©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural and Functional Studies of the Intracellular Tyrosine Kinase MATK Gene and Its Translated Product (*)

(Received for publication, June 21, 1994; and in revised form, October 14, 1994)

Shalom Avraham Shuxian Jiang Setsuo Ota Yigong Fu Bijia Deng Lisa L. Dowler (1) Robert A. White (1) Hava Avraham (§)

From the Division of Hematology/Oncology, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215 and the Section of Genetics, Children's Mercy Hospital, UMKC School of Medicine, Kansas City, Missouri 64108

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We recently cloned the cDNA which encodes a novel megakaryocyte-associated tyrosine kinase termed MATK. In this study, we have cloned and characterized the human MATK gene as well as the murine homolog of human MATK cDNA and performed functional studies of its translated product. Comparison of the deduced amino acid sequences of human and murine MATK cDNAs revealed 85% homology, indicating that MATK is highly conserved in mouse and human. The human gene consists of 13 exons interrupted by 12 introns. The genetic units which encode the SH3 and SH2 domains are located on separate exons. The putative ATP binding site (GXGXXG) is localized on exon 7, and the entire catalytic domain is subdivided into seven exons(7, 8, 9, 10, 11, 12, 13) . Somatic cell hybrid analysis indicated that human MATK gene is located on chromosome 19 while the murine Matk gene is located on chromosome 10. The immediate 5`-flanking region was highly rich in GC sequences, and potential cis-acting elements were identified including several SP1, GATA-1, APRE, and APRE1. Antisense oligonucleotides directed against MATK mRNA sequences significantly inhibited megakaryocyte progenitor proliferation. Functional studies indicated that MATK can phosphorylate the carboxyl-terminal conserved tyrosine of the Src protein. These results support the notion that MATK acts as a regulator of p60 in megakaryocytic cells and participates in the pathways regulating growth of cells of this lineage.


INTRODUCTION

Several Src-related protein tyrosine kinases are known to function in the regulation of proliferation and maturation of hematopoietic cells(1, 2, 3, 4) . Fyn(5) , Lck(6) , and Zap-70 (7) play important roles in T-cell receptor signaling. A similar signaling mechanism exists in B-cells and involves Lyn(8) . Deficient expression of the tyrosine kinase ATK/MPK results in the syndrome of human x-linked agamma-globulinemia(9, 10) . Furthermore, the oncogenic potential of the Src family protein tyrosine kinases was found to be associated with their enzymatic activation(4, 5, 6, 11, 12) . This enzymatic activity is primarily regulated through the phosphorylation of a conserved carboxyl-terminal tyrosine residue (3, 4, 11, 12) . Phosphorylation of this residue reduces kinase activity, while dephosphorylation by protein tyrosine phosphatases increases kinase activity. Phosphorylation of the regulatory tyrosine residue appears to involve Csk, a recently identified intracellular protein tyrosine kinase distinct from the known members of the Src family. Csk was initially purified from rat brain (13) and later cloned from human and chicken tissues(14, 15, 16) . The protein lacks an autophosphorylation site within its kinase domain and a carboxyl-terminal equivalent of Tyr-527. Csk phosphorylates several Src family protein tyrosine kinases at their carboxyl-terminal tyrosines thereby altering their enzymatic function(17, 18, 19, 20) . Csk-deficient mouse embryos yields a lethal phenotype(21, 22) .

We have recently identified and characterized a novel intracellular tyrosine kinase, termed MATK, (^1)which shares 50% homology to Csk and is predominantly expressed in cells of megakaryocytic lineage and brain(23) . The MATK cDNA clone encodes a polypeptide of 507 amino acids. Sequence comparisons also indicate that MATK contains Src homology (SH) region 2 and region 3 domains but lacks the NH(2)-terminal myristylation signal, the negative regulatory tyrosine (Tyr-527), and the autophosphorylation site (Tyr-416) corresponding to those found in Src. Expression of MATK mRNA was up-regulated in megakaryocytic cells induced to differentiate by the phorbol ester PMA(23) .

In the present report, we have cloned and characterized the human MATK gene and the mouse homolog of the human MATK cDNA. We have determined the exon-intron organization of the human MATK gene and have mapped its putative transcription initiation site. The putative promoter region was sequenced, and potential cis-acting elements were identified. The chromosomal location of human MATK and murine Matk was determined. In addition, functional studies of the MATK protein were performed. We observed that MATK can phosphorylate the carboxyl-terminal tyrosine of Src. Furthermore, the generation of megakaryocyte colonies (CFU-MK) from marrow CD34 progenitor cells treated with MATK antisense oligonucleotides was significantly reduced compared to the sense-treated CD34 cells or untreated control cells. These studies suggest that MATK may play an important role in signal transduction pathways of megakaryocytic cells, particularly those involved in their growth and maturation.


EXPERIMENTAL PROCEDURES

Materials

Chemical reagents were purchased from Sigma. The -EMBL-3 human genomic library was kindly obtained from Dr. Stuart Orkin (Children's Hospital, Harvard Medical School, Boston). The gtll mouse brain cDNA library was obtained from Clontech (Palo Alto, CA). Restriction endonucleases, modifying enzymes, terminal deoxynucleotidyl transferase, random priming kits, and Sephadex G-25 quickspin columns were purchased from Pharmacia Biotech Inc. and New England Biolabs (Beverly, MA). The primers for polymerase chain reaction (PCR), RNA-PCR, and for sequencing, were synthesized by an automated DNA synthesizer (Applied Biosystems, model 394). The PCR and RNA-PCR kits were obtained from Perkin-Elmer Cetus. Sequenase and random primer kits were obtained from U. S. Biochemical Corp. (Cleveland, OH) and RNA isolation kits from Stratagene (La Jolla, CA). The antibodies for Csk were kindly obtained from Dr. Andre Veillette (McGill University, Montreal, Canada).

Cells

Human bone marrow was obtained by aspiration from the iliac crest of normal donors following informed consent as described previously(24) . After two washes with sterile 1 times phosphate-buffered saline, the cells were resuspended in RPMI 1640 medium with 7.5% platelet poor plasma (PPP), penicillin/streptomycin (P/S), and L-glutamine, seeded onto T-75 tissue culture flasks (Corning, Corning, NY), and incubated at 37 °C in 5% CO(2). CD34 bearing marrow progenitor cells were purified from heparinized bone marrow aspirates using immunomagnetic beads coated with anti-CD34 monoclonal antibody as described(24) . The CD34 cell population was 95-98% pure as judged by labeling with fluorescein-conjugated CD34 antibodies after an overnight recovery in RPMI plus 7.5% PPP.

The CMK cell line (provided by Dr. T. Sato) (25) and the Dami cell line (provided by Dr. S. Greenberg) (26) have authentic properties of cells of megakaryocytic lineage. The CMK and Dami cell lines were cultured in RPMI 1640 medium with 10% fetal calf serum. The TPA301 cell lines were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. HeLa cells were obtained from the ATCC and cultured in RPMI 1640 medium with 10% fetal calf serum. Megakaryocytic cells were induced to differentiate by treatment with PMA. PMA was dissolved in dimethyl sulfoxide and stored at -20 °C until use, when it was diluted in RPMI 1640 medium and used at 10 ng/ml.

