from a rat L2 yolk sac tumor cell cDNA library that taken together define a 987-base pair (bp') cDNA that encodes a M,. = 18,600 proteoglycan peptide core (16, ...
Vol. 263,No. 15, Issue of May 25, pp. 7287-7291,1988 Printed in U.S . A .
THEJOURNALOF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterizationof a cDNA That Encodes the Peptide Core of the Secretory Granule Proteoglycan of Human Promyelocytic Leukemia HL-60Cells* (Received for publication, September 28, 1987)
Richard L. StevensSSV,Shalom AvrahamSQII, Michael C. GartnerSS, Gail A. P. BrunsS**, K. Frank AustenSS, and JohnH. Weis$§ From the $Departmentof Medicine, Harvard Medical School, the §Department of Rheumatology and Immunology, Brigham and Women’s Hospital,and the **Genetics Division, Children’s Hospital, Boston, Massachusetts02115
A cDNA that encodes the peptide core of the secre- tory granulesof different types of rat and mouse mast tory granuleproteoglycan of the human promyelocytic cells possess small peptide cores that arerich in serine leukemic cell line, HL-60, has been isolated and ana- and glycine, we propose that thisHL-60 cell-3 derived lyzed. When human genomic DNA was digested and cDNA encodes the peptide core of the proteoglycan probed under conditions of low stringency with a rat that is expressed in the secretory granulesof this hucDNA that encodes a M, = 18,600 serinelglycine-rich man promyelocytic cell. proteoglycan peptide core in L2 yolk sac tumor cells (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985)Proc. Natl. Acad. Sci. U. S. A. 82, 1321-1325) and basophilic leukemia-1 cells (AvraDespite the fact that there are atleast 12 distinct proteins ham, S., Stevens, R. L., Gartner, M. C., Austen, K. F., in the human that exist as proteoglycan peptide cores, the Lalley, P. A., and Weis, J. H. (1988) J. Biol. Chern. complete primary structure and/or chromosomal location of 263, 7292-7296), a number of DNA fragments were only a fewof these proteoglycans are presently known. A identified. A HL-60 cell-derived cDNA library was cDNA that encodes the dermatan sulfate proteoglycan that therefore screened underconditions of low stringency resides in theextracellular matrix aroundfibroblasts has been with the rat probe to identify and isolate a human homologue of this rat proteoglycan peptide core. cloned from a human embryonic fibroblast cell line (1).Its Analysis of the resultinghuman cDNA clones indicated nucleotide sequence predicts a peptide core of M? = 40,000 that theproteoglycan peptide core that is expressed in with three potential glycosaminoglycan initiation sites and HL-60 cells is M , = 17,600 and contains an 18-amino three potential sites for N-linked oligosaccharides. In certain acid glycosaminoglycan attachment region that con- circumstances, cell surface glycoproteins can exist as proteosists primarily of alternatingserineand glycine. glycans. For example, the transferrin receptor (2) and the Northern blot analysis of total RNA probed with the invariant chain of the class I1 antigens (3)have been reported human cDNA revealed that themajor message for this to be proteoglycans on the plasma membrane of human skin proteoglycan peptide core in HL-60cells is -1.3 kilo- fibroblasts and human lymphoid tissues, respectively. The base pairs in size. When a Southern blot of digested deduced amino acid sequence of the cDNAs that encode the human genomic DNA was probed withthe human transferrin receptor (4) and the invariant protein (5) have cDNA, three bands of approximately 6, 9,and 12 kil- revealed serine-glycine glycosaminoglycan initiation sites. obase pairs were detected. However, when the South- Another proteoglycan which contains chondroitin sulfate glyern blot was probed with the XrnnI + 3’ fragment of cosaminoglycanshas been described on the surface of a human this human cDNA, one prominent band was detected, melanoma cell (6). The gene that encodes its M , > 240,000 