Supplemental material

Material and methods

Murine strains

Procedures involving animals and their care were conducted in conformity

with guidelines of the Institutional Animal Care and Use Committee, San Raffaele

Hospital, Milan, Italy, in compliance with national (D.L. No. 116, G.U. Suppl. 40,

Feb. 18, 1992, Circolare No. 8, G.U., 14 Lug. 1994) and international laws and

policies (EEC Council Directive 86/609, OJ L 358, 1 DEC.12, 1987; NIH Guide for

the Care and use of Laboratory Animals, U.S. National Research Council, 1996).

Mapping of the par locus

Mapping of the par locus was achieved by genotyping a set of 50 par/par mice

from an inter-subspecific F2 progeny. We localized the par locus on mouse

chromosome 18 between markers D18Mit140 and D18Mit141. High resolution

mapping of the par locus was achieved through molecular genotyping of 1150

informative haplotypes from two inter-subspecific F2 progenies (data not shown). We

mapped the par locus within a 630 kb genetic interval delineated by D18Par547, a

microsatellite marker inside of exon 1 of Cidea, the locus for cell death-inducing

DNA fragmentation factor a and by microsatellite 27.MMHAP68FLA6.seq at the

telomeric end. Matching our data with the SNPs sequence databases we confirmed

that indeed the chromosomal segment in which the par mutation occurred derived

from the C57BL/6 clade of strains.

Mapping of the Emv66 locus

The MEV/2TyJ linkage testing stock contains over 30 stable MuLV proviral

integrations fixed in its genome (Taylor and Frankel, 1993). Therefore, we created a

congenic strain on the FVB/NJ background (FVB.MEV-Emv66) by 10 consecutive

backcrosses to isolate the Emv66 insertion mutation. The onset and progression of the

FVB.MEV-Emv66/Emv66 mutant phenotype remained unchanged from that observed

on the MEV parental background. Southern blots were used to follow the 6.8 kb

Emv66-specific PvuII fragment and to select against the other MEV-derived Emv

proviruses (data not shown). The absence of any other MuLV ecotropic proviruses in

the FVB strain background allowed us to clone the Emv66 insertion site by inverse

PCR using primers in the proviral LTR.

The genotype of Afg3l2+/+, Afg3l2+/Emv66 and Afg3l2Emv66/Emv66 mice could be

unambiguously determined by amplifying the wild-type allele using primers R25F

and R25R, and the Emv66 mutant allele using primers LTRF and R25R. The Emv66

proviral insertion is not detected in the parental MEV/2TyJ strain or in the

AKXD14/TyJ or C58/J strains (data not shown), which were originally used to

develop the MEV stocks (Taylor and Rowe, 1989; Taylor and Frankel, 1993).

Southern hybridization, Inverse PCR and genotyping

Genomic DNA was prepared from mouse spleens for Southern blot analysis

essentially as previously described (Taylor and Frankel, 1993). A 518 bp PCR

fragment from the gp70 gene of the AKV provirus (F-5’-

TGTATGTTGGCCCTCCACGG-3’ and R-5’-TGGGTCATGTCCAGAGACGT-3’)

was used as a probe to detect an Emv66-specific 6.8 kb PvuII fragment. Inverse PCR

to identify the 3’ flanking sequence of the MuLV insertion was performed essentially

as described (Cox et al., 1993). Briefly, 5 µg of genomic DNA from Afg3l2Emv66/Emv66

mutant mouse was digested with NlaIII, extracted with phenol/chloroform, ethanol

precipitated and ligated overnight. The self-ligated circular DNA was amplified with

outwardly facing primers within the viral long terminal repeats U3-LTRF (5’-

CCAGAAACTGTCTCAAGGTTCC-3’) and U3 LTRR (5’-

GTGCTTGACCACAGATATCCTG-3’). PCR products were separated on 1%

Agarose/TBE gels and the 571 bp mutant-specific band was excised and purified

using Qiaquick columns (Qiagen) for Big Dye Terminator cycle sequencing on an

Applied Biosystems 3700.

For routine typing, genomic DNA from both strains was isolated from tail tips as

previously described (Taylor and Frankel, 1993). Genotyping for the Afg3l2Emv66/Emv66

mutants was performed by PCR using primers U3-LTRF and R25R (5’-

TGGATTCTGCACATCTCTTAACCC-3’) to PCR amplify a 317 bp product

corresponding to the 3’ junction of the proviral insertion. The wild-type allele (258

bp) was amplified using primers R25F (5’- GGAACTGACCATATCTGGTTGTCTG-

3’) and R25R flanking the MuLV insertion within intron 14 of the Afg3l2 gene.

Genotyping for the Afg3l2par/par mutants was performed using oligonucleotide primers

exon10-F (5’-CTGGTTCAATGGTCTTTAGGG-3’) and exon10R (5’-

CCCACAGCATCAATCTCATCA-3’) that amplify a 268 bp PCR product containing

the mutation. Heterozygous mice were distinguished from wt by DHPLC analysis

(Transgenomics Limited, Crewe, UK).

