Organization of the mouse microfibril-associated glycoprotein-2
Neal G. Copeland,
Debra J. Gilbert,
Nancy A. Jenkins,
J. Michael Shipley
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Barnes-Jewish Hospital at Washington University School of Medicine,
216 S. Kingshighway Blvd., St. Louis, Missouri 63110, USA
Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA
Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, USA
Received: 27 August 1999 / Accepted: 2 November 1999
Abstract. A 1.4-kb EST clone encoding mouse microfibril-
associated glycoprotein-2 (MAGP-2), identified by its similarity
with the reported human cDNA, was used to screen a mouse 129
genomic bacterial artificial chromosome (BAC) library. The
mouse gene contains 10 exons spanning 16 kb, located on the
distal region of Chromosome (Chr) 6. The exons range in size from
24 to 963 bp, with the ATG located in exon 2. The tenth and
largest exon contains 817 bp of 3Ј untranslated sequence, including
a B2 repetitive element. Northern analysis demonstrates abundant
expression of MAGP-2 mRNA in skeletal muscle, lung, and heart.
Sequence analysis of additional cDNA clones suggests that the two
mRNA forms of MAGP-2 in the mouse arise from alternative
polyadenylation site usage. The promoter does not contain an ob-
vious TATA box, and the sequence surrounding the start site does
not conform to the consensus for an initiator promoter element.
Additionally, the mouse promoter contains 22 copies of a CT
dinucleotide repeat sequence located ∼155 bp 5Ј to exon 1.
Microfibrils are a structural component of the extracellular matrix
of a number of tissues (Rosenbloom et al. 1993). In tissues such as
lung, ligaments, and arterial vessels, these microfibrils form an
organized network that is associated with elastin to give rise to
elastic fibers. In other tissues, such as the ciliary zonules of the
eye, periodontal ligament, skeletal muscle, and kidney, these mi-
crofibrils are free of elastin, yet otherwise appear morphologically
identical to those in elastic tissue (Cleary and Gibson 1983). Com-
positional differences between microfibrils, as they relate to their
functional diversity, are the subject of investigation in many labo-
Many of the structural components of microfibrils have been
identified. The major constituent of microfibrils are fibrillins 1 and
2, 350-kDa proteins organized in head-to-tail arrays (Sakai et al.
1986; Sakai et al. 1991; Zhang et al. 1994; Reinhardt et al. 1996).
Other microfibrillar proteins that have been identified include
MAGP-1 (Gibson et al. 1986) and MAGP-2 (Gibson et al. 1996).
Both have been localized to microfibrils by immunoelectron mi-
croscopy. The restricted pattern of expression of MAGP-2 sug-
gests that it is not a component of all microfibrils and that it
probably serves a function different from that of MAGP-1 within
In this report, we have characterized the structure of the mouse
MAGP-2 gene and compared it with that of the human gene
(Hatzinikolas and Gibson 1998). While the mouse and human
MAGP-2 proteins are similar in sequence, the promoters for the
two genes share little in common. The presence of two mRNA
species for MAGP-2 in the mouse raised the possibility that more
than one isoform of the protein might be synthesized. We have
characterized both mRNA species and determined that they do not
code for different variants of the protein.
Materials and methods
Characterization of an EST clone for mouse MAGP-2.
pressed sequence tag (EST) clone encoding mouse MAGP-2 (GenBank
accession #AA153960) was identified by its similarity with the human
MAGP-2 cDNA (Gibson et al. 1996) by use of the blastn program and was
sequenced in its entirety. The GenBank accession number for the complete
cDNA sequence is AF180805. Comparison of the mouse and human
MAGP-2 proteins was done with the GeneWorks program, version 2.5
(Oxford Molecular). The B2 repetitive element located in the 3Ј untrans-
lated region of the mouse cDNA was identified with the Repeat Masker
Cloning the mouse MAGP-2 gene.
The MAGP-2 EST insert was used
to screen a mouse 129 genomic BAC library (Genome Systems, St. Louis,
Mo.). A single clone was identified, digested with EcoRI, BamHI, or SacI,
and fragments were ligated into pBluescript SK(−). Exon-containing re-
combinants were identified by colony hybridization and characterized by
restriction mapping, PCR, and DNA sequencing. The gap between the SacI
and BamHI fragments was verified as 285 bp PCR and DNA sequencing.
In addition, ∼800 bp of the 5Ј flanking region of the gene was sequenced.
Interspecific mouse backcross mapping.
Interspecific backcross prog-
eny were generated by mating (C57BL/6J × M. spretus)F
C57BL/6J males as described (Copeland and Jenkins 1991). A total of 205
mice were used to map the Magp2 locus with a probe derived from
bp 1–1049 of the cDNA. A major fragment of 10.5 kb was detected in
HindIII-digested C57BL/6J DNA, and a major fragment of 7.2 kb was
detected in HindIII-digested M. spretus DNA. The presence or absence of
the 7.2-kb HindIII M. spretus-specific fragment was followed in backcross
mice. A description of the probes and restriction fragment length polymor-
phisms (RFLPs) for the loci linked to Magp2 including Atp6e, M6pr, and
Slc2a3 (formerly Glut3) has been reported previously (Puech et al. 1997).
Recombination distances were calculated with Map Manager, version
Northern blot analysis.
A mouse multiple-tissue Northern blot contain-
ing poly A-selected RNA was purchased from Clontech and hybridized
with a probe from 1–250 bp of the cDNA according to the manufacturer’s
Correspondence to: J.M. Shipley, e-mail: email@example.com
Mammalian Genome 11, 191–195 (2000).
© Springer-Verlag New York Inc. 2000