Genetic mapping of the mouse Rab7 gene and pseudogene and of the
human RAB7 homolog
Laura De Gregorio,
Tommaso A. Dragani,
Carmelo B. Bruni,
Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘‘L. Califano’’ and Centro di Endocrinologia ed Oncologia Sperimentale ‘‘G. Salvatore’’
del Consiglio Nazionale delle Ricerche, Universita` degli Studi di Napoli ‘‘Federico II’’, Via S. Pansini 5, 80131, Napoli, Italy
Division of Experimental Oncology A, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milano, Italy
Received: 27 October 1997 / Accepted: 1 January 1998
Abstract. Rab proteins are small GTP-ases localized to distinct
membrane compartments in eukaryotic cells and regulating spe-
cific steps of intracellular vesicular membrane traffic. The Rab7
protein is localized to the late endosomal compartment and con-
trols late steps of endocytosis. We have isolated, by library screen-
ing, the 5Ј region, including the promoter, of the mouse Rab7 gene
and a Rab7 pseudogene. We have mapped, by genetic linkage
analysis, the mouse Rab7 gene on Chromosome (Chr) 6 and the
Rab7-ps1 pseudogene on Chr 9, where the Rab7 gene has been
previously reported to map. By radiation hybrid mapping, we have
located the human RAB7 gene on Chr 3, in a region homologous
to the mouse Chr 6, where the Rab7 gene maps.
The Rab family of Ras-related GTP-ases appears to be essential for
the regulation of intracellular membrane traffic in mammalian
cells. Rab proteins are anchored to the cytoplasmic surface of
specific intracellular membrane compartments via a geranyl-
geranyl group that is added to the C-terminal cysteines post-
translationally, and that is important for their function (Magee and
Newman 1992). Each Rab protein regulates one (or more) specific
steps of intracellular membrane traffic in eukaryotic cells, prob-
ably by assembling the general docking/fusion machinery (Pfeffer
Rab7 cDNAs have been isolated from several mammalian spe-
cies (Bucci et al. 1988; Chavrier et al. 1990; Vitelli et al. 1995,
1996; Davies et al. 1997), and the Rab7 protein has been localized
to the late endosomal compartment (Chavrier et al. 1990). This
compartment is involved in some human diseases such as the
Chediak-Higashi syndrome (Burkhardt et al. 1993) or the vacuolar
degeneration of epithelial cells induced by Helicobacter pylori
(Papini et al. 1997). Recently the function of Rab7 has been in-
vestigated by several laboratories and compared with that of other
Rab proteins (Feng et al. 1995; Me´resse et al. 1995; Papini et al.
1997; Vitelli et al. 1997). All these studies demonstrated that while
Rab5 controls early steps of endocytosis (Bucci et al. 1992), Rab7
is involved in the regulation of the late endocytic pathway. More-
over, it has been demonstrated that Rab7 is essential for cellular
vacuolation induced by the Helicobacter pylori cytotoxin VacA
(Papini et al. 1997).
As an aid in the further characterization of Rab7, we decided
to investigate the chromosomal localization of the gene. We iso-
lated in mouse the 5Ј-region of the gene and a pseudogene. We
report here that, in contrast to what was previously published
(Barbosa et al. 1995), the mouse Rab7 gene is present on Chr 6, not
on Chr 9, where instead the Rab7 pseudogene maps. We have also
found that the human gene maps on Chr 3, in agreement with a
recent report (Davies et al. 1997), and more precisely at 3q21-q25,
a region homologous to mouse Chr 6.
Materials and methods
Isolation of genomic clones.
A partial Sau3AI adult Balb/c mouse liver
genomic library in EMBL3 was purchased from Clontech (Clontech Labo-
ratories, Palo Alto, Calif.). In total,2×10
recombinant clones were
screened by the in situ plaque hybridization technique (Maniatis et al.
1982) using as a probe either a HincII-BglII fragment (that comprises the
first part of the coding region) from mouse cDNA (Vitelli et al. 1995) or
a HindIII-HincII fragment (5Ј untranslated region) from the rat cDNA
(Bucci et al. 1988). These probes were uniformly labeled by random prim-
ing (Feinberg and Vogelstein 1983) at a specific activity of3×10
g. The filters were hybridized and washed under stringent conditions as
described (Church and Gilbert 1984). Clones which gave a positive signal
on duplicate filters were purified through three consecutive cycles, and
then phage DNA was prepared (Maniatis et al. 1982).
Restriction maps and DNA sequence.
Restriction maps were deter-
mined by single and double digestion on the entire phage DNA or on DNA
fragments subcloned in the vector pGem4Z. DNA sequencing was per-
formed according to the dideoxy chain terminating method.
Primer extension assay.
Primer extension analysis was performed es-
sentially as previously described (Stern et al. 1988). Briefly, oligonucleo-
tides were diluted at a concentration of 2 ng/l and combined 1:1 with
hybrid buffer (250 m
K-HEPES, 500 m
KCl, pH 7.0). Total RNA
(10–20 g) from mouse liver or NIH3T3 cells was added, and the samples
were incubated for 1 min at 85°C and then allowed to cool down to 45°C.
The extension reaction was performed exactly as described (Stern et al.
1988). The reaction samples were precipitated with ethanol and resus-
pended in loading buffer (80% formamide, 0.03% bromophenol blue, and
xylene cyanol dyes) and analyzed on an 8% denaturing polyacrylamide gel.
The primers used in the elongation reaction were Rab7-4 (5ЈAGGTTC-
GACTCCGGCTCCAGCAGGCTCCTC-3Ј) and Rab7-7 (5Ј-
Mouse genetic mapping.
Details on the interspecific testcross popula-
tion of 106 male (C3H/He × M. spretus) × C57BL/6J, designed HSB, have
already been reported (Manenti et al. 1994). The marker loci D9Mit2 and
D9Mit8 were identified by PCR-based analysis of simple sequence length
polymorphism (SSLP; Dietrich et al. 1994). Ms6-4 and Rpl32 were iden-
tified by Southern blot hybridization of respectively HinfI and TaqI-
digested DNAs with probes 33.6 (Jeffreys et al. 1987) and rpL32 (Meyuhas
The nucleotide sequence data reported in this paper have been submitted to
the EMBL/GenBank Data Libraries and have been assigned the accession
numbers Y13361 (Rab7 pseudogene) and Y13362 (5Ј region of the Rab7
Correspondence to: C.B. Bruni
Mammalian Genome 9, 448–452 (1998).
© Springer-Verlag New York Inc. 1998