Antisense Oligonucleotide Synthesis and Cell Treatment

Modified 18-mer oligonucleotides were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, Texas), precipitated, and resuspended in RPMI 1640 as described previously(24, 27) . MATK antisense AS1 5`-AAC CAG AGA GCC TCG CCC CGC-3` corresponded to nucleotides +4 to +24. All experiments were carried out with the corresponding sense 5`-GCG GGG CGA GGC TCT CTG GTT-3` and scrambled sequence controls.

CD34 cells were incubated at a concentration of 1 times 10^6 cells/ml in serum-deprived medium. Medium contained iron-saturated human transferrin (300 µg/ml), insulin (100 ng/ml), calcium chloride (28 µg/ml), deionized bovine serum albumin (2%), 6.14 mg of oleic acid, and 7.4 mg of dipalmitoyl lecithin in 10 ml of RPMI. Incubation medium was supplemented with recombinant human interleukin-3 (100 units/ml) (R& Systems, Minneapolis, MN). Oligonucleotides were used at a concentration of 10 mmol/liter (70 µg/ml). After 16 h of incubation at 37 °C, 5 mmol/liter oligonucleotides were added. Cells were further incubated for an additional 6 h and then washed in RPMI 1640 before plating or RNA extraction, or preparation of total lysates for Western blot analysis as described(23) .

Colony Assays

Cells were placed in the fibrin clot culture system as described(28, 29) . Cells were seeded at a concentration of 500 cells/0.5 ml in culture containing 10% PPP and interleukin-3 (100 units/ml). Cultures were incubated for 12 days. Fibrin clots were fixed for 5 min with 10% neutral formalin and reacted with platelet glycoprotein IIIa (GpIIIa) fluorescein-conjugated monoclonal mouse antibodies to human GpIIIa (1:1000 dilution) (Dako, Carpinteria, CA) for 30 min. The numbers of positive CFU-MK were counted.

Reverse Transcription-Polymerase Chain Reaction

RNA extracted from a pellet containing 1 times 10^5 CD34 cells was reverse transcribed at 42 °C for 40 min in a final volume of 50 µl as described(23) . The 5`- and 3`-specific primers were added in final concentrations each of 5 ng/50 µl. The mixture was subjected to 30 amplification cycles using the Perkin-Elmer thermal cycler set as follows: denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min, and extension at 72 °C for 2 min.

The sequence of the MATK upstream primer was 5`-GCG GGG CGA GGC TCT CTG GTT-3` (corresponding to position +265 to +285 bp). The nucleotide sequence of the downstream primer was 5`-TGC GAG CAC ACC CGC CCC AAG-3` (corresponding to position +430 to +450 bp). Primers for the beta-actin message were: upstream primer 5`-ATG GAT GAT GAT ATC GCC GCG-3` and downstream primer was 5`-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG GCC-3` (30) . Primers specific for the c-kit message as well as the probe for c-kit were used as described previously(31) . The amplification products were detected by an overnight hybridization to synthetic P-ATP-labeled oligomer probes for: MATK probe 5`-GCC GTC ATG ACG AAG ATG CAA-3` and beta-actin probe 5`-GAG GAG CAC CCC GTG CTG CTG A-3`. The PCR products were analyzed as described previously (23) .

Screening of Human Liver Genomic Library

A genomic library derived from a Sau3A1 digest of human liver DNA (32) was used to isolate 15-18 kilobase (kb) genomic DNA clones containing the gene that encodes MATK. The human genomic library (6 times 10^5 recombinants/screening) containing inserts ligated into the BamHI site of the bacteriophage EMBL3 was probed at 42 °C with an [alpha-P]dCTP (3,000 Ci/mmol; DuPont NEN)-labeled 2.0-kb MATK cDNA probe in hybridization buffer (50% (v/v) formamide, 0.75 M NaCl, 75 mM sodium citrate, 5 times Denhardt's buffer, 0.1% (w/v) sodium dodecyl sulfate, 1 mM EDTA, 10 mM sodium phosphate, and 100 µg/ml salmon sperm DNA carrier). The nitrocellulose filters were washed under conditions of high stringency (63 °C; 30 mM NaCl, 3 mM sodium citrate, 0.1% sodium deodecyl sulfate, 1 mM EDTA, and 10 mM sodium phosphate, pH 7.0). Three distinct human genomic DNA clones (designated HG-matk-1, HG-matk-2, and HG-matk-3) were isolated, and DNA was prepared (33, 34) from each. These human genomic clones were digested singularly or with various combinations of restriction endonucleases. The DNA fragments were fractionated by electrophoresis in 1% agarose gels and were transferred to nylon filters (Nytran Plus, Schleicher & Schuell) (35) . The resulting DNA blots were probed under the above conditions of high stringency with the P-labeled 686-bp HindIII 3`-probe, the 639-bp 5` BglII probe, and whole MATK cDNA. Human genomic DNA and mouse genomic DNA were isolated (36) from the human CMK cell line and mouse liver, respectively. Samples were digested (10 µg/sample) separately with EcoRI, BamHI, HindIII, and XbaI for 4 h at 37 °C. The fragments were resolved by agarose gel electrophoresis and were transferred to Nytran Plus membranes. The resulting DNA blots were analyzed for hybridization under conditions of high stringency, with 5` BglII fragment or HindIII 3`-fragment of human MATK cDNA as probes. A randomized and oligo(dT)-primed mouse brain cDNA library (Clontech) (6 times 10^5 recombinants/screening) was screened using the same procedure and conditions as described above for human genomic library.

Nucleotide Sequencing of a Human Genomic Clone That Encodes MATK

HG-matk-1, HG-matk-2, and GH-matk-3 were digested with SalI, BglII, HindIII, XbaI, and EcoRI, or with a combination of these restriction enzymes, to generate distinct DNA fragments of the clone which were approximately 8.0 and 4.0 kb in size, respectively. These DNA fragments were subcloned into Bluescript plasmid and sequenced in both orientations by the chain termination method (37, 38) and by automatic sequencing using the Pharmacia ALF sequencer. The nucleotide sequence of the first and last 250 nucleotides of each genomic fragment was determined using T7 and T3 primers (Stratagene). Based on the nucleotide sequences of the genomic fragment being analyzed, two oligonucleotides of 21 nucleotides in length were synthesized and used as primers to determine the contiguous nucleotide sequence of the next 200-250 nucleotides in each direction of the double-stranded DNA. The exon-intron organization and the putative promoter region of the human MATK gene were determined by this approach. The sizes of the introns were determined by two methods, either by restriction digest mapping of DNA from the HG-matk clones and hybridization with specific oligonucleotide of 21 nucleotides, or by PCR using sense and antisense specific primers that are localized between each set of two exons. The resulting DNA sequences were analyzed at the Molecular Biology Computer Research Resource facilities at the Dana Farber Cancer Institute (Boston, MA) for the extent of their homology to other reported DNA sequences in the GenBank data base. Data base searches were run against deposited sequences in GenBank and EMBL.

