF? Here, we tested the effects of exposure to UV-C, cold and heat on HRF at 7, 14, 21 and 28 days post germination (dpg). We found that exposure at 14 and 21 dpg resulted in a higher increase in HRF as compared to exposure at 7 dpg; longer exposure to UV-C resulted in a higher frequency of HR, whereas prolonged exposure to cold or heat, especially at later developmental stages, had almost no effect on somatic HRF. Exposure at 7 dpg had a positive effect on somatic growth of plants; plants exposed to stress at this age had larger leaves. The analysis of HRF in the progeny showed that the progeny of plants exposed to stress at 7 dpg had an increase in somatic HRF and showed larger sizes of recombination spots on leaves. The progeny of plants exposed to UV-C at 7 dpg and the progeny of plants exposed to cold or heat at 28 dpg had larger leaves as compared to control plants. To summarize, our experiments showed that changes in somatic and transgenerational HRF depend on the type of stress plants are exposed to, time of exposure during development and the duration of exposure.

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Section: Plants Used For Experimentsmentioning

confidence: 99%

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

Rahavi

Kovalchuk

2013

Physiol Mol Biol Plants

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“…The recombination markers also were successfully used in rice (Oryza sativa) (S. Toki, personal communication). Apart from the GUS gene as marker, the luciferase gene was used, permitting noninvasive and sensitive assays of recombination (Kovalchuk et al, 2003;Ilnytskyy et al, 2004). Equally important is the development of specific recombination lines, the use of which allowed answering specific questions about the mechanism of SHR.…”

Section: Multiple Variations Of the Shr Setupmentioning

confidence: 99%

In Planta Somatic Homologous Recombination Assay Revisited: A Successful and Versatile, but Delicate Tool

Puchta

Höhn

2012

Plant Cell

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Marker-transgene-dependent lines of Arabidopsis thaliana measuring somatic homologous recombination (SHR) have been available for almost two decades. Here we discuss mechanisms of marker-gene restoration, comment on results obtained using the reporter lines, and stress how caution must be applied to avoid experimental problems or false interpretation in the use of SHR reporter lines. Although theoretically possible, we conclude that explanations other than SHR are unlikely to account for restoration of marker gene expression in the SHR lines when used with appropriate controls. We provide an overview of some of the most important achievements obtained with the SHR lines, give our view of the limitations of the system, and supply the reader with suggestions on the proper handling of the SHR lines. We are convinced that SHR lines are and will remain in the near future a valuable tool to explore the mechanism and influence of external and internal factors on genome stability and DNA repair in plants.

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“…This system subsequently was modified by switching to assayable marker genes, such as uidA (b-GLUCURONIDASE [GUS]) (Swoboda et al, 1994) and LUCIFERASE (LUC) (Ilnytskyy et al, 2004;Molinier et al, 2004), eliminating the need for selection and tissue culturing. Reporter lines containing LUC were shown to be more sensitive than GUS in recombination assays.…”

Section: Somatic Homologous Recombination Reporter Lines: Lack Of Sufmentioning

confidence: 99%

“…Analysis of several independent reporter lines carrying these marker genes indicated that the average somatic recombination frequency detected with the LUC transgene was ;10-fold higher (10 25 ) in Arabidopsis thaliana and tobacco (Nicotiana tabacum) plants compared with GUS recombination frequencies (Ilnytskyy et al, 2004). Using such GUS-or LUC-based SHR reporter lines, it was shown that abiotic and especially biotic stresses lead to enhanced somatic recombination (Kovalchuk et al, 1998(Kovalchuk et al, , 2001a(Kovalchuk et al, , 2001b(Kovalchuk et al, , 2003Lucht et al, 2002;Filkowski et al, 2004;Ilnytskyy et al, 2004;Molinier et al, 2004Molinier et al, , 2005Molinier et al, , 2006Schuermann et al, 2005). Using such a system, it was also discovered that plants can remember a stress perceived in one generation for several following generations.…”

Section: Somatic Homologous Recombination Reporter Lines: Lack Of Sufmentioning

confidence: 99%

“…For example, a study using two transgenic lines carrying a single-copy transgene in either direct (line 1406) or inverted (line 1415) repeats of GUS fragments in Arabidopsis showed that UV-C, bleomycin, and xylanase all increased somatic recombination frequency, and the orientation of GUS fragments did not influence the recombination frequency . Another study using similar single-copy lines carrying the GUS gene fragments in direct (A11) or inverted (A651) orientations in Arabidopsis showed that background spontaneous recombination frequency was much higher in line A11 compared with A651 (1.90 versus 0.21 spots, respectively) (Ilnytskyy et al, 2004). However, the fold induction upon UV-C treatment was higher in line A651 compared with A11 (4.4-versus 2.1-fold induction, hence leading to 4 versus 0.9 total spots, respectively).…”

Section: Reporter Lines With Inverted Partial Repeatsmentioning

confidence: 99%

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Reevaluation of the Reliability and Usefulness of the Somatic Homologous Recombination Reporter Lines

et al. 2012

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A widely used approach for assessing genome instability in plants makes use of somatic homologous recombination (SHR) reporter lines. Here, we review the published characteristics and uses of SHR lines. We found a lack of detailed information on these lines and a lack of sufficient evidence that they report only homologous recombination. We postulate that instead of SHR, these lines might be reporting a number of alternative stress-induced stochastic events known to occur at transcriptional, posttranscriptional, and posttranslational levels. We conclude that the reliability and usefulness of the somatic homologous recombination reporter lines requires revision. Thus, more detailed information about these reporter lines is needed before they can be used with confidence to measure genome instability, including the complete sequences of SHR constructs, the genomic location of reporter genes and, importantly, molecular evidence that reconstituted gene expression in these lines is indeed a result of somatic recombination.

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Luciferase-based transgenic recombination assay is more sensitive than β-glucoronidase-based

Cited by 30 publications

References 46 publications

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

In Planta Somatic Homologous Recombination Assay Revisited: A Successful and Versatile, but Delicate Tool

Reevaluation of the Reliability and Usefulness of the Somatic Homologous Recombination Reporter Lines

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