indicating that a single gene encodes this protein in thepeptide core is predicted to reside on chromosome 15 (7). human. Analysis of the DNA from humanlmouse and A number of effector cells have recently beenshown to human/hamster somatic cell hybrids probed with the contain proteoglycans in their secretory granules which are human cDNA demonstrated that thegene that encodes this molecule resides on human chromosome 10. Be- distinct from those that reside on the plasma membranes of cause the proteoglycans that are present in the secre- cells, as well as those that reside in extracellular matrices (8). In the case of mouse (9), rat (10-lZ), and human (13) mast *This workwas supported by Grants AI-23401,AI-23483, AI- cells, these secretory granule-localized proteoglycans are 24158, and HL-36110 from the National Institutes of Health; Grant highly sulfated and resistant to proteolytic degradation. HepIM-412 (to J. H. W.) from the American Cancer Society; and by arin proteoglycans (10-12), chondroitin sulfate diB (IdUAgrants from the Rothschild Foundation and the American Heart 2S0, + GalNAc-4-S04)/heparin hybrid proteoglycans (14), Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be and chondroitin sulfate E (GlcUA + GalNAc-4,6-diS04)prohereby marked “advertisement” in accordance with 18U.S.C. Section teoglycans (9, 15) have been purified from rat serosal mouse cells, rat basophilic leukemia-1 cells, and mouse bone marrow1734 solely to indicate this fact. The nucleotide seqwnce(s) reported in this paper has been submitted derived mast cells, respectively. Because the peptide cores of to theGenBankTM/EMBLDataBankwith accession number(s) these proteoglycans are similar to each other in their small 503223. M , and high serine and glycine content, it has been concluded 11 Established Investigator of the American Heart Association. To whom reprintrequests should be addressed at: Harvard Medical that theregion of their peptide cores where the glycosaminoglycans are attached lack those amino acids that would be School, The Seeley G. Mudd Bldg., Rm. 608, 250 Longwood Ave., attacked by most proteolytic enzymes (15). Recently, two Boston, MA 02115. 11 Fogarty International Fellow. partial cDNAs (designated pPG-1 and pPG-6) were isolated
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Sequence of HL-60 Cell Proteoglycan Peptide Core
from a rat L2 yolk sac tumor cell cDNA library that taken together define a 987-base pair (bp') cDNA that encodes a M , = 18,600proteoglycan peptide core (16, 17). The deduced amino acid sequence of this ratcDNA revealeda proteoglycan peptide core that contained a 49-amino acid region of alternating serine and glycine. Using a gene-specific 489-bp Ssp1 + 3' fragment (designated pPG-M) of pPG-1, we demonstrated that mouse myelomonocytic cells,rat andmouse mast cells, and rat natural killer cells possess substantial amounts of a 1.0-1.3 kb mRNA that encodes a molecule similar to the rat L2 cell proteoglycan peptide core (18, 19). The promyelocytic leukemia cell line, HL-60, is a transformed human cell that synthesizes chondroitin sulfate proteoglycans and stores these proteoglycans in its secretory granules (20, 21). Under certain in uitro conditions this cell can be induced to differentiate into cells that resemble neutrophils (22), monocytes-macrophages (23), eosinophils (24), and basophils (25). In thepresent study, we have used the rat L2 cell-derived cDNA, pPG-1 (16), toisolate a cDNA (designated cDNA-H4) from a HL-60 cell cDNAlibrary. cDNA-H4 encodes a M , = 17,600protein which contains aserine-glycine repeat sequence. As assessed by Northern and Southern blot analyses, a single gene encodes the -1.3-kb mRNA for this proteoglycan peptide core. By probing DNA from a panel of human/mouse and human/hamster hybrid cells with cDNAH4, we have identified that thegene that encodes the HL-60 cell proteoglycan peptide core resides on chromosome 10.