To determine the transcription initiation site within the human MATK gene, primer extension analyses were performed as described by Lee and co-workers (39) with few modifications. CMK cells were lysed in the presence of guanidinium isothiocyanate, and total RNA and poly(A) RNA were obtained by CsCl density gradient centrifugation (40) and oligo(dT)-cellulose(41) , respectively. A sample of 36 oligonucleotides (5 pmol) (5`-GCG GTC CCG GCT GCA CAA CTT GGA GCG AGT TGC TCC-3`), which corresponded to residues +1 to +36 and a sample of a second primer of 24 oligonucleotides (5`-GCT CAG GGG GCG CCC CCG AGC CGC-3`), which corresponded to residues +87 to +110 of the antisense strand of MATK cDNA, were separately end labeled with [-P]ATP using T4 polynucleotide kinase to a specific activity of >3 times 10^8 counts/min/µg(42) . The samples were suspended in 50 mM Tris-HCl (pH 8.0) containing 3.5 mM MgCl(2), 10 mM dithiothreitol, 50 mM KCl, 100 µg/ml bovine serum albumin, and 0.5 mM samples of each dNTP. CMK-derived poly(A) mRNA (3 µg) and 400 units of avian myeloblastosis virus reverse transcriptase (Life Technologies, Inc.) were added, and the samples were incubated at 45 or 65 °C for 1 h. At the end of the incubation period, the samples were extracted with phenol and precipitated with ethanol, and the size of prominent radiolabeled DNA fragments extended onto the primers were determined. For a negative control, primer extension analyses were performed with RNA from TPA301 cells as a template.

Chromosomal Localization of the Mouse Matk Gene

Genomic DNAs for C57BL/6J, Mus spretus, and the (C57BL/6J X M. spretus) F(1) X M. spretus backcross DNA panel were obtained from the Jackson Laboratory, Bar Harbor, ME(43) . Southern blots and hybridizations were performed as described previously(44) . Genomic DNAs of C57BL/6J and M. spretus were digested with 29 different restriction enzymes. The Southern blots were probed with a mouse 1.8-kb Matk cDNA fragment labeled with P using a Decaprime II Kit (Ambion). Digestion of the backcross DNA panel with HincII, Southern blotting, and hybridizations were carried out as described(44) .

Chromosomal Localization of the Human MATK Gene

Genomic DNAs from the NIGMS Hybrid Mapping Panel 2 were obtained from the NIGMS Genetic Mutant Cell Repository (Corriel Cell Institute for Medical Research, Camden, NJ). Mapping Panel 2 consists of DNA isolated from 24 human/rodent cell hybrids retaining one or two human chromosomes. All but two of the hybrids retain a single intact human chromosome. In addition, Mapping Panel 2 includes DNA samples isolated from human, mouse, and chinese hamster cell lines (rodent parental cell lines). Approximately 5 µg of DNA was digested for each sample with restriction enzymes overnight. Southern blots and hybridizations were carried out as described previously(44) .

Biochemical Analyses

For preparation of proteins for immunoprecipitation, proteins from CMK and Dami cells were lysed in modified RIPA buffer(20, 23) . One µg of appropriate antibody was added to each protein sample (10 µg). Antibodies used were rabbit anti-p60 (Upstate Biotechnology, NY), rabbit anti-MATK antibodies (23) which recognize the NH(2)-terminal region of the human MATK gene product, and rabbit anti-Csk serum generated against a TrpE fusion protein containing residues 182-450 of rat p50 Csk (a generous gift from Dr. Andre Veillette, McGill University, Montreal, Canada)(19) . In some experiments, we used p60 partially purified enzyme (Upstate Biotechnology, NY). After absorbing to protein G-Sepharose (Pharmacia), immunoprecipitates were processed as described previously(23, 45) . In a kinase assay, immunoprecipitate obtained from 10 µg of total proteins, 4.5 µg of acid-treated enolase(45) , and 4 mmol/0.74 MBq of [-P]ATP were included in 25 µl of kinase assay buffer. 5-(p-Fluorosulfonylbenzoyl) adenosine (FSBA) was added in a final concentration of 1.0 mM as indicated. Phosphorylation was allowed to proceed at 25 °C for 20 min, and phosphoproteins were resolved by SDS-polyacrylamide (10%) gel electrophoresis as described(45) . Metabolic labeling, immunoprecipitation, and immunoblots were carried out as described (23) .


RESULTS

Molecular Cloning of the Full-length Murine Matk cDNA

Southern blot analysis of human (Fig. 1) and mouse (data not shown) genomic DNA digested with EcoRI, HindIII, BamHI, or XabI and probed under conditions of high stringency with either 5` BglII fragment (639 bp in size) or HindIII 3`-fragment (689 bp in size) of human MATK cDNA as probes, revealed a single band in each lane, indicating that the human MATK gene and the mouse Matk gene are highly homologous and are single genes. Therefore, a randomized and oligo(dT)-primed mouse adult brain cDNA library was screened under conditions of high stringency for the full-length mouse cDNA of Matk using the human MATK cDNA as a probe. Thirty clones were isolated. Two of these clones were sequenced in both directions, and 10 additional clones were partially sequenced. Sequence analysis of these clones revealed identical sequences. The 1.9-kb full-length cDNA has an open reading frame of 466 amino acid residues and possesses 85% homology with the human MATK (Fig. 2). A 1.9-kb Matk transcript was detected in murine brain and megakaryocytes using the mouse Matk cDNA as a probe (data not shown). RNase protection assay demonstrated only one protected fragment using the mouse Matk cDNA and RNA isolated from mouse megakaryocytes as a template (data not shown).


Figure 1: Southern blot analysis of human genomic DNA using 5`- or 3`-specific fragment of human MATK cDNA as probes. Aliquots of 10 µg of HindIII, EcoRI, BamHI, or XbaI digested human genomic DNA were separated on 1% agarose gel and transferred. Molecular sizes (kb) as determined by migration of -HindIII fragments are shown.




Figure 2: Alignment of the predicted amino acid sequences (single-letter code) of the mouse Matk and human MATK gene translated products. Amino acid residues found to be observed between mouse Matk and human MATK are boxed.



Chromosomal Localization of the Murine Matk Gene

Chromosomal localization of Matk in the mouse-Southern blots of C57BL/6J and M. spretus DNAs were digested with 29 different restriction enzymes and probed with a mouse Matk 1.8-kb cDNA. A HincII restriction fragment length polymorphism (RFLP) was detected (Fig. 3A). The alleles for this HincII RFLP consists of a 16.5 kb band, characteristic of C57BL/6J and a 9.7 kb band which is found in M. spretus. These alleles were characterized in 94 DNAs from the C57BL/6J X M. spretus backcross panel. Results of the haplotype analysis from this mapping data indicate that the Matk gene co-localizes with D10Mit22 (MIT anonymous DNA fragment 22) and is linked to Iapls3-28 (intra-cisternal A particle LTR sequence 3-28) on mouse chromosome 10 (Fig. 3B). The Matk locus mapped between Iapls3-28 and D10Mit65 (MIT anonymous DNA fragment 65) and the calculated distances are: Iapls3-28-3.2 ± 1.8 cM-Matk-6.4 ± 3.5 cM-D10Mit65.


