Analysis of the nad6 t-element

A

At5S-5

At5S-Mega.H

nad6

At5S-Mega.R

(-179)

(-17/-16)

(+40)

5S rRNA

5’ 3’

nad6 mRNA

nad6 pre-mRNA

nad6 t-element5S rRNA

Atnad6-6Atnad6-Endo.3’.R

Atnad6-Endo.3’.H

nad6 t-element

ma

rke

rmt

RN

A

250/242 bp

500/501/489 bp

404 bp

331 bp

190 bp

147 bp

111/110 bp

I

B

primersused for primer

namelocation of the primer relative to the 5‘-terminal nucleotide of 5S rRNA (+1)*/stop codon of nad6 (-1)** and in nc_001284

synthesis of cDNA first strand

Atnad6-6 +40** to +23** (76,602 to 76,619)

PCR amplification

At5S-Mega.H

-2* to +22* (361,181 to 361,158)

PCR amplification

Atnad6-Endo-3‘.R

+37** to +11** (76,605 to 76,631)

C

transcript ends identified in the PCR productsPCR product

size of PCR product

5‘ end of nad6 t-element relative to the stop codon (-1) and in nc_001284

3‘ end of 5S rRNA relative to 5‘ end (+1)

I 172 bp -15 (76,656) +118

D

list of mapped individual endsclone 5‘ end position in the mitochondrial

genome (nc_001284)5‘ end position in respect to the TAA codon (-1)

Non-encoded nucleotides

#28 76,675 -34 G

#3 76,657 -16 AA**

#1 76,656* -15*

#5 76,656* -15*

#8 76,656* -15*

#9 76,656* -15*

*A single adenosine at the ligation site could be either assigned to nad6 or 5S rRNA, so that the nad6 t-element either ends at position -16 or -15. If the 5S rRNA is assumed to end at position 361,062 as noted in the database entry (nc_001284) and as always found in the mapping experiments of the cox1 t-element, the adenosine is derived from 5S rRNA. Thus the 5’ end of the nad6 t-element is located at position -15.

** If the 5S rRNA 3’ end is located at position 361,062 (see above), there are three non-encoded As. Additionally, if the 5’ end of the nad6 t-element is located at position -15, four non-encoded As are present.

E

transcript ends identified in the PCR productsPCR product

size of PCR product

5‘ end of 5S rRNA relative database entry (+1)

3‘ end of nad6 t-element relative to the stop codon (-1) and in nc_001284

II 164/163/162 -2/-1/+1 +40 (76,602)

ma

rke

rmt

RN

A

250/242 bp

500/501/489 bp

404 bp

331 bp

190 bp

147 bp

111/110 bp

II

G

H

F

primersused for primer

namelocation of the primer relative to the 5‘-terminal nucleotide of 5S rRNA (+1)*/nad6 stop codon (-1)** and in nc_001284

synthesis of cDNA first strand

At5S-5 +118* to +100* (361,062 to 361,080)

PCR amplification

Atnad6-Endo.3‘.H

-15** to +19** (76,656 to 76,623)

PCR amplification

At5S-Mega.R

+107* to +82* (361,073 to 361,098)

list of mapped individual endsclone 3‘ end position in the mitochondrial

genome (nc_001284)3‘ end position in respect to the TAA codon (-1)

Non-encoded nucleotides

#16 76,615/76,614* +27/+28*

#11 76,602 +40 CC

#14 76,602 +40 CC

#13 76,602 +40 CCA

*A single nucleotide at the ligation site could be either assigned to nad6 or 5S rRNA. Thus the nad6 t-elements ends at position +27 or+28.

K

Supplementary Figure 29. Analysis of the nad6 t-element.

A modified CR-RT-PCR using endogenous 5S rRNA as adapter molecule was carried to determine the 5’ end (B-E) and

the 3’ end (F-K) of the t-element downstream of the nad6 transcript (A). The site of the endonucleolytic cleavage of the

pre-mRNA is marked by a pair of scissors. Primers indicated in tables (C) and (G), respectively, were used and the PCR

products were separated by agarose gel electrophoresis (B and F). The products (marked by an arrow and detailed in

tables (D) and (H)) were excised, cloned and analyzed by sequencing. The ends identified within the individual clones are

listed in tables (E) and (K), respectively.