sequence was determined by the method of Hopp and Woods (30). RNA and DNA Blot Analysis-Total RNA (-5 pg/lane) from HL60 cells was denatured in formaldehyde/formamide, electrophoresed in formaldehyde/agarose gels, and transferred to Zetabind (Cuno Inc., Meriden, CT) (31). The Northern blots were incubated at 37 "C for 24 h in hybridization buffer containing nick-translated pPG-1. After washing the blots under the above conditions oflow stringency, autoradiography was performed using Kodak XAR film. Followinga 4-h incubation a t 70 "C in order to remove the rat probe, each blot was incubated for 24 h at 42 "C in hybridization buffer containing nick-translated cDNA-H4. Blots were then washed under conditions of high stringency before being analyzed. Human genomicDNA(-10pg) from Raji cells (line CCL 86; American Tissue Type Collection) was digested for 4 h a t 37 "Cwith BamHI, HindIII,or EcoRI, and theresulting fragments were resolved by agarose gel electrophoresis. Following transfer to Zetabind (321, the Southern blots were analyzed for hybridization under the above standard conditions of low and high stringency using nick-translated pPG-1, pPG-M, or cDNA-H4 and itssubcloned fragments as probes. Chromosomal Localization of the Human Gene That Encodes the Secretory Granule Proteoglycan Peptide Core of HL-60 Cells-For the chromosomal localization of the human gene that encodes cDNA-H4, DNA from 5 different human/mouse (lines 13C2,24B2,1711, 46C2TG, and 175) and 12 different human/hamster(lines 35A2, 35A4, 35B5, 35C1, 35D3, 35D5, 3534, 35F1,35F3, 35F5, 8935, and 95A4) somatic cell hybrids (33-35) were digested with BanHI. The resulting fragments were resolvedby agarose gel electrophoresis, and the Southern blots were analyzed under conditions of high stringency using cDNA-H4 as a probe. The percent discordance of the cDNAH4 probe to each human chromosome was determined as described in Table I; a discordant fraction of 0.00 indicates the chromosomal localization of this gene.
EXPERIMENTALPROCEDURES
Construction and Screening of a HL-60 Cell-derived cDNA Library-HL-60 cells (line CCL 240; American Tissue Type Collection, Rockville, MD) werelysed in the presence of guanidine isothiocyanate (Bethesda Research Laboratories, Gaithersburg, MD), and total RNA was purified by the CsCl density gradientcentrifugation technique of Chirgwin et al. (26). The poly(A)' RNA that was obtained by oligo(dT)-cellulose (Collaborative Research, Waltham, MA) chromatography (27) was converted into cDNA (28). The resultingcDNAs were blunted with T4 DNA polymerase (Biolabs, Beverly, MA), the internal EcoRI sites were methylated, and thecDNAs were ligated to EcoRI polylinkers. After selection of cDNAs of >500 bp by Sepharose CL-4B (Pharmacia) chromatography, the cDNAs were ligated to dephosphorylated X g t l O . Escherichia coli (strain C600 Hfl) were infected with the resulting recombinant bacteriophages resulting in a library with a complexity >1 X lo6. The HL-60 cell-derived cDNA library was probed a t 37 "C with [(u-~'P]~CTP (3000 Ci/mmol; New England Nuclear, Boston, MA), nick-translated pPG-1 (16) in hybridization buffer (50% formamide, 5 X SSC (0.15 M NaCI, 15 mM sodium citrate), 2 X Denhardt's buffer, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 100 pg/ml salmon sperm DNA carrier, and 10 mM sodium phosphate). The filters were washed a t 37 "C under conditions of low stringency of 1.0 X SSC, 0.1% SDS, 1 mM EDTA, and 10 mM sodium phosphate, pH 7.0. Approximately 500,000 recombinants in the library were plated to isolate the clone designated cDNA-H4. The HL-60 cell-derived cDNA library (-500,000 recombinants) were rescreened using cDNA-H4 as theprobe. Thirty clones that hybridized under conditions of high stringency (55 "C; 0.2 X SSC, 1mM EDTA, 0.1% SDS, and 10 mM sodium phosphate, pH 7.0) with cDNA-H4 were isolated from the secondary screening of the library. The individual HL-60 cell-derived cDNAs and their subcloned fragments were inserted into M13 mp18 and mp19 (Amersham, Arlington Heights, IL) and sequenced by the dideoxy chain termination method of Sanger et al. (29). Both strands of cDNA-H4 were sequenced. The hydropathicity plot of the predicted amino acid
' The abbreviations used are: bp, base pairs; cDNA-H4, the HL-60 cell-derived cDNA that encodes the proteoglycan peptide core of this human promyelocytic leukemia cell; pPG-1, the cDNA that encodes the peptide core of the proteoglycan that is expressed in rat L2 yolk sac tumor cells; pPG-M, the 3' portion of pPG-1 thatrecognizes the specific rat gene that encodes the L2 cell proteoglycan peptide core; SDS, sodium dodecyl sulfate; and SSC, 0.15 M NaCI, 15 mM sodium citrate.
