Cloning and characterization of murine Fanconi anemia group A gene:
Fanca protein is expressed in lymphoid tissues, testis, and ovary
Henri J. van de Vrugt,
Ngan Ching Cheng,
Yne de Vries,
Martin A. Rooimans,
Jan de Groot,
Rik J. Scheper,
Maureen E. Hoatlin,
Department of Clinical Genetics and Human Genetics, Free University Medical Centre, Amsterdam, The Netherlands
Department of Pathology, Free University Medical Centre, Van de Boechorststraat 7, NL-1081 BT, Amsterdam, The Netherlands
Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201, USA
Received: 5 September 1999 / Accepted: 3 December 1999
Abstract. Fanconi anemia (FA) is an autosomal recessive disor-
der in humans characterized by bone marrow failure, cancer pre-
disposition, and cellular hypersensitivity to cross-linking agents
such as mitomycin C and diepoxybutane. FA genes display a care-
taker function essential for maintenance of genomic integrity. We
have cloned the murine homolog of FANCA, the gene mutated in
the major FA complementation group (FA-A). The full-length
mouse Fanca cDNA consists of 4503 bp and encodes a protein
with a predicted molecular weight of 161 kDa. The deduced Fanca
mouse protein shares 81% amino acid sequence similarity and
66% identity with the human protein. The nuclear localization
signal and partial leucine zipper consensus motifs found in the
human FANCA protein were also present in the murine homolog.
In spite of the species difference, the murine Fanca cDNA was
capable of correcting the cross-linker sensitive phenotype of hu-
man FA-A cells, suggesting functional conservation. Based on
Northern as well as Western blots, Fanca was mainly expressed in
lymphoid tissues, testis, and ovary. This expression pattern corre-
lates with some of the clinical symptoms observed in FA patients.
The availability of the murine Fanca cDNA now allows the gene
to be studied in experimental mouse models.
Fanconi anemia (FA) is an autosomal recessive chromosomal in-
stability syndrome characterized by a diversity of clinical symp-
toms including skeletal abnormalities, progressive bone marrow
failure, and a marked predisposition to cancer. FA cells show
spontaneous chromosomal instability and an elevated sensitivity to
cross-linking agents such as diepoxybutane (DEB) and mitomycin
C (MMC; Auerbach et al. 1998). Somatic cell fusion studies have
indicated at least eight complementation groups, suggesting the
involvement of this many genes (Joenje et al. 1997). Presently
three FA genes have been cloned, FANCC (Strathdee et al. 1992),
FANCA (Lo Ten Foe et al. 1996; The FA/Breast cancer consor-
tium 1996), and FANCG (de Winter et al. 1998), and two FA
genes have been mapped, FANCD to 3p22-26 (Whitney et al.
1995) and FANCE to 6p21-22 (Waisfisz et al. 1999b). For the
FANCC gene murine, rat and cow homologs have been reported
(Wevrick et al. 1993; Ching et al. 1997). Identified FA genes do
not show significant homology to each other, and no sequence
motifs have been found that could provide a clue to their function.
FANCA is the gene affected in the predominant FA comple-
mentation group, representing over 60% of the FA patients world-
wide (Buchwald 1995; Joenje 1996). Recently it was reported that
the N-terminal part of the FANCA protein directly interacts with
the FANCG protein (Garcia-Higuera et al. 1999; Waisfisz et al.
1999a). Further studies are required to elucidate a currently con-
troversial interaction between FANCA and FANCC. Current data
suggest that the FANCA protein is phosphorylated and subse-
quently transported into the nucleus. Nuclear localization seems to
be essential for complementation of cross-linker sensitivity
(Kupfer et al. 1997; Kruyt and Youssoufian 1998; Yamashita et al.
1998; Na¨f et al. 1998). Here we report the cloning and partial
characterization of the murine homolog of the FANCA cDNA.
Materials and methods
The 5Ј 1.5-kb of the human FANCA cDNA was
labeled (Stratagene Random primer labeling) and hybridized to Hybond
N+ filters of a murine teratocarcinoma cDNA library (Stratagene) at 53°C
in hybridization buffer (0.5
sodium phosphate, pH 6.8 (NaPi), 7% SDS,
ethylenediamine tetraacetic acid (EDTA), 0.1 mg/ml salmon sperm
DNA) for 16 h. Filters were washed at 53°C in 40 m
NaPi, 1% SDS. After
rescreening, murine Fanca clone 1 was identified. Clone 1 was used to
screen two ZAP ®II con A-stimulated FVB murine lymphoblast cDNA
libraries, kindly provided by J. Allen, at 65°C for 16 h. Filters were washed
as described. After single plaque purification, the cDNAs were obtained in
) by in vivo excision, following manufacturer’s
Nucleotide sequencing and analysis.
After subcloning of cDNA frag-
ments in pSK
, sequencing was performed by cycle sequencing as de-
scribed previously (Lo Ten Foe et al. 1996; de Winter et al. 1998). Nucleo-
tide readings of larger restriction fragments were completed by specifically
designed Cy 5.5 labeled primers (Isogen). Sequence data were compiled
and analyzed by HIBIO DNASIS™ for Windows®, Version 2. Protein
sequences were aligned by Clustal W.
Construction of full-length cDNA.
To construct the full-length murine
cDNA, a fragment from nucleotide (nt) 2393–3092, including exon 27, was
obtained by PCR [2.5 units TAQ polymerase, 3 m
HCl pH 8.4, 50 m
KCl, 0.3 m
dNTPs (Gibco BRL)] with plaque-
purified lambda phage clone 5 as template [primer p27+: 5Ј TCAT-
GTCCTGGGCTTGGCTGCTCTTGC 3Ј and primer p27−: 5Ј TCCTTGT-
TTCCTGTGCGGCCACCA 3Ј (Life Technologies, Rockville, MD, USA)].
The PCR product was digested with BamHI (nt 2457) and SacI (nt 2794)
and cloned directionally in pSK
. Double-strand sequencing of the PCR
fragment was performed. The full-length cDNA was constructed with the
following endonucleases (Gibco-BRL) and partial cDNA clones. Clone 1
GenBank/EMBL database accession numbers. The nucleotide sequence
data reported in this paper have been submitted to Genbank and have been
assigned the accession number AF208116 for the full-length mouse cDNA,
and numbers AF208117, AF208118, AF208119, AF208120, AF208121,
AF208122, AF208123 for the isolated cDNA fragments.
Correspondence to: F. Arwert; e-mail: email@example.com
Mammalian Genome 11, 326–331 (2000).
© Springer-Verlag New York Inc. 2000