©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Characterization of the Human Megakaryocyte Growth and Development Factor (MGDF) Gene (*)

(Received for publication, September 23, 1994)

Ming-shi Chang (§) Jennifer McNinch Rita Basu John Shutter Rou-yin Hsu Chris Perkins Vernon Mar Sid Suggs Andy Welcher Luke Li Hsieng Lu Tim Bartley Pam Hunt Frank Martin Babru Samal Jakob Bogenberger

From the Department of Developmental Biology, Amgen Inc., Thousand Oaks, California 91320

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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, (^1)the megakaryocyte growth and development factor. In this report, we describe the isolation, analysis, and chromosomal localization of the MGDF gene.


MATERIALS AND METHODS

Isolation of Human Genomic Clone

A human placenta genomic library (1 times 10^6 plaque-forming units) in the Fix II vector (Stratagene, La Jolla, CA) was screened with a radioactive probe consisting of a mixture of six canine MGDF oligomers, each 24 nucleotides in length (6). Phage DNA from positive plaque was isolated and sequenced (10).

Analysis of the 5`-Untranslated Region

Rapid amplification of 5` cDNA end (RACE) system (Life Technologies, Inc.) was utilized to analyze the 5`-untranslated region. First strand cDNA was synthesized from human fetal liver or testis mRNA using an antisense primer complementary to nucleotides 1590-1613. Following tailing, PCR amplification was performed using the kit's anchor primer (Life Technologies, Inc.) and a nested antisense primer (nucleotides 1565-1589). Amplified fragments were directly sequenced with the model 373A DNA sequencer (Applied Biosystems Inc., Foster City, CA).

Chromosomal Localization

The chromosomal location of the MGDF gene was determined by fluorescence in situ hybridization at BIOSLab Inc. (New Haven, CT). Phage DNA of the genomic clone MGDF-1 was labeled with digoxigenin-dUTP and hybridized to human metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes. Specific signal was detected with antidigoxigenin-fluorescein isothiocyanate. Chromosomes were then counterstained with propidium iodide.

Ribonuclease Protection Assay

[alpha-P]UTP-labeled antisense probe was transcribed from a linearized plasmid containing human MGDF cDNA using the Riboprobe Core System (Promega Inc., Madison, WI).

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).


RESULTS AND DISCUSSION

Genomic Structure of the Human MGDF Gene

The canine MGDF genomic clone was isolated using a probe based on the amino-terminal sequence of the MGDF protein purified from aplastic canine serum (6). Several positive human genomic clones from a placenta genomic library were then identified by screening with a mixture of six canine MGDF oligonucleotides. One of the genomic clones, MGDF-1, contained a 17-kb insert of which 8 kb has been sequenced. The MGDF gene consists of seven exons and six introns (Fig. 1). The sequences of the intron-exon junctions conform to the consensus sequence of eukaryotic splice junctions (Fig. 2A). The intron-exon junctions unique to the two cDNA variants described below are shown in Fig. 2(B and C). The MGDF protein is encoded by exons 3-7. The nucleotide sequence of the coding region was identical to that of the cDNA clone reported previously (6). Comparison of the MGDF gene and the erythropoietin gene indicates that their genomic structures are similar within most of the coding region but diverge at the last exon.


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.



Chromosomal Location of the MGDF Gene

The gene for MGDF was localized to human chromosome 3q26.3 by fluorescence in situ hybridization analysis as described under ``Materials and Methods.'' Several lines of evidence have suggested that gene rearrangements in the region of chromosome 3q26 are associated with abnormal hematopoiesis (11-14). Twenty percent of patients presenting with 3q26 abnormalities display several megakaryopoietic anomalies, including increased megakaryocyte numbers frequently associated with the presence of numerous micromegakaryocytes, and elevated platelet counts. The clinical manifestation seen in these patients may be the result of improper regulation of the MGDF gene.

Analysis of the 5`-Untranslated Region of the MGDF Transcript

Previously reported MGDF cDNA clone contained up to 213 nucleotides of 5`-untranslated sequence (9). To determine the extent of 5`-untranslated sequence of the human MGDF gene, 5` RACE was performed as described under ``Materials and Methods.'' The mRNAs from human fetal liver and testis were used in reverse transcription and PCR amplification. The nucleotide sequence of amplified PCR fragments from both fetal liver and testis contained an identical 5` end, extending the 5`-untranslated region to nucleotide 1 shown in Fig. 3. This extends the 5`-untranslated region by 328 nucleotides upstream and reveals an additional exon (exon 1). This result is consistent with the presence of the 2.1-kb transcript observed in Northern blot analysis. (^2)A smaller 5` RACE product was also observed in which exon 2 was deleted. The predicted size of a transcript lacking exon 2 is consistent with the 1.8-kb transcript also observed in Northern blots. No evidence for transcription start sites or alternate splicing within exon 3 was observed in probe protection experiments.


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.



Identification of Potential Regulatory Elements

The nucleotide sequence of the 5`-noncoding region is shown in Fig. 3. At the 5`-flanking region, several potential regulatory elements were identified. These regulatory elements include SP-1, AP-2, and nuclear factor NF-kappaB. In addition, there are several Ets and GATA elements present downstream of exon 1, which may not be biologically relevant. No TATA box or CAAT motif is present upstream of exon 1. Lack of these motifs has been observed in the promoters of both constitutively expressed genes (15) and highly regulated genes (16-18). Like the c-Mpl receptor gene (19), the MGDF gene contains a G-C-rich region upstream of exon 1, which may be involved in MGDF gene regulation (20, 21).

Splice Variants

In addition to the previously described full-length cDNA, two cDNA variants were isolated from human fetal liver. One variant (LPPQ) has a deletion of 4 amino acids at the junction of exon 6 and 7 but maintains the same reading frame (Fig. 2B). A second variant is produced by an internal splice within exon 7 causing a frameshift (Fig. 2C). The amino acid sequence encoded by this variant is shown in Fig. 4.


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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17071[GenBank].

§
To whom correspondence should be addressed: 14-1-B-219, Amgen Inc., 1840 DeHavilland Dr., Thousand Oaks, CA 91320-1789.

(^1)
The abbreviations used are: MGDF, megakaryocyte growth and development factor; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; kb, kilobase(s).

(^2)
A. Welcher, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Robert Bosselman and Larry Souza for their support, Joan Bennett for preparation of the manuscript, and Maureen Bunting, Deron Johnson, and Danette Barron for preparation of the figures.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.