RESULTS
Southern Blot Analysis of Human Genomic DNA Probed with pPG-1-To determine if the rat L2 cell-derived cDNA, pPG-1, could be used to identify any human gene sequence, Southern blots of digested human genomic DNA were probed with nick-translated pPG-1 or pPG-M. While no hybridization occurred when the blot was probed under conditions of high stringency with either pPG-1or pPG-M, or probed under
1
4.4-
2
3
y
FIG. 1. Southern blot analysis of human genomicDNA from Raji cells incubated with BamHI (lane I ) . Hind11 (lane 2 ) , a n d EcoRI (lane 3)and probed under conditions of low stringency with pPG-1. The migration positions of the M, = 23,000, 9,400, 6,600, and 4,400 fragments ofHind111 digested X DNA are indicated.
Sequence of HL-60 Cell Proteoglycan Peptide Core A
5'
X
A
3'
7289
Rot Probe
Human Probe
(offi-1)
(4)
CDNA-H4 I
5'
X
A
3'
CDNA-H8 f
5' X
A
X
A
3'
CDNA-H 19
5'
3'
CDNA-H2 I
10 200 rnGAAclncrr~mGAATccAACAPGAlcaxcGrCIGmACT F E L L P G E S N K I P R L R T 72 genes that possessed DNA sequences related to pPG-1) ruled out an attempt toprobe a genomic phage library or a cosmid GAcCIT~CcAAPG~PGAAlcCPEG42TIGAATcGrATc~CcA 248 library with this probe. When the HL-60 cell-derived cDNA D L F P K T R I Q D L N R I F P 88 library was screened with pPG-1 under conditions oflow stringency, a -650-bp cDNA was obtained that was desig296 CITl"~GAC?wTCT~TCAGGClTCGGCTccGGCTccGGCl" L S E D Y S G S G F G S G S G S 104 nated cDNA-H4. Using cDNA-H4 as a probe, another -500,000 recombinants in the cDNA library were screened 344 GGAna~l"mmGGCrncInm~mGAAcpGcaT?w under conditions of higher stringency and 30 additional G S G S G S G F L T E M E Q D Y 120 cDNAs were isolated. The restrictionmap and the DNA 2€sL 392 ( T v \ ~ G l ' A G A c G A A ~ ~ G c T ~ C 4 T G A c M c C l " A a : ~ C T T fragments of cDNA-H4, as well as some of the other HL-60 Q L V D E S D A F H D N L R S L 136 cell-derived cDNAs that were sequenced, are depicted in Fig. 2A. The consensus nucleotide sequence and predicted amino G A c ~ A A T c R : o o C ~ G p c A G c C 4 Z ~ T n ; ~ c A A ~ G G A l T 4 440 acid sequence are depicted in Fig. 2B. While cDNA-H4 lacked D R N L P S D S Q D L G Q H G L 152 a poly(A) tail, it containedall of the coding information 488 G A A ~ W T m k T G T J n ? m ~ necessary for the peptide core of this secretory granule proE E D F M L+** 1% teoglycan. The initiation site for translation is defined by an ATG codon that resides 3' of a TAG stop codon and 5' of a 544 607 4 (TI CKnT TAA stop codon in the same reading frame. Thirty one and FIG. 2. A , restriction map and nucleotide sequencing strategy of cDNA-H4 and its related cDNAs. X and A refer to the peptide. Stop codons are indicated by ***. The numbers on the right sites within each cDNA which are susceptible to XmnI and AccI, respectively. The arrows indicate the direction and length of each subcloned fragment of cDNA that was sequenced. B , consensus nucleotide sequence of the HL-60 cell-derived cDNAs, and the predicted amino acid sequence of the translated proteoglycan peptide core. The arrow indicates the putative site of cleavage of the signal
and left indicate the amino acid and the nucleotide in the respective sequence. The XmnI and AccI restriction sites are indicated. The 5' end of cDNA-H12 was 4 bp longer and the5' end of cDNA-H19 was 14 bp shorter than cDNA-H4. cDNA-H8 differed from cDNA-H4 in that it had an extra thymidine (shown in parentheses) at the 3' end of its cDNA.