Figure 3: Matk maps to chromosome 10 in mouse using a C57BL/6J X M. spretus backcross. A, HincII restriction enzyme pattern for C57BL/6J (B) and for (C57BL/6J X M. spretus) F(1) heterozygote (BS) genomic DNAs probed with Matk cDNA. The major difference between the two DNAs and molecular sizes of fragments in kb are indicated. B, haplotype analysis of molecular markers in backcross progeny. Inheritance of chromosome 10 markers in backcross mice from a C57BL/6J X M. spretus backcross showing linkage and relative position of Matk. Gene names and references to these loci can be found in GBASE. The first two columns indicate the number of backcross progeny with no recombinations. The following columns indicate recombinational events between adjacent loci (signified by a change from an open box to a closed box). The number of recombinants are listed below each column and crossing-over percentage between adjacent loci is indicated.



Cloning, Sequence Analysis, Genomic Organization, and Chromosomal Localization of the Human MATK Gene

We previously observed that expression of human MATK mRNA in megakaryocytes and brain was specific and abundant(23) . To determine the exon-intron organization of the human MATK gene and to identify the potential tissue-specific response elements, we screened approximately 6 times 10^5 total recombinants from a human liver genomic library in -EMBL-3 for genomic clones under conditions of high stringency with the 686-bp P-labeled 3`-gene-specific fragment (HindIII 3`) of the human MATK cDNA (Fig. 4). We isolated a 15-kb genomic DNA clone, termed -HG-matk-1. In addition, using probes derived from both the 3`-end and the 5`-end, respectively, of the human MATK cDNA (686-bp HindIII 3`-fragment; 639-bp 5` BgIII fragment), we isolated an additional two genomic DNA clones of 18-kb genomic DNA clone (termed -HG-matk-2), and 15-kb genomic DNA clones (termed HG-matk-3).


Figure 4: Restriction map and exon/intron organization of the human MATK gene. BI, BgII, D, H, K, N, PsI, PvII, and S refer to the sites within the human MATK gene that are susceptible to BalI, BglII, DdeI, HindIII, KPNI, NarI, PstI, PvuII, SmaI, respectively. The 13 boxes indicate the 13 exons. The dotted, shaded, and striped areas within exons 4-7 correspond to the SH3 and SH2 domains and the putative ATP-binding site, respectively. The arrows indicate the region of genomic clones -HG-matk-2 which were sequenced.



A restriction map of each genomic clone was constructed by digesting the phage DNA with a panel of restriction enzymes separately or in various combinations: SalI, BglII, HindIII, XbaI, and EcoRI. The DNA blots were probed under conditions of high stringency with either the 5` BglII fragment, the BglII HindIII fragment, or the HindIII 3`-fragment of the human MATK cDNA. In parallel, a blot was prepared of human liver DNA that had been digested with the same panel of restriction enzymes. When this DNA blot was probed with the 686-bp HindIII 3`-gene-specific fragment or the 639-bp 5` BglII gene-specific fragment of the human MATK cDNA, the pattern of hybridization was identical to that obtained with -HG-matk-2 (data not shown), indicating that this clone probably contained the entire gene that encodes human MATK. The restriction enzyme map of the human MATK gene was constructed, and the nucleotide sequences of -HG-matk-1, -HG-matk-2, and -HG-matk-3 were determined according to the strategy depicted in Fig. 4. Based on the nucleotide sequences of the genomic fragments analyzed, two oligonucleotides of 21 nucleotides in length were synthesized and used as primers to determine the contiguous nucleotide sequence of the next 200-250 nucleotides in each direction of the double-stranded DNA. No mismatches were found between the genomic sequence shown in Fig. 5and the cDNA. The exon-intron organization and the putative promoter region of the human MATK gene were determined by this approach.


Figure 5: Nucleotide and deduced amino acid sequence of the human MATK gene. The nucleotides are numbered relative to the putative transcription initiation site. The amino acids are numbered relative to the translation initiation site. Only the sequence of introns adjacent to splice junctions is shown in lower-case letters. Putative regulatory elements are underlined and labeled. The nucleotide sequence of the 5`-flanking region, the exon/intron junction, and the 13 exons are depicted. The arrow indicates the putative transcription initiation site. The putative ATP-binding site GXGXXG in exon 7 is underlined. The catalytic domain is boxed in exons 7-13. The SH3 domain and SH2 domain are boxed in exons 4-6. The polyadenylation site in exon 13 is underlined.*** refers to stop codon.



Based on the nucleotide sequences of its 8.0- and 4.0-kb subcloned fragments, the human MATK gene is comprised of 13 exons that span about 8 kb of DNA (Fig. 5). The gene is approximately 8 kb from the putative transcription initiation site to the end of exon 13. Our genomic -HG-matk-2 clone contained an additional 6 kb of 5`-flanking sequences and 4 kb of sequences downstream of exon 13. Exon 1 contained the 5`-untranslated sequence, and exon 2 contained the putative translation initiation site. The sequence encoding the SH3 domain was localized on exon 4, while the sequence encoding the SH2 domain was localized on exons 5 and 6. The putative ATP-binding site (GXGXXG) (46) was localized on exon 7, and the entire catalytic domain was localized on exons 7-13. The intron splice junctions were sequenced for each exon, and an additional 369 bp of 5`-flanking sequence was characterized. The sequence -1 to -270 bp was highly rich in GC content.

The putative transcription initiation site was identified by primer extension analysis. A single-stranded DNA that corresponded to the antisense nucleotide sequence of MATK cDNA (nucleotide residue +1 to +36) was used in the primer extension reaction with RNA prepared from CMK cells as the template. About 190 nucleotides were extended onto the primer resulting in a DNA product of 220 nucleotides in length (Fig. 6). Therefore, the putative transcription initiation site is 360 nucleotides upstream of the translation initiation site. Interestingly, a highly GC-rich region is located just upstream to the putative transcription initiation site. Additional primer extension experiments with a different antisense nucleotide sequence were performed to confirm the putative transcription initiation site of the human MATK gene (data not shown).


Figure 6: Determination of the 5`-end of the mRNA which encodes MATK by primer extension. The primer extension reaction was performed using control TPA301 RNA (lane 1) and CMK RNA (lane 2). The radiolabeled DNA HinfI fragment (as well as their size) which are generated by times 174-P-5`-end-labeled are indicated in the left lane (M). The arrow indicates the size of the oligonucleotide that is polymerized onto the primer.



