Molecular phylogeny of Fv1
Marilyn R. Lander,
Sisir K. Chattopadhyay,
Herbert C. Morse III
Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 7, Room 304, MSC
0760, Bethesda, Maryland 20892-0760, USA
Laboratoire Ge´nome et Populations, Universite´ de Montpellier II, Montpellier, France
Georgetown University Medical School, Washington, DC, USA
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
Received: 9 January 1998 / Accepted: 17 August 1998
Abstract. Alleles at the Fv1 gene of inbred mice confer resistance
to infection and spread of vertically or horizontally transmitted
murine leukemia viruses (MuLV). The nucleotide sequence of Fv1
bears similarity to the gag of a human endogenous retrovirus,
HERV-L, but is more closely related to the gag-coding sequence
of a newly described class of HERV-L-related mouse endogenous
retroviruses designated MuERV-L. Both observations suggest an
origin of Fv1 from endogenous gag sequences. The molecular
definition of Fv1 provided an opportunity to determine the phy-
logeny of the gene among wild mice and its relation to MuERV-L.
PCR primers, chosen to include most of the coding region of Fv1
for both the n and b alleles, were used to amplify sequences from
animals of the genus Mus, which were then sequenced. Closely
related products were obtained from almost all animals examined
that evolved after the separation from Rattus, in which the ho-
mologous gene was shown to be absent. A phylogenetic tree gen-
erated with Fv1 sequence data differs noticeably from that devel-
oped with sequence data from other genes. In addition, non-
synonymous changes were found to be present twice as frequently
as synonymous changes, a fact that departs from the standard
behavior of a structural gene. These observations suggest that the
Fv1 gene may have been subjected to possible horizontal transfers
as well as to positive Darwinian selection.
Fv1 is one of several genetic loci in inbred mice that restrict the
replication and spread of various murine leukemia viruses
(MuLV). Alleles at Fv1 control the sensitivity of cells to different
subgroups of MuLV (Lilly 1970; Pincus et al. 1971a, 1971b; Hart-
ley et al. 1977). These viruses are classified as N-tropic if they
replicate best in Fv1
cells or B-tropic if they replicate best in Fv1
cells. Alleles of Fv1 are expressed codominantly. A phenotypically
null allele, Fv1
, was identified in certain wild mice sensitive to all
retroviruses (Hartley and Rowe 1975; Lander and Chattopadhyay
1984; Kozak 1985). Two Fv1
cell lines are SC-1 cells from mice
trapped in Bouquet Canyon, Calif. (Hartley and Rowe 1975) and
dunni cells from Mus dunni (Lander and Chattopadhyay 1984).
Fv1 n and b alleles act on these viruses at a point after entry into
the cell and reverse transcription of the viral RNA genome but
before entry into the nucleus and integration into the host genome
(Jolicoeur and Baltimore 1976; Sveda and Soeiro 1976).
Recently, Best et al. (1996) reported the cloning of Fv1, which
comprises a small intronless open reading frame (ORF). They
suggested its origins lie within the gag region of an endogenous
retrovirus with sequence similarity to the HERV-L family of hu-
man endogenous retroviruses (Cordonnier et al. 1995). The murine
homolog of HERV-L, designated MuERV-L, has now been cloned
and found to contain an ORF in the gag and pol genes (Be´nit et al.
1997). The predicted Gag protein of MuERV-L shares 43% iden-
tity with the Fv1 ORF product and bears a number of structural
features. These studies suggest that MuERV-L may be the endog-
enous element that gave rise to the Fv1 gene of inbred mice.
Best et al. (1996) also demonstrated that Fv1-related sequences
were present in the genome of Mus dunni but not in the rat,
suggesting that the putative integration event for this retrovirus-
like sequence occurred early in Mus speciation. Here we present
analyses of Fv1-related sequences of the genus Mus.
Materials and methods
DNA samples for analyses of Fv1.
DNAs were prepared from estab-
lished cell lines or tissues of mice, rats, and humans collected by Chat-
topadhyay and Lander (NIH) and described previously. Other samples
were prepared from the collection of wild-derived mouse strains main-
tained in Montpellier or were purchased from The Jackson Laboratory (Bar
Harbor, Me.; Table 1).
PCR amplification and determination of Fv1 sequences.
actions were performed with the primers listed in Fig. 1A with 200 ng of
cellular DNA and 0.05 m
primers and Taq polymerase. Denaturing was
at 95°C for 5 min, followed by 35 cycles of 94°C for 1 min, 56°C for 45
s, and 72°C for 30 s with extension at 72°C for 7 min. Products were
separated on 0.7% agarose gels. Amplified PCR products were cloned with
the TA cloning kit (Invitrogen, San Diego, Calif.) and conditions recom-
mended by the manufacturer. Sequencing of cloned Fv1 products was
performed manually and by machine with standard techniques.
Southern blot analysis of Fv1 sequences.
Southern blot hybridization
analyses of cellular DNA were performed with two probes: cloned se-
quences between the P1 primers amplified from the C57BL/6 (B6) Fv1
gene, which was labeled with
P by random primer extension; and a
synthetic probe corresponding to the coding sequences for the unique car-
boxyterminal 22 amino acids of the b allele of Fv1. Two oligonucleotides
of 36 bases 5Ј and 51 bases 3Ј that overlapped for 14 bases were annealed
and filled in with Klenow (Life Technologies, Gaithersburg, Md.) under
conditions recommended by the supplier. The purified 66 bp fragment was
P. DNA separated on agarose gels was transferred to
nitrocellulose, baked for2hat80°C, and hybridized with probes at 65°C
in 3 × SSC and 0.2% each Ficoll, PVP, and BSA, 5 m
EDTA, 0.1% SDS,
50 g/ml sonicated herring sperm DNA, and 10% dextran sulfate for
18–20 h. Membranes were washed three times for 20 min at RT with 2 ×
SSC and once with 0.15 × SSC and 0.1% SDS at 65°C for 20 min.
Sequence alignment was done visually with a standard
text editor. Putative insertions and deletions were placed so as to minimize
Correspondence to: H.C. Morse III
Mammalian Genome 9, 1049–1055 (1998).
© Springer-Verlag New York Inc. 1998