Sequence of HL-60Cell Proteoglycan Peptide Core
7290
154 untranslated bp were located 5' and 3', respectively, of the open reading frame of the nucleotides that encodes this proteoglycan peptide core. Based on the position of the 3' stop codon, the deduced M,of this proteoglycan peptide core was M,= 17,600. As assessed by hydropathicity plot (data not shown) and the deduced amino acid sequence (Fig. 2B), the human proteoglycan peptide core contained a typical hydrophobic signal peptide. cDNA-H4 possessed an 18-amino acid region that, except for 1phenylalanine residue, wasa sequence of alternating serine and glycine. The nucleotide sequence that encodes this serine-glycine repeat region (including the position of the phenylalanine residue) was confirmed using cDNA-H8 and cDNA-H21. Likewise, as assessed by agarose gel electrophoresis of their XmnIIAccI fragments (the region of the cDNA that encodes the serine-glycine repeat sequence (Fig. 2)). all five of the examined HL-60 cell-derived cDNAs contained the same sized serine-glycine repeat sequence (data not shown). cDNA-H8 and cDNA-H21 were also used to confirm the 3' TAA stop codon. RNA BlotAnalysis-Nospecific hybridization occurred when a Northern blot containing totalRNA fromHL-60 cells was probed with nick-translatedpPG-1 or pPG-M under conditions of high stringency, nor did hybridization occur when the blot was probed with pPG-M under conditions of low stringency (data notshown). Although pPG-1 hybridized under conditions oflow stringency predominantly to ribosomal sized RNA, some weak hybridization to an -1.3-kb transcript was observed (Fig. 3). When the same Northern blot was washed and reprobed with cDNA-H4 under conditions of high stringency, substantial amounts of an -1.3-kb RNA were detected (Fig. 3). cDNA-H4 also hybridized under conditions of high stringency to lesser amounts of -6-kb and -3-kb sized RNAs. Genomic Complexity of the Gene Encoding the HL-60 Cellderived Proteoglycan Peptide Core-HumangenomicDNA was digested with BamHI, HindIII, and EcoRI, and the fragments were analyzed for hybridization with cDNA-H4. When the Southern blot was probed under conditions of high stringency with cDNA-H4, three to four fragments hybridized with almost equal intensity to the full length cDNA no matter which restriction enzyme was used (Fig. 44).For example, in
C
B
A
. "7
4
3
4.