The region upstream of the putative transcriptional initiation site was sequenced to identify potential cis-acting elements which might be involved in the regulation of MATK gene expression. Analysis of DNA sequences 369 bp proximal to the putative transcription initiation site revealed several potential cis-acting elements proximal to the putative promoter region (Fig. 5). Computer analyses of the putative promoter region did not identify a classical TATA box. A highly GC-rich region was found close to the beginning of exon 1, a feature typical for a selected group of genes lacking a classical TATA box(33, 47) . Potential cis-acting regulatory sequences were identified as GATA-1, ``GC box,'' Sp1, APRE, and APRE1 (see Fig. 5for details). Hamster, human, and mouse DNAs were digested with BamHI, HindIII, and PstI to identify specific RFLP patterns for each species. A unique PstI RFLP for MATK was identified in human DNA from the parental cell lines used to prepare human/rodent cell hybrids (Fig. 7). DNAs from the parental and the somatic hybrid cell lines were digested with PstI, Southern blotted, and probed. Analysis indicated that the human-specific PstI pattern was observed only in cell line 19 which contains human chromosome 19 (Fig. 7).


Figure 7: Mapping of human MATK to Chromosome 19. PstI-digested genomic DNAs from hamster (h), human (H), and mouse (M) as well as 24 human/rodent somatic cell hybrids (labeled 1-22, X, and Y) probed with MATK cDNA. The human-specific RFLP is indicated with arrowheads and is seen in the human control lane and lane 19.



Degradation of MATK mRNA by Antisense Oligonucleotides

To address the role of MATK in megakaryocytopoiesis, we exposed purified CD34 cells to antisense oligonucleotides. We first assessed the stability of MATK transcripts and the half-life of the protein by exposing CMK cells to actinomycin D (5 ng/ml) for 15, 30, or 60 min. MATK mRNA was stable for about 30 min after actinomycin D addition and then destabilized. No MATK protein was observed after treatment with actinomycin D after 8 h as determined by Western blot analysis (data not shown).

Using these kinetics, we then incubated CD34 cells in serum-free medium containing growth factors and modified sense, antisense, or scrambled oligonucleotides. Equal numbers of cells (1 times 10^5 cells) were used for total RNA extraction and subsequent PCR analysis as described(24) . The remaining cells were seeded in cultures to assess the biologic effects of oligonucleotide treatment. The MATK antisense encompassing the second amino acid to the eighth amino acid resulted in a significant decrease in MATK mRNA levels while the sense oligonucleotide had no effect (Fig. 8A). Controls for efficient reverse transcription and mRNA stability were performed by amplification of actin and c-kit transcripts. These results indicated that MATK antisense oligonucleotides bound specifically to MATK mRNA, resulting in its degradation. Furthermore, no expression of MATK protein was observed in CD34 cells treated with MATK antisense oligonucleotides, while there was no effect on MATK protein expression in CD34 untreated or treated with sense or scrambled oligonucleotides (Fig. 8B).


Figure 8: A, expression of MATK mRNA after treatment of CD34 cells to sense, antisense, or scrambled antisense oligonucleotides. CD34 cells from bone marrow were isolated and treated with other respective oligonucleotides as described under ``Experimental Procedures.'' RNA was extracted and analyzed for MATK, c-kit, and actin transcripts as described. Autoradiographs were exposed for 18 h at -80 °C. B, expression of MATK protein after treatment of CD34 cells to sense, antisense, or scrambled antisense oligonucleotides. CD34 cells were treated with oligonucleotides as described under ``Experimental Procedures.'' Total lysates were prepared and products were analyzed by SDS-polyacrylamide gel electrophoresis. MATK p60 protein was analyzed by Western blot using anti-MATK antiserum (dilution 1:100).



Effect of MATK Antisense Oligonucleotides on in Vitro Megakaryocytopoiesis

The proliferation of the megakaryocytic cell line, CMK, in the presence of MATK antisense and sense oligonucleotides, was assayed by [^3H]thymidine incorporation and cell viability. This approach has been successfully used to address the function of regulatory genes such as c-myb, growth factors such as interleukin-11, and the putative cytokine receptor c-mpl in hematopoiesis(24, 48, 49) . A myb antisense oligonucleotide (5`-GTG CCG GGG TCT TCG GGC -3`) served as a positive control due to its known inhibitory effects on generation of megakaryocyte colonies (CFU-MK)(49) . HeLa cells served as negative controls which do not express MATK. Kinetic and dose-response studies using the oligonucleotides were performed to determine the optimal conditions to assess their effects on megakaryocyte growth and proliferation using CMK cells as a model system. These studies indicated that the optimal concentration of antisense oligonucleotide was 70 µg/ml added for 48 h. The sense or antisense oligonucleotides (70 µg/ml) were then added to the cultures of the human megakaryocytic cell line CMK. These experiments indicated that the MATK antisense oligonucleotides inhibited proliferation of CMK cells to a similar degree (about 50%) as the myb antisense construct. No effects on HeLa cell growth were noted (Table 1).



To further address the role of MATK in the regulation of megakaryocytopoiesis in a more physiological model system, we exposed purified bone marrow CD34 cells to MATK antisense and sense oligonucleotides. The CD34 cells were isolated using immunomagnetic beads as described(24) . CD34 cells were incubated at a concentration of 1 times 10^6 cells/ml in serum-deprived medium containing growth factors and synthetic sense or antisense oligonucleotides. 1 times 10^5 cells were used for total RNA extraction and subsequent PCR analysis while the remaining cells were seeded in plasma-clot cultures to test the effects of sense/antisense oligonucleotide treatment on CFU-MK. The generation of megakaryocyte colonies (CFU-MK) from CD34 progenitor cells treated with MATK antisense was reduced significantly (about 50%) compared to the sense-treated CD34 and control untreated cells (Table 1). These results indicate that MATK antisense oligonucleotides specifically inhibited in vitro megakaryocytopoiesis using primary marrow progenitor cells.

Phosphorylation of p60 by MATK and Csk

p60 protein was incubated with 1 µM[-P]ATP and 3 nM MnCl(2) in the absence or presence of MATK p60 or Csk p50, subjected to SDS-PAGE, and then located by autoradiography. As shown in Fig. 9, p60 was phosphorylated by MATK or Csk in a time-dependent manner. The degree of p60 phosphorylation by MATK was similar to that by Csk. MATK was capable of phosphorylating a synthetic peptide corresponding to the last 25 amino acid residues, indicating that MATK phosphorylates Src at carboxyl-terminal tyrosine residues (data not shown).


Figure 9: Phosphorylation of p60 by MATK and Csk. MATK p60 and Csk p50 were immunoprecipitated with specific antibodies as described under ``Experimental Procedures.'' Purified p60 (2 units) were incubated with or without immunoprecipitates of MATK and Csk in a reaction system containing 1 µM [-P]ATP and 3 mM MnCl(2). Samples were taken at the indicated times and subjected to SDS-PAGE followed by autoradiography. The labeled protein was then excised from the gel, and its radioactivity was counted in scintillation fluid.



