Variations in the maize Ac transposase transcript level and the Ds excision frequency in transgenic wheat callus lines

Genome ◽  
1999 ◽  
Vol 42 (6) ◽  
pp. 1234-1241 ◽  
Author(s):  
Shigeo Takumi ◽  
Koji Murai ◽  
Naoki Mori ◽  
Chiharu Nakamura
Keyword(s):  
Microprojectile Bombardment ◽  
Transcript Level ◽  
Gus Gene ◽  
Large Variability ◽  
Transposase Gene ◽  
Ds Excision ◽  
Ac Transposase ◽  
Callus Lines ◽  
Transgenic Callus ◽  
Wheat Callus

To investigate the excision of a maize transposable element in wheat cells, plasmid DNAs containing a Dissociation (Ds) element located between a rice actin 1 gene promoter and a beta-glucuronidase (GUS) gene (gus) were introduced into wheat callus lines by microprojectile bombardment, and transient GUS expression was assayed. The gus-expressing cells after Ds excision were detected only when the Activator (Ac) transposase gene was co-transformed. To further examine a relationship between the amount of Ac mRNA and the Ds excision frequency, the Ds-containing plasmids were introduced into 15 independent transgenic callus lines transformed with the Ac transposase gene. Ten lines expressed the Ac transposase gene under the control of either the cauliflower mosaic virus 35S promoter or the Ac native promoter. The gus gene expression that indicated the Ds excision was observed only in the transgenic callus lines stably expressing the Ac transposase gene. The number of blue spots reflecting the frequency of Ds excision was variable among them. Northern-blot analysis also showed a large variability in the amount of Ac transposase transcripts among the lines. It was however noted that the excision frequency was decreased at a high level of the Ac transposase transcripts, supporting the hypothesis that Ds excision is inhibited above a certain level of the Ac transposase as observed in maize and transgenic tobacco.Key words: transposon, Ds excision, Ac transposase transcript level, transgenic callus, wheat.

Variations in the maize Ac transposase transcript level and the Ds excision frequency in transgenic wheat callus lines

Genome ◽  
1999 ◽  
Vol 42 (6) ◽  
pp. 1234-1241 ◽  
Author(s):  
Shigeo Takumi ◽  
Koji Murai ◽  
Naoki Mori ◽  
Chiharu Nakamura
Keyword(s):  
Transcript Level ◽  
Transgenic Wheat ◽  
Callus Lines ◽  
Wheat Callus

Hygromycin-resistant calli generated by activation and excision of maize Ac/Ds transposable elements in diploid and hexaploid wheat cultured cell lines

Genome ◽  
1996 ◽  
Vol 39 (6) ◽  
pp. 1169-1175 ◽  
Author(s):  
Shigeo Takumi
Keyword(s):  
Transposable Elements ◽  
Cell Lines ◽  
Hexaploid Wheat ◽  
Microprojectile Bombardment ◽  
35S Promoter ◽  
Hygromycin B ◽  
Transposase Gene ◽  
Cultured Cell ◽  
Ac Element ◽  
Ac Transposase

To investigate the activation and transposition of maize transposable elements in wheat cultured cells, plasmid DNAs containing the maize Ac/Ds elements located between the CaMV 35S promoter and a hygromycin B resistance gene (hph) were introduced into two wheat (Triticum aestivum and Triticum monococcum) cultured cell lines by microprojectile bombardment. In the first experiment, hph was activated by excision of the Ac element, which encodes transposase, in the two wheat cell lines. In the second experiment, the Ds element was excised by a stabilized Ac element, lacking inverted repeats of the Ac element and located on another plasmid, and therefore leading to activation of hph. After selection of bombarded cells by hygromycin B, many resistant calli were recovered in both wheat cell lines. The integration of hph and the Ac transposase gene was confirmed by PCR and genomic Southern analysis. The stable expression of hph and the transposase gene was also assessed by Northern blot and reverse transcriptase PCR analysis, respectively. Moreover, characteristic sequence alterations were found at Ac/Ds excision sites. These findings indicate that the maize Ac/Ds transposable elements are activated and excised by expression of the Ac transposase gene in both diploid and hexaploid wheat cells. Key words : transposable elements, excision site, transgenic wheat callus, particle bombardment, Ac/Ds.