"2
3
1
2
3
.. 2.32.0-
TABLE I Segregation pattern of c D N A - H ~with DNA from humnn/rodent somatic cell hybrids The DNA from different human/hamster and human/mouse somatic hybrid celllines and the DNA from the controls were analyzed for their hybridization to cDNA-H4. The column designations are: +/+, both hybridization to cDNA-H4 and the specific human chromosome are present; -/-, hybridizationto cDNA-H4 and the chromosomearebothabsent; +/-, hybridization is presentbut the chromosome is absent;and -/+, hybridization is absent but the chromosome is present. For calculation of the discordant fraction for each chromosome,the sum of the +/- and -/+ columns are divided by the sum of the +/+, -/-, +/-, and -/+ columns. (The 19q+ categoryrepresents the der 19 translocationchromosomesfor the hybrid clones derivedfrom fusions withleukocytes from the two different X/19 translocationcarriers. The X and Xq- categories represent the intact X and the der X translocationchromosomes (33.) Human chromosome
1 2 3 4
5 6 7
a
9 10 11 12 13
Discordant Segregation pattern
+I+
+
+/-
1 3 3 2
8 8 6
4
3 6 2 0 8 3 2 5
14
6
15 16 17 18 19 and 19q+ 20 21 22 X and Xq-
4
Y
3 2 5 6 3 3 3 2 0
-I+
fraction
7
1
0.47
4
5 3 5
8 5
4 4
0.31 0.40 0.57 0.29
7
2 6
0 3 3 1 3 2 3
6 5 9 6
7
4
0 3 1 2
2 3
0 5 5 3 1 3 3 5 3 2 3
4
4
6 4
2 6
9
7
7 7 4
8 6 6 7
0.47
0.24 0.53 0.69 0.00 0.47
4
0.40 0.29 0.33 0.25 0.40 0.50 0.29 0.53 0.60 0.56 0.36 0.63
0
0.44
4
1 3 3 2 7
6 5 3
the case of BamHI-digested DNA, cDNA-H4 hybridized to DNA fragments of approximately 12,9, and 6 kb. When this Southern blot of BamHI-digested human DNA was probed with the 5' + XmnI fragment of cDNA-H4, two fragments of 12 and 6 kb were detected (Fig. 4B). However, when the blot was probed with the XmnI + 3' fragment of cDNA-H4, there was hybridization to only one prominent fragment of 9 kb (Fig. 4C), indicating that a single gene encodesthis human proteoglycan peptide core. Chromosomal Location of the Gene That Encodes the HL60 Cell-derivedProteoglycan Peptide Core-The Southern blots of BamHI-digested DNA from 5 human/mouse and 12 human/hamster somatic cell hybrids probed with cDNA-H4 revealed hybridization to three DNA fragments of approximately 12, 9, and 6 kb only in those mouse cell lines (lines 13C2, 24B2, 1711, 46C2TG, and 175) and hamster cell lines (lines 35C1,35D5, and 35F3) that contained human chromosome 10. When the discordant fraction was calculated for each human chromosome, a value of 0.00 was obtained for chromosome 10 (Table I).
DISCUSSION FIG.4. Southern blot analysis of human genomic DNA incubated with BamHI, (lane I ) , H i d 1 1 (lane 2 ) . and EcoRI A cDNAwas isolated from a HL-60cell-derived cDNA (lane 3), and probed under conditions of high stringency with cDNA-H4 ( A ) ,its 5' XmnI fragment ( B ) ,or its XmnI 4 3' library that presumably encodes a human secretory granule in this study fragment (C). The migration positions of the M,= 23,000 to 2,000 proteoglycan peptide core. HL-60 cells were used Hind111 fragments of X DNA are indicated. because large numbers of these human promyelocytic leuke-
Sequence of HL-60 Cell Proteoglycan Peptide mia cells can be obtained in culture and because they are known to synthesize chondroitin sulfate proteoglycans that are storedin secretory granules (20,21). Screening of the HL60 cell-derived cDNA library with the rat cDNA, pPG-1 (16), resulted in the isolation of an -650-bp cDNA that was designated cDNA-H4. Using cDNA-H4, 30 additional cDNAs were isolated from the HL-60 cell-derived cDNA library. The complete nucleotide sequence of cDNA-H4 and the partial nucleotide sequences of four of the other cDNAs were determined (Fig. 2 A ) . cDNA-H4, H8, and HZ1 all possessed a serine-glycine repeat region that consisted of 18 amino acids (Fig. 2B). Two proteins have been