Download Supplemental material Material and methods Murine strains

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’- GGAACTGACCATATCTGGTTGTCTG3’) 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
CGAGCCTCAATCTTCAAAGTTCAC-3’)
and
were
14R
12F
(5’(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 membraneassociated 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 5:221232.
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 AFG3L2deficient 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
respectively.
par/par
and Afg3l2
Emv66/Emv66
,
wild type
par/par
A
B
C
D
E
F
Figure 9
Emv66/Emv66
C
par/par
C
Emv66/Emv66
C
par/par
C
C
B
C
Emv66/Emv66
A
kD
39 kD
47.5
32
20 kD
25
porin
16.5
P-
P+
brain
liver
Figure 10