(Received for publication, September 23, 1994)
From the
The megakaryocyte growth and development factor (MGDF) is a cytokine that regulates megakaryocyte development and is a ligand for the MPL receptor. In this study, we describe the genomic structure of the human MGDF gene. The MGDF gene was found to consist of seven exons and six introns spanning 8 kilobases. The protein is encoded by exons 3 through 7. The human MGDF gene has been mapped to chromosome 3q26.3. In addition to the previously described full-length cDNA, two cDNA variants were isolated from human fetal liver. Comparison of these two cDNA sequences with the genomic sequence indicates that they arise by differential splicing.
Megakaryocytes are the cellular source of platelets and arise
from a common bone marrow precursor cell population. The process of
megakaryopoiesis is controlled by a number of cytokines (1-4), among which the ligand for the cellular oncogene
c-Mpl plays a major role (5). The cDNA sequence of this ligand
has been recently reported by several groups (6-9). We
have named it MGDF, ()the megakaryocyte growth and
development factor. In this report, we describe the isolation,
analysis, and chromosomal localization of the MGDF gene.
Protection assays were performed using the RPA II Ribonuclease Protection Assay kit (Ambion Inc., Austin, TX). Briefly, 1 µg of mRNAs from various tissues (Clontech, Palo Alto, CA) were hybridized with gel-purified probe at 45 °C for 18 h. Nonhybridizing RNA was then digested with RNase A (5 units/ml) and RNase T1 (200 units/ml) for 30 min at 37 °C. Protected fragments were resolved on a 6% polyacrylamide, 7 M urea gel and visualized on a PhosphorImager with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Figure 1:
Genomic organization of the human MGDF
gene. A, restriction enzyme mapping of the gene. Only BamHI (B), EcoRI (E), XbaI (X), and PstI (P) sites are shown. B, schematic diagram of the human MGDF gene. The length and
position of each exon are indicated. C, relationship of the
genomic DNA to the cDNA(- - -) and protein (). The dashedlines indicate (from left to right) the
start and the end of the cDNA, and the start and the end of the coding
region.
Figure 2: Sequences of the intron-exon junctions of the human MGDF gene. A, exon sequences are shown in uppercaseletters, and intron sequences are in lowercaseletters. The encoded amino acids are indicated below the exon sequences. The length of each intron is shown in parentheses. The intron-exon junctions of exon 7 in the two alternate splice variants are shown in B and C.
Figure 3: Sequence of the 5`-noncoding region of the human MGDF gene. The 5` upstream sequence along with the first 12 bases of coding region are shown. The potential regulatory element are underlined. The primers used in RACE to determine the 5`-untranslated region are overlined. The boundaries of exons and introns are indicated by bentarrows. The 5` end of the previously reported cDNA (21) are indicated by a dot.
Figure 4: Deduced amino acid sequence of the exon 7 splice variant. COOH-terminal portions different from full-length MGDF are underlined. Amino acids (Gly-165 and Cys-170) that are conserved between this splice form, full-length MGDF, and erythropoietin, are italicized.
RNase probe protection
experiments were performed to analyze splice variations of the human
MGDF gene. An RNA antisense probe containing 12 nucleotides of exon 3,
all of exons 4-6, and 127 nucleotides of exon 7 yielded three
different protected fragments (Fig. 5), consistent with the
major splice variants found among cDNA clones. These protected
fragments were present in similar relative frequencies in all tissues
we examined that express MGDF, i.e. liver (fetal and adult),
kidney, and testis. A protected fragment of 519 nucleotides corresponds
to the full-length transcript. A fragment of 392 nucleotides is derived
from the LPPQ variant, while the exon 7 splice
variant gives rise to the 473-nucleotide fragment (Fig. 5). Both
splice variants have also been found in canine and murine MGDF cDNAs
(data not shown).
Figure 5: Ribonuclease protection assays show splice variants of MGDF. Figure shows a schematic representation of the splice variations and of expected protected fragments using an antisense probe derived from full-length cDNA spanning from the 3` end of exon 3 through exon 7.
Transient expression of the full-length MGDF cDNA
resulted in the secretion of biologically active MGDF. Identical
experiments with the LPPQ and the exon 7 splice
variants of MGDF showed that the proteins were expressed but not
secreted (data not shown). The biological activities of these molecules
are unknown.
In summary, we have characterized the human MGDF gene. This gene consists of 7 exons and 6 introns spanning 8 kb. The gene was mapped to chromosome 3q26.3, a region previously shown to be associated with abnormal megakaryopoiesis. Several splice variants have been identified. The biological functions and significance of these splice variants remain to be explored.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17071[GenBank].