To exclude the possibility that the apparent action of MATK was mediated by enhancement of the autophosphorylating activity of p60, we examined whether MATK phosphorylated p60 without kinase activity. For this, p60 was treated with an ATP analogue, FSBA, which is known to inactivate p60 by reacting with lysine 295(20) . Almost all the kinase activity of p60 was destroyed by incubation with 1.0 mM FSBA for 60 min at 30 °C. Residual FSBA was quenched by beta-mercaptoethanol. The inactivated p60 was then incubated with or without MATK, the degree of phosphorylation was analyzed by SDS-PAGE as described above, and the radioactivity in the phosphoprotein corresponding to p60 was counted (Table 2, Fig. 10). The results showed that MATK phosphorylated the inactivated p60. These studies indicate that MATK indeed catalyzed the phosphorylation of a tyrosine residue on c-src distinct from the autophosphorylation site.




Figure 10: Phosphorylation of FSBA-treated p60 by MATK. p60or FSBA-treated p60 were incubated with or without MATK in a reaction mixture as described in Table 2. Samples were subjected to SDS-PAGE followed by autoradiography.



Phosphorylation of p60 by MATK or Csk resulted in a decrease in their abilities to phosphorylate enolase. The effects were apparent when activities were measured in the presence of a limited (1 µM) and an excess (10 µM) amount of ATP (data not shown).


DISCUSSION

In this study we have characterized the human MATK gene, cloned the murine Matk cDNA, and performed functional studies of its translated product. Comparison of the deduced amino acid sequences of human MATK and murine Matk cDNAs revealed 85% homology, indicating that MATK is highly conserved in human and mouse (Fig. 2). Somatic cell hybrid analysis indicated that human MATK gene is localized on chromosome 19 while the murine Matk gene is localized on chromosome 10 within a region which is homologous to human chromosome 19 (Fig. 3, and 7).

While this article was in preparation, molecular cloning of murine Ntk from mice fetal thymus (50) and Ctk from mice adult brain (51) were reported. Sequence analysis of murine Matk revealed >99% homology with Ctk (differences in amino acids 105 and 106 due to shifting of G and C nucleotides in this position) and 100% homology with Ntk, indicating that the reported cDNA Ctk or Ntk represent the murine homolog of human MATK cDNA. The reported mouse Ntk cDNA (50) has an extra coding region of 41 amino acids like human MATK cDNA including the translation initiation site in the same position. The sequence upstream of the translation initiation site of mouse Ntk is different from the sequence upstream of the translation initiation site of mouse Matk and Ctk and is probably due to different exon usage (52) and tissue-specific selection of the transcription initiation site and the translation initiation site, suggesting tissue-specific regulation of mouse Matk gene. Additional findings support the conclusion that mouse Matk, Ctk, and Ntk are the murine homologs of human MATK. Southern blot analysis of human and mouse genomic DNA digested with several enzymes and hybridized with cDNA fragments from the 5`- or 3`-region of human MATK cDNA, revealed hybridization to a single band (Fig. 1). In RNase protection assays using mouse brain mRNA as a template, full protection was demonstrated (data not shown). Matk in the mouse was mapped to chromosome 10 in a region which is homologous to human chromosome 19(53) . The Matk gene co-localized with D10Mit22 which is 1 cM proximal to the Amh gene (anti-Muellerian Hormone). This region of mouse chromosome 10 also has some homology to human chromosome 21(53) . We have also mapped the human MATK gene by using DNAs from human/rodent somatic cell hybrid lines to human chromosome 19 (Fig. 7). Our results indicated the exact localization of MATK was chromosome 19 p13.3 based on human-mouse chromosomal homology and is in full agreement with the reported (54) assignment of the HYL locus determined by fluorescent in situ hybridization.

To determine the underlying molecular mechanisms of MATK regulation, we cloned, sequenced, and analyzed the genomic structure of the human MATK gene. This gene consists of 13 exons which span a genomic distance of about 8 kb compared with genomic loci of the coding region of Csk (55) and of Src (56, 57, 58) spanning genomic distance of 4.9 kb (exons 2-12) and 15 kb (exons 2-12), respectively. The structure of exon-intron junctions is in agreement with established consensus sequences(59) . The first exon encodes the 5`-untranslated region. The second exon encodes the following 24 amino acid sequence that contains the NH(2) terminus of the MATK protein. Comparing the genomic structure of MATK shows similarity with the exon-intron organization of Csk (55) suggesting that the MATK intron-exon structure is intermediate between the Src-family (56, 57, 58) and the fes/fer group(60, 61, 62, 63) . This homology is in agreement with the localization of the MATK gene in a phylogenetic tree close to the Csk gene based on sequence homology within the catalytic domain as suggested by Brauninger et al.(55) for the Csk gene.

Primer extension reactions were performed and revealed that 360 bp upstream of the translation initiation site is the putative transcription initiation site in the MATK gene. The human MATK gene does not contain a classical TATA box. A GC-rich region was found upstream of the putative transcription initiation site. These GC-rich motifs, which could serve as Sp1 sites, were identified in the putative MATK promoter in close vicinity to the putative transcription initiation site.

A number of nucleotide sequences that correspond to known cis-acting elements that enhance or suppress transcription of other genes were identified in the 396-bp sequence upstream of the transcription initiation site, including GATA-1, APRE, several Sp1 sites, and APRE1 (64) . However, it remains to be determined if these motifs play a functional role in the regulation of transcription of MATK in megakaryocytes, CD34 marrow cells, or brain.

Recently, molecular cloning of the human intracellular protein tyrosine kinase (HYL) cDNA was reported(54) , which appears to be identical to human MATK cDNA. Sequence analysis of the human MATK gene is in complete agreement with the human MATK and HYL cDNA sequences. Furthermore, the human MATK gene is located on human chromosome 19 (Fig. 7), which is the same chromosomal localization as reported for the HYL gene(54) . Using 5`-gene-specific and 3`-gene-specific probes for the human MATK gene, a single band was identified by Southern blot analysis of human and mouse genomic DNA (Fig. 1). In addition, RNase protection analysis using the above antisense RNA probes with mRNA from PMA-treated CMK cells as a template, only one band fully protected was demonstrated (data not shown). Taken together, these results indicate that MATK and HYL are the same gene.

High levels of p60 are found in terminally differentiated cells such as platelets(65, 66, 67, 68, 69, 70) , suggesting a role in normal cell function that is not related to cell proliferation. Since the regulation of c-src activation is important in platelet function, and since MATK shares homology with Csk, we studied whether MATK could serve as a regulator of p60 in megakaryocytes, the precursors to platelets. Our prior studies provided evidence that suggested a potential physiologic function of MATK, based on its restricted expression in CD34 marrow cells and megakaryocytes. MATK expression appeared to be up-regulated during PMA stimulation, suggesting that MATK could participate in the process of megakaryocyte maturation and/or platelet production. The results presented here demonstrate that p60 in megakaryocytes can be regulated negatively by MATK. In our reaction system, MATK could phosphorylate p60. These results are similar to the reported effects of Csk and Ctk/Ntk on p60 which appear to phosphorylate the COOH terminus(50, 51) . Taken together, our data suggest MATK may act to modulate Src activity in cells of megakaryocytic lineage.

