Hartung et al. 10.1073/pnas.0705998104.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Table 1
SI Table 2
SI Table 3




SI Figure 5

Fig. 5. mRNA expression of the respective genes interrupted by T-DNA insertions. RT-PCR of the mutant lines. The location of the primer binding sites used for RT-PCR are given, primer sequences are shown in SI Table 1. None of the mutants exhibited expression spanning over the insertion locus, whereas in front of and behind the insertion sites comparable amounts of cDNA, as in the Col-0 control, were detected (lanes 1/1R, 3/3R, 4/4R, 6/6R, 7/7R, and 8/8R). The homozygous RecQ mutants were fully fertile and did not differ in their visual phenotype from wild-type Col-0 plants, whereas the TOP3a mutant was lethal in its homozygous state.





Table 1. Primers used for RT-PCR analysis of the T-DNA insertion lines

Primer

Locus

Sequence 5´- to 3´-

Tm

, °C
 

AtRECQ4A

  

1

 

ATG AGT AGG AGT CAC CTC C

53

1R

 

TAG ATT CGA ACT GCT CAG CTG

55

2

 

GTC CTG ATC GTG TTG GAC AG

57

2R

 

GAA TAA GAG ACA CAA GTG GAG

55

3

 

GCC TGA TGT CCG CTT TGT TA

55

3R

 

CCA AGG CCA TTG ATT TCA AG

53

 

AtRECQ4B

  

4

 

CGA TGG AAC ACT GCC CTT C

55

4R

 

CAT GAG TTG GAG AAT CTG TC

53

5R

 

CAA CAT CTT CTT TCA CGC TG

53

6

 

CGT GCC TTT GTA CAG AAA C

53

6R

 

GAG GTG CAA AGT GTC TTG TC

55

 

AtTOP3A

  

7

 

GAT ATT AAG AAG ACA TTG GAG G

55

7R

 

CAG CCT CAG CAA ACA ATT G

51

8

 

CAT TAC CTA GCA TGT GTT TC

51

8R

 

TTC CTG AGT GCC ATA TCT G

51

9

 

ACG AAT TGT CCC TCA CGG G

55

9R

 

GCT CTC CAG TTG CAG AGA C

55

LB

GABI T-DNA

GAC CAT CAT ACT CAT TGC TG

55

LB1

SALK T-DNA

TCG GAA CCA CCA TCA AAC AG

53

The oligonucleotides used as primers for the RT-PCR possess a similar melting temperature (Tm) that has been calculated using the formula: [2*(AT) + 4*(GC) - 5].





Table 2. Fresh weight of MMS- and cis-platin-treated plants

Plant line

MMS

cis

-platin

60 ppm

100 ppm

5 µm

10 µm

FW

SD

FW

SD

FW

SD

FW

SD

Col-0

63.1

7.7

30.9

7.9

75.6

12.9

24.9

8.0

recq4A-4

39.8

4.9

9.4

2.5

15.4

3.7

9.9

2.9

recq4B-1

63.2

6.6

26.4

11.3

75.9

12.6

32.0

10.5

recq4B-2

60.5

5.0

25.2

10.0

72.8

8.4

24.1

10.5

recq4A-4/4B-2

41.6

10.6

8.3

1.9

16.9

4.4

8.2

1.9

Each experiment was performed at least four times; the fresh-weight percentage given results from the mean of these experiments. For each fresh weight ten plantlets were treated with MMS or cis-platin and weighted together to randomize individual plant growth. The weight then was compared with the weight of ten untreated plantlets from the same line. The significantly reduced fresh-weight data of recq4A-4 and the double mutant are shown in bold. SD, standard deviation; FW, fresh weight.





Table 3. HR capacity of the T-DNA insertion lines in 651 and IC9C background, respectively

651 background

GM

BLE

mIF

spp

SD

spp

SD

fold

SD

 

 

 

 

 

 

 

Control

1.5

0.5

53.6

12.6

36.3

8.0

recq4a-4

4.2

0.9

26.0

7.1

6.3

1.4

recq4b-1

0.7

0.2

3.3

0.8

5.0

1.6

recq4b-2

0.3

0.1

4.5

2.0

14.6

4.8

recq4a/4b

1.0

0.4

7.7

3.3

8.6

2.8

IC9C background

GM

BLE

mIF

spp

SD

spp

SD

fold

SD

 

 

 

 

 

 

 

Control

0.4

0.1

6.0

2.1

15.6

5.6

recq4A-4

3.0

0.5

8.3

2.5

2.8

0.9

recq4B-1

0.18

0.08

1.9

0.9

10.8

4.0

recq4B-2

0.1

0.04

1.1

0.6

10.6

5.8

recq4A-4/4B-2

0.8

0.1

3.0

1.0

4.0

1.6

Each experiment was performed at least four times and the number given for sectors per plant (spp) is the mean of these experiments. The mean induction factor (mIF) results from averaging the IF of each independent experiment. Per experiment, an individual IF was defined as the relation between the mean HR events per plant of the sample treated with bleomycin (BLE) and the mean HR events per plant of the untreated sample. SD, standard deviation.