scholarly journals Regulation of Activator/Dissociation Transposition by Replication and DNA Methylation

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1723-1733
Author(s):  
Francesca Ros ◽  
Reinhard Kunze
Keyword(s):  
Dna Methylation ◽  
Transposable Elements ◽  
Strong Evidence ◽  
Complementary Strand ◽  
Regulatory Effect ◽  
Ds Excision ◽  
Dna Strand ◽  
Petunia Protoplasts ◽  
Ac Transposase ◽  
High Binding Affinity

Abstract In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids (“chromatid selectivity”). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.

Trans-activation of a maize Ds transposable element in transgenic wheat plants expressing the Ac transposase gene

1999 ◽  
Vol 98 (6-7) ◽  
pp. 947-953 ◽  
Author(s):  
S. Takumi ◽  
K. Murai ◽  
N. Mori ◽  
C. Nakamura
Keyword(s):  
Transposable Element ◽  
Transgenic Wheat ◽  
Transposase Gene ◽  
Ac Transposase

Repression of the Ac-transposase gene promoter by Ac transposase

1996 ◽  
Vol 9 (6) ◽  
pp. 911-917 ◽  
Author(s):  
Marcelo Fridlender ◽  
Kate Harrison ◽  
Jonathan D.G. Jones ◽  
Avraham A. Levy
Keyword(s):  
Gene Promoter ◽  
Transposase Gene ◽  
Ac Transposase

scholarly journals AGROBACTER1UM-MED1ATED TRANSFORMATION OF APPLE

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 661e-661
Author(s):  
Rudaina Alrefai ◽  
Schuyler S. Korban ◽  
Leslie L. Domier
Keyword(s):  
Binary Vector ◽  
Gus Activity ◽  
Hygromycin Resistance ◽  
Gus Gene ◽  
Regeneration Medium ◽  
Helper Plasmid ◽  
Integrative Transformation ◽  
Transgenic Lines ◽  
Callus Lines

Two strains of Agrobacterium tumefaciens carrying a disarmed Ti-binary vector, pZA-7, were used as vectors for transformation of apple leaf segments, EHA101:pZA-7 carries a helper plasmid derived from pTiB0542, and C58Z707:pZA-7 carries a helper plasmid derived from pTiC58. The binary vector provides two selection markers, neomycin phosphotransferase (nptII) and hygromycin resistance genes, and a screening marker, β-glucuronidase (GUS) gene. Preliminary experiments were conducted to determine the effect of different concentrations of kanamycin, carbenicillin, and cefotaxime on regeneration of apple leaf sections and inhibition of A. tumefaciens strains. In vitro-derived leaf sections of `Royal Gala' apple were grown on a regeneration medium containing thidiazuron and NAA; these were then dipped into a suspension culture of A. tumefaciens and transferred to a fresh regeneration medium. Callus lines exhibiting kanamycin and hygromycin resistance were obtained mostly with agrobacterium strain EHA101:pZA-7. Expression of GUS activity was also determined in putative transformed calli. Southern blot analysis was used for confirming integrative transformation in transgenic lines.

Stably Transformed Callus Lines from Microprojectile Bombardment of Cell Suspension Cultures of Wheat

1991 ◽  
Vol 9 (8) ◽  
pp. 743-747 ◽  
Author(s):  
Vimla Vasil ◽  
Sherri M. Brown ◽  
Diane Re ◽  
Michael E. Fromm ◽  
Indra K. Vasil
Keyword(s):  
Cell Suspension ◽  
Microprojectile Bombardment ◽  
Cell Suspension Cultures ◽  
Suspension Cultures ◽  
Callus Lines

A 200-bp constructed inducible PR-1a promoter fusion to the Ac transposase gene drives higher transposition of a Ds element than the native PR-1a promoter fusion drives

Plant Science ◽  
1997 ◽  
Vol 130 (1) ◽  
pp. 73-86 ◽  
Author(s):  
Yuh-Chyang Charng ◽  
Chia Ma ◽  
Jenn Tu ◽  
Tsong-Teh Kuo
Keyword(s):  
Transposase Gene ◽  
Promoter Fusion ◽  
Ds Element ◽  
Ac Transposase ◽  
Gene Drives

Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants

1993 ◽  
Vol 239 (3) ◽  
pp. 354-360 ◽  
Author(s):  
Ko Shimamoto ◽  
Chikara Miyazaki ◽  
Hisako Hashimoto ◽  
Takeshi Izawa ◽  
Kimiko Itoh ◽  
...  
Keyword(s):  
Transposable Element ◽  
Transgenic Rice ◽  
Transposase Gene ◽  
Rice Plants ◽  
Stable Integration ◽  
Transgenic Rice Plants ◽  
Ac Transposase ◽  
Maize Transposable Element

285 TRANSCRIPT LEVEL OF Hsp70 AND IGF2R GENES IN BOVINE INDIVIDUAL BLASTOCYSTS DERIVED FROM OOCYTES MATURED WITH FBS OR FATTY ACID-FREE BSA

2007 ◽  
Vol 19 (1) ◽  
pp. 258
Author(s):  
E. Warzych ◽  
E. Pers ◽  
A. Buszka ◽  
T. Strabel ◽  
D. Lechniak
Keyword(s):  
Fatty Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Embryo Quality ◽  
Rna Extraction ◽  
Transcript Level ◽  
Large Variability ◽  
Protein Supplements ◽  
Relative Standard

The efficiency of in vitro embryo production in cattle varies between 30 and 40% of blastocysts derived from oocytes matured in vitro. Despite a rigorous selection, some embryos at blastocyst stage displaying normal morphology are not competent to develop after hatching (Maddox-Hyttel et al. 2003 Reproduction (Suppl. 61), 103–116). Therefore, a lot of attention has been focused on embryo quality. Supplements to culture media are one of the factors significantly contributing to this phenomenon. Serum (FBS) and albumin (fatty acid-free BSA, fafBSA) are widely used protein supplements; however, their effect on embryo quality is still variable (Rizos et al. 2003 Biol. Reprod. 68, 236–243). The aim of the present study was to investigate whether good-quality blastocysts (hatched or expanded) derived from oocytes matured in media supplemented with FBS or fafBSA differ in transcript level of 2 genes: heat shock protein (Hsp70) and receptor for insulin-like 2 factor (IGF2R). Bovine Day 8 blastocysts were produced in vitro from oocytes aspirated from slaughterhouse ovaries after maturation in TCM-199 medium supplemented with 10% FBS or 6% fafBSA, as previously described (Makarevich and Markkula 2002 Biol. Reprod. 66, 386–392). On Day 8 post-insemination (pi), good-morphology blastocysts were allocated into 3 groups: (1) hatched, (2) expanded of excellent quality, and (3) expanded of good quality, and individually frozen in liquid nitrogen. Each embryo was processed individually through RNA extraction and cDNA synthesis. Transcript quantitation protocol included: real-time PCR with SYBR Green I, β-actin gene as an internal standard, and relative standard curve method. Data analysis was performed by 2-way ANOVA. In each reaction, an equivalent of 0.125 embryo (2.5 �L of cDNA) was used, and 43 blastocysts were analyzed. All analyzed embryos were positive for the Hsp70 transcript, whereas IGF2R mRNA was detected in only 58% of blastocysts regardless of the maturation medium. A large variation in relative abundance (RA) was observed among individual embryos: coefficients of variation were 114.5 and 323.6% for IGF2R and Hsp70, respectively. Due to the distribution of Hsp70 RA, log transformation was performed. Real-time PCR data revealed a maximum 100-fold variation for the reference gene. Hatched blastocysts were characterized by a significantly lower RA for both analyzed genes. The 2 classes of expanded blastocysts did not differ in transcript level. With regard to protein supplements, only the RA for Hsp70 gene was significantly affected. This transcript was more abundant in embryos derived from fafBSA-supplemented IVM medium. The present results confirmed previously the described phenomenon concerning a large variability in mRNA content in single pre-implantation embryos. Moreover, because embryos able to hatch significantly differed in RA from their expanded counterparts, it is possible to relate embryo quality to transcript level.

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