To further determine whether MATK might play a role in the regulation of megakaryocytopoiesis, we used an antisense approach. Exposure of CD34 marrow cells to MATK antisense oligonucleotides resulted in significant inhibition of megakaryocyte progenitor formation in vitro. These results indicate that the MATK-encoded protein likely transduces signals for survival, proliferation, and/or maturation in megakaryocyte progenitors.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants R01 HL51456 and R01 HL46668. 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.

§
To whom correspondence should be addressed: Div. of Hematology/Oncology, New England Deaconess Hospital, One Deaconess Rd., Boston, MA 02215. Tel.: 617-632-0119; Fax: 617-424-6237.

(^1)
The abbreviations used are: MATK, megakaryocyte-associated tyrosine kinase; PTK, protein tyrosine kinase; SH, Src homology; PCR, polymerase chain reaction; PPP, platelet-poor plasma; P/S, penicillin/streptomycin; PMA, phorbol 12-myristate 13-acetate; GpIIIa, glycoprotein IIIa; RFLP, restriction fragment length polymorphism; bp, base pair(s); kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; FSBA, 5-(p-fluorosulfonylbenzoyl) adenosine.


ACKNOWLEDGEMENTS

We thank Dr. Jerome E. Groopman for critical reading of the manuscript and very helpful discussion. We thank Dr. Andre Veillette, McGill University, Montreal, Canada for supplying Csk antibodies, Dr. Stuart Orkin, Children's Hospital, Harvard Medical School, Boston, MA for supplying -EMBL 3 human genomic library, and Linda Pasztor for valuable discussions on human gene mapping. We also thank Lucy Rowe, Joe Nadeau, and Ed Birkenmeier of The Jackson Laboratory for supplying the DNA panel and for performing analysis of linkage data and Laura Gatson for her technical assistance. We thank Patricia DeLapp for her help in preparing the manuscript.