RT-PCR

Total RNA was prepared from brain, spinal cord, kidney, liver and heart of 2-3

week old Afg3l2Emv66/Emv66, Afg3l2+/Emv66 and Afg3l2+/+mice by Trizol method

(Invitrogen) and reverse transcribed using random decamers and oligo-dT primers in

the RETROscript first strand synthesis kit (Ambion) according to the manufacturer’s

instructions.

Primers used to detect Afg3l2 cDNA were 12F (5’-

CGAGCCTCAATCTTCAAAGTTCAC-3’) and 14R (5’-

AAGCCACCGTCTTCTTCTCCTCAG-3’) amplifying a 271 bp product; 13F (5’-

CGAACAAGCGATTGAGCGAG-3’) and 15R (5’-

TCTGGGTAACCTTCCTCAAGTCG-3’) amplifying a 332 bp product; and 13F and

MLVR (5’-GGTGGTCAGTAGGACGGTGTA-3’) amplifying a 336 bp product from

the Emv66 mutant-specific spliced mRNA.

Mitochondrial protein synthesis

Isolated Mitochondria were resuspended in 50 µl translation buffer (20 mM

TrisHCl pH 7.2, 0.6 M sorbitol, 150 mM KCl, 15 mM KH2PO4, 12.5 nM MgSO4, 4

mM ATP, 0.5 mM GTP, 5 mM α-ketoglutarate, 5 mM Phosphoenol-pyruvate, 3

mg/ml fatty acid-free BSA, 0.012 mg/ml of all amino acids except methionine) in the

presence of pyruvate kinase (2.4 U/ml). To achieve maximal energization,

mitochondria were incubated for 2 min at 30°C. Mitochondrial translation products

were labeled by adding 5 µCi [35S] methionine (1069 Ci/mmol, ICN) and subsequent

incubated at 30°C for 30 min. The incorporation of [35S] methionine was stopped by

the addition of 20 mM cold methionine and 50 µg/ml puromycin (stock solution: 1

mg/ml in H20). Mitochondria were re-isolated by centrifugation for 12 min at 9000g,

washed with 250 µl 0.6 M sorbitol, 1 mM EDTA and 5 mM methionine. Translation

products were analyzed by 15% SDS-PAGE and autoradiography (Langer et al.,

1995).

References

Cox GA, Cole NM, Matsumura K, Phelps SF, Hauschka SD, Campbell KP, Faulkner

JA, Chamberlain JS (1993) Overexpression of dystrophin in transgenic mdx

mice eliminates dystrophic symptoms without toxicity. Nature 364:725-729.

Langer T, Pajic A, Wagner I, Neupert W (1995) Proteolytic breakdown of membrane-

associated polypeptides in mitochondria of Saccharomyces cerevisiae.

Methods Enzymol 260:495-503.

Taylor BA, Rowe L (1989) A mouse linkage testing stock possessing multiple copies

of the endogenous ecotropic murine leukemia virus genome. Genomics 5:221-

232.

Taylor BA, Frankel WN (1993) A new strain congenic for the Mtv-7/Mls-1 locus of

mouse chromosome 1. Immunogenetics 38:235-237.

Figure legends

Supplemental Figure 1 Reduction of myelinated fibers in spinal cord.

Semithin sections of spinal cord of Afg3l2par/par mice and controls at P14. Relative to

controls, the number of myelinated axons in mutants is dramatically reduced in the

anterior funiculus (B), in the antero-lateral funiculus (D) and in the fasciculus gracilis

(F). Bar represents 20 µm.

Supplemental Figure 2 Protein synthesis is not impaired in Afg3l2-deficient

mitochondria.

(A) Mitochondria-encoded proteins were synthesized from control (C) and AFG3L2-

deficient brain mitochondria in the presence of [35S] methionine at 30° for 30 minutes.

Newly synthesized proteins were analyzed by SDS PAGE and autoradiography. A

specific blocker of mitochondrial protein synthesis (puromycin) was used as negative

control. P+ = puromycin added, P- = no puromycin. (B) Western blot analysis on

complex I revealed by anti-39 kD (nuclear encoded subunit) and anti-20 kD

(mitochondrial encoded subunit) antibodies. Anti-porin antibody was used to verify

equal loading.

Supplemental movies

Movie 1 and 2 show the severe phenotype of Afg3l2 par/par and Afg3l2 Emv66/Emv66,

respectively.

A B

C D

E F

Figure 9

wild type par/par

Figure 10

A

P- P+

47.5

32

25

16.5

kD

Emv66/Emv66

C C

39 kD

20 kD

liverbrain

porin

Emv66/Emv66

Emv66/Emv66

C C Cpar/par

par/par

C

B