Molecular cloning, gene expression, and identification of a splicing
variant of the mouse parkin gene
Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Received: 5 May 1999 / Accepted: 11 February 2000
Abstract. We have isolated mouse cDNA clones that are homolo-
gous to human Parkin gene, which was recently found to be re-
sponsible for the pathogenesis of autosomal recessive juvenile par-
kinsonism (AR-JP). One of these cDNA clones had the 1,392-bp
open reading frame encoding a protein of 464 amino acids with
presumed molecular weight of 51,615. The amino acid sequence of
mouse parkin protein exhibits 83.2% identity to human Parkin
protein, including the ubiquitin-like domain at the N-terminus
(identity ס 89.5%) and the RING finger-like domain at the C-
terminus (identity ס 90.6%). Two other clones had the 783-bp
open reading frame encoding a truncated protein of 261 amino
acids without RING finger-like domain. It was proved to be a
novel splicing variant by 3Ј-RACE method.
Northern blot analysis revealed that mouse parkin gene is ex-
pressed in various tissues including brain, heart, liver, skeletal
muscle, kidney, and testis. It is notable that mouse parkin gene
expression appears evident in 15th day mouse embryo and in-
creases toward the later stage of development. These mouse parkin
cDNA clones will be useful for elucidating the essential physi-
ological function of parkin protein in mammals.
Recently, we have identified a novel gene, named Parkin, as a
pathogenic gene of a familial Parkinson’s disease, autosomal re-
cessive juvenile parkinsonism (AR-JP) (Kitada et al. 1998). The
human Parkin gene encodes a protein of 465 amino acids (MW ס
51,652) with two distinct domains, including a ubiquitin-like do-
main at the N-terminus and a RING finger-like domain at the
C-terminus. Moreover, the Parkin gene consists of 12 exons and
appears to span over 1 Mb in size. Various mutations, including
large deletions and point mutations, have been found in AR-JP
patients of different ethnic origins (Kitada et al. 1998; Hattori et al.
1998a, 1998b; Abass et al. 1999).
A major transcript of 4.5 kb was detected in a wide variety of
human tissues including brain, skeletal muscle, and heart (Kitada
et al. 1998). However, it is not yet known how the AR-JP patients
suffered with selective degeneration of dopaminergic neurons in
the substantia nigra (Takahashi et al. 1994; Yamamura et al. 1994,
To aid the studies on physiological function of Parkin protein
and its gene regulation, we performed an isolation of mouse ho-
molog of human Parkin gene, providing initial characterization of
the mouse parkin gene and its expression.
Materials and methods
Isolation of mouse parkin cDNAs.
A gt 10 library (Clontech) con-
taining mouse skeletal muscle cDNA (BALB/c males, 8–12 weeks) was
screened with a
P-labeled probe prepared with the random primer exten-
sion kit (NEN™ Life Science Products) and the human skeletal muscle
cDNA clones including the open reading frame of Parkin gene as the
template. Recombinant phage (6 × 10
) was grown on a bacterial loan
(C600hfl) of a culture plate (220-mm dish) for 18 h at 37°C and then
replica plated onto two duplicate nylon filters (GeneScreen Plus; NEN™
Life Science Products). After fixation of the filters by baking for 5 min and
UV-crosslinking at 12 × 10
, prehybridization, hybridization, and
posthybridization were performed according to described procedures
(Sambrook et al. 1989). Seven positive clones were obtained from2×10
plaques (four culture plates) by a third screening.
Sequencing of mouse parkin cDNAs.
The cDNA inserts of positive
clones were amplified by vector-specific primers (F10 inner, 5Ј-AGC CTG
GTT AAG TCC AAG CTG-3Ј; R10 inner, 5Ј-GAA GGT CCC ATT TTT
CGT TTT C-3Ј). PCR amplification was carried out in 10-l reactions,
each of which contained 100 ng phage DNA as template, PCR buffer [50
Tris-HCl (pH 9.2 at 25°C), 14 m
, 1.75 m
of each dNTP, 0.5
of each primer, and 0.35 U of DNA polymerase
of Expand Long Template (Boehringer Mannheim). PCR conditions were
35 cycles at 94°C for 30 s, 50°C for 1 min, 68°C for 4 min. The amplified
fragments were sequenced by primer walking method. Cycle sequencing
was performed by using the vector-specific primers and several primers for
walking and a commercial kit (ABI PRISM labeling kits) according to the
manufacturer’s instruction manual (Perkin-Elmer) on the ABI model 377
DNA sequencer (Applied Biosystems).
Rapid amplification of cDNA ends (RACE).
cDNA library (Clontech) of mouse skeletal muscle (BALB/c males, age
9–11 weeks) was screened to clone 3Ј-ends of the three different types of
positive cDNA clones and to study whether they were from mature
mRNAs. Several PCR primers were designed from the partial sequences of
SKM2, SKM4, and SKM5, any of which started for 3Ј-side including
(designed from the common sequence of seven positive clones,
nt696–nt716), 5Ј-TTA AAT GTG GAG CAC ACC CAA-3Ј,P
peculiar sequence of SKM4, nt1085–nt1106), 5Ј-AAG CAC CGC ACC
TCA TGA CTT T-3Ј, and P
(from the peculiar sequence of SKM2),
5Ј-TGT CCG TAG GTG TCA CAC ACT-3Ј (Fig. 1). 3Ј-RACE was per-
formed with these primers and the (3Ј-)adaptor primer 1,5Ј-CCA TCC
TAA TAC GAC TCA CTA TAG GGC-3Ј. Furthermore, nested PCR was
performed to refine these PCR products with these primers and the
(3Ј-)nested adaptor primer 2,5Ј-ACT CAC TAT AGG GCT CGA GCG
GC-3Ј. PCR conditions were almost the same as those of the cDNA inserts
of positive clones.
Assembling and alignment of sequence data of seven
positive clones were carried out with Autoassembler DNA sequence as-
sembly software (Applied Biosystems). Homology search against known
Correspondence to: N. Shimizu; E-mail: firstname.lastname@example.org
The nucleotide sequence of cDNA for mouse parkin protein has been
deposited in DDBJ database (accession number AB019558).
© Springer-Verlag New York Inc. 2000Mammalian Genome 11, 417–421 (2000).