REFERENCES

  1. Ullrich, A., and Schlessinger, J. (1990) Cell 6, 203-212
  2. Pawson, T., and Gish, G. D. (1992) Cell 71, 359-362 [Medline] [Order article via Infotrieve]
  3. Cooper, J. A. (1990) in Peptides and Protein Phosphorylation (Kemp, B., ed) CRC Press, Boca Raton, FL
  4. Bolen, J. B., Rowley, R. B., Spana, C., and Tsygankov, A. Y. (1992) FASEB J. 6, 3403-3409 [Abstract/Free Full Text]
  5. Samelson, L. E., Philips, A. F., Loung, E. T., and Klausner, R. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4358-4362 [Abstract]
  6. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. B. (1988) Cell 55, 301-308 [Medline] [Order article via Infotrieve]
  7. Chan, A., Iwashima, M., Turck, C. W., and Weiss, A. (1992) Cell 71, 649-662 [Medline] [Order article via Infotrieve]
  8. Yamanashi, Y., Kakiuchi, T., Mizuguchi, J., Yamamoto, T., and Toyoshima, K. (1991) Science 251, 192-194 [Medline] [Order article via Infotrieve]
  9. Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S., Belmont, J. W., Cooper, M. D., Conley, M. E., and Witte, O. N. (1993) Cell 72, 279-290 [Medline] [Order article via Infotrieve]
  10. Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., Edvard Smith, C. I., and Bentley, D. R. (1993) Nature 361, 226-233 [CrossRef][Medline] [Order article via Infotrieve]
  11. Hunter, T. (1987) Cell 49, 1-4 [Medline] [Order article via Infotrieve]
  12. Cantley, L. C, Auger, K. R., Carpenter, C., Duckworth, B., Granziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302 [Medline] [Order article via Infotrieve]
  13. Kaplan, J. M., Mardon, G., Bishop, J. M., and Varmus, H. E. (1988) J. Mol. Cell. Biol. 8, 2435-2441
  14. Nada, S., Okada, M., MacAuley, A., Cooper, J. A., and Nakagawa, H. (1991) Nature 35, 69-72
  15. Partanen, J., Armstrong, E., Bergman, M., Makela, T. P., Hirvonen, H., Hueber, K., and Alitalo, K. (1991) Oncogene 6, 2013-2018 [Medline] [Order article via Infotrieve]
  16. Sabe, H., Knudsen, B., Okada, M., Nada, S., Nakagawa, H., and Hanafusa, H. (1989) Proc. Natl. Acad. Sci. U. S. A. 89, 2190-2194 [Abstract]
  17. Okada, M., Nada, S., Yamanashi, Y., Yamamoto, T., and Nakagawa, H. (1991) J. Biol. Chem. 266, 24249-24252 [Abstract/Free Full Text]
  18. Bergman, M., Mustelin, T., Oetken, C., Partanen, J., Flint, N. A., Amrein, K. E., Autero, M., Burn, P., and Alitalo, K. (1992) EMBO J. 11, 2919-2924 [Abstract]
  19. Chow, L. M. L., Fournel, M., Davidson, D., and Veillette, A. (1993) Nature 365, 156-160 [CrossRef][Medline] [Order article via Infotrieve]
  20. Okada, M., and Nakagawa, H. (1989) J. Biol. Chem. 264, 20886-20893 [Abstract/Free Full Text]
  21. Nada, S., Yagi, T., Takeda, H., Tokunaga, T., Nadagawa, H., Ikawa, Y., Okada, M., and Aizawa, S. (1993) Cell 73, 1125-1135 [Medline] [Order article via Infotrieve]
  22. Imamoto, A., and Soriano, P. (1993) Cell 73, 1117-1124 [Medline] [Order article via Infotrieve]
  23. Bennett, B. D., Cowley, S., Jiang, S., London, R., Deng, B., Grabarek, J., Groopman, J. E., Goeddel, D. V., Avraham, H. (1994) J. Biol. Chem. 269, 1068-1074 [Abstract/Free Full Text]
  24. Methia, N., Louache, F., Vainchenker, W., and Wendling, F. (1993) Blood 82, 1395-1401 [Abstract]
  25. Sato, T., Fuse, A., Eguchi, M., Hayashi, Y., Sugita, K., Nakazawa, S., Minato, K., Shima, Y., Komori, I., Sunami, S., Okimoto, Y., and Nakajima, H. (1987) Exp. Hematol. 15, 495-502
  26. Greenberg, S. M., Rosenthal, D. S., Greeley, T. A., Tantravahi, R., and Handin, R. I. (1988) Blood 72, 1968-1974 [Abstract]
  27. Small, D., Levenstein, M., Kim, E., Carow, C., Amin, S., Rockwell, P., Witte, L., Burrow, C., Ratajczak, M. Z., Gewirtz, A. M., and Civin, C. I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 459-463 [Abstract]
  28. McLeod, D., Shreeve, M., and Axelrad, A. A. (1976) Nature 261, 492-494 [Medline] [Order article via Infotrieve]
  29. Vainchenker, W., Bouget, J., Guichard, J., and Breton-Gorius, J. (1979) Blood 54, 940-947 [Abstract]
  30. Avraham, H., Vannier, E., Chi, S. Y., Dinarello, C. A., and Groopman, J. E. (1992) Int. J. Cell Cloning 10, 70-75 [Abstract]
  31. Ratajczak, M. Z., Luger, S. N., Deriel, K., Abraham, J., Calabretta, B., and Gewirtz, A. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1710-1715 [Abstract]
  32. Aegerter-Shaw, M., Cole, J. L., Klickstein, L. B., Wong, W. W., Fearon, D. T., Lalley, P. A., and Weis, J. H. (1987) J. Immunol. 138, 3488-3494 [Abstract/Free Full Text]
  33. Avraham, S., Austen, K. F., Nicodemus, C. F., Gartner, M. C., and Stevens, R. L. (1989) J. Biol. Chem. 264, 16719-16726 [Abstract/Free Full Text]
  34. Craik, C. S., Choo, Q.-L., Swift, G. H., Quinto, C., MacDonald, R. J., and Rutter, W. J. (1984) J. Biol. Chem. 259, 14255-14264 [Abstract/Free Full Text]
  35. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517 [Medline] [Order article via Infotrieve]
  36. Blin, N., and Stafford, D. W. (1976) Nucleic Acids Res. 3, 2303-2308 [Abstract]
  37. Chen, E. Y., and Seeburg, P. H. (1985) DNA 4, 165-170 [Medline] [Order article via Infotrieve]
  38. Wingender, E. (1988) Nucleic Acids Res. 16, 1879-1902 [Medline] [Order article via Infotrieve]
  39. Lee, J. L., Calzone, F. J., Britten, R. J., Angerer, R. C., and Davidson, E. H. (1986) J. Mol. Biol. 188, 173-183 [Medline] [Order article via Infotrieve]
  40. Chirgin, J. M. Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  41. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1408-1412 [Abstract]
  42. Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560 [Medline] [Order article via Infotrieve]
  43. Rowe, L. B., Nadwau, J. H., Turner, R., Frankel, W. N., Letts, V. A., Eppig, J. T., Ko, M. S. H., Thurston, S. J., and Birkenmeier, E. H. (1994) Mamm. Genome 5, 253-274 [Medline] [Order article via Infotrieve]
  44. White, R. A., Peters, L. L., Adkison, L. R., Korsgren, C., Cohen, C. M., and Lux, S. E. (1992) Nature Genet. 2, 80-83 [Medline] [Order article via Infotrieve]
  45. Hunter, T., and Cooper, J. A. (1985) Annu. Rev. Biochem. 54, 897-930 [CrossRef][Medline] [Order article via Infotrieve]
  46. Patel, G., Kreider, B., Rovera, G., and Reddy, E. P. (1993) Mol. Cell Biol. 13, 2269-2276 [Abstract]
  47. Lemarchandel, V., Ghysdael, J., Mignotte, V., Rahuel, C., and Romeo, P.-H. (1993) Mol. Cell Biol. 13, 668-676 [Abstract]
  48. Kobayashi, S., Teramura, M., Sugawara, I., Oshimi, K., and Mizoguchi, H. (1993) Blood 81, 889-893 [Abstract]
  49. Gewirtz, A. M., and Calabretta, B. (1988) Science 242, 1303-1306 [Medline] [Order article via Infotrieve]
  50. Chow, L. M. L., Jarvis, C., Hu, Q., Nye, S. H., Gervais, F. G., Veillette, A., and Matis, L. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4975-4979 [Abstract]
  51. Klages, S., Adam, D., Class, K., Fargnoli, J., Bolen, J. B., and Penhallow, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2597-2601 [Abstract]
  52. Breitbart, R. E., Andriadis, A., and Nadal-Ginard, B. (1987) Annu. Rev. Biochem. 56, 467-495 [CrossRef][Medline] [Order article via Infotrieve]
  53. Taylor, B. A., Frankel, W. N., Burmeister, M., and Bryda, E. (1993) Mamm. Genome 4, S154-S163
  54. Sakano, S., Iwama, A., Inazawa, J., Ariyama, T., Ohno, M., and Suda, T. (1994) Oncogene 19, 1155-1161
  55. Brauninger, A., Karn, T., Strebhardt, K., and Rubsamen-Waigmann, H. (1993) Oncogene 8, 1365-1369 [Medline] [Order article via Infotrieve]
  56. Tanaka, A., Gibbs, C. P., Arthur, R. R., Anderson, S. K., Kung, H.-J., and Fujita, D. J. (1987) Mol. Cell Biol. 7, 1978-1983 [Medline] [Order article via Infotrieve]
  57. Anderson, S. K., Gibbs, C. P., Tanaka, A., Kung, H.-J., and Fujita, D. J. (1985) Mol. Cell Biol. 5, 1122-1129 [Medline] [Order article via Infotrieve]
  58. Parker, R. C., Mardon, G., Lebo, R. V., Varmus, H. E., and Bishop, J. M. (1985) Mol. Cell Biol. 5, 831-838 [Medline] [Order article via Infotrieve]
  59. Mount, S. M. (1982) Nucleic Acids Res. 10, 459-472 [Abstract]
  60. Semba, K., Kamata, N., Toyoshima, K., and Yamamoto, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6497-6501 [Abstract]
  61. Vandenbark, G. R., deCastro, C. M., Taylor, H., Dew-Knight, S., and Kaufman, R. E. (1992) Oncogene 7, 1259-1266 [Medline] [Order article via Infotrieve]
  62. Johnson, D. E., Lu, J., Chen, H., Werner, S., and Williams, L. T. (1991) Mol. Cell Biol. 11, 4627-4634 [Medline] [Order article via Infotrieve]
  63. Matsushima, H., Wang, L.-H., and Shibuya, M. (1986) Mol. Cell Biol. 6, 3000-3004 [Medline] [Order article via Infotrieve]
  64. Ptashne, N. (1988) Nature 335, 683-689 [CrossRef][Medline] [Order article via Infotrieve]
  65. Ferrel, J. E., Jr., and Martin, G. S. (1988) Mol. Cell Biol. 8, 3603-3610 [Medline] [Order article via Infotrieve]
  66. Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 901-905 [Abstract]
  67. Nakamura, S., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091 [Abstract/Free Full Text]
  68. Dhar, A., Paul, A. K., and Shukla, S. D. (1990) Mol. Pharmacol. 37, 519-525 [Abstract]
  69. Dhar, A., and Shukla, S. D. (1993) Br. J. Hematol. 84, 1-7 [Medline] [Order article via Infotrieve]
  70. Dhar, A., and Shukla, S. D. (1994) J. Biol. Chem. 269, 9123-9127 [Abstract/Free Full Text]

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