found which contain a similar type of serine-glycine repeat region, namely the peptide cores of the Drosophila biologic clock proteoglycan (36, 37) and the rat L2yolk sac tumor cell proteoglycan (16). Since the coding region of the ratL2 cell proteoglycan peptide core cDNA is identical with that of the secretory granule proteoglycan of the rat basophilic leukemia-1 cell cDNA (see Ref. 38), we conclude that the659-bp cDNA isolated from the HL-60 cell encodes the peptide core of the chondroitin sulfate proteoglycan that is expressed in this human promyelocytic leukemia cell. Based on the position of the initiation and stop codons in the nucleotide sequence, the M , of the translatedproteoglycan peptide core is 17,600. The location of the 3’ stop codon in the DNA sequence was determined in both directions in cDNA-H4 and in one direction in cDNA-HS and in cDNAH21 to ensure that thiswas indeed the end of the’open reading frame. The peptide core of cDNA-H4 has atypical hydrophobic signal peptide, and, based on the -3, -1 rule (39) for cleavage of similar types of signal peptides, it is likely that the first 27 amino acids of the translated peptide core are removed in the endoplasmic reticulum. The lack of any asparagine-irrelevant amino acid-serine/threonine sites in this proteoglycan peptide core indicates that no N-linked oligosaccharides are bound to theHL-60 cell proteoglycan. Northern blot analysis of HL-60 cell-derived total RNA, probed under conditions of high stringency with cDNA-H4, revealed aprominent -1.3-kb species of RNA and lesser amounts of two larger sized RNA species (Fig. 3). It is not yet known if these larger sized RNAs represent incompletely spliced precursor molecules, are RNAs that contain additional exons, or are RNAs that arederived from related genes which encode homologous but distinct proteins. Southern blot analysis of human DNA digested with either BamHI, EcoRI, or H i d 1 1 revealed three to four fragments of DNA whichhybridized under conditions of high stringency with c D N A - H ~(Fig. 4). All of these fragments segregated to human chromosome 10 (Table I). This finding is in contrast to the many fragments in the rat genome (18) or human genome (Fig. 1) that hybridize under conditions of high and low stringency, respectively, to the ratcDNA. This apparent difference in complexity of the human and rat genome may be the result of nonspecific cross-hybridization of the much longer serine-glycine repeat region of the ratprobe relative to the human probe (147 nucleotides versus 54 nucleotides) to a variety of unrelated sequences. Nevertheless, the observation 3’ portion of cDNA-H4 hybridizes to a that the XnnI
-
Core
7291
single fragment of human genomic DNA (Fig. 4) indicates that in the human, as in the rat (18),a single gene of >6 kb encodes the peptide core of the proteoglycan that is stored in secretory granules of this effector cell. Acknowledgments-We thank M. A. Bourdonand E. Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA) for providing the rat L2 cell-derived cDNA, pPG-1. REFERENCES 1. Krusius, T., and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 7683-7687 2. Fransson, L.-O., Carlstedt, I., Coster, L., and Malmstrom, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,5657-5661 3. Giacoletto, K. S., Sant, A. J., Bono, C., Gorka, J., O’Sullivan, D. M., Quaranta, V., and Schwartz, B. D. (1986) J . Exp. Med. 1 6 4 , 1422-1439 4. Schneider. C.. Owen..M. .J.., Banville. , . D.. ,and Williams. J. G. (1984)Nature 3 11,675-678 Claesson, L., Larhammar, D., Rask, L., and Peterson, P. A. (1983) Proc. Natl. Acad. Sei. U. S. A. 8 0 , 7395-7399 Bumol, T. F., and Reisfeld, R. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 7 9 , 1245-1249
9. 10. 11. 12. 13. 14.
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