GENOMICS

l&788-794

(1992)

Structure of the Gene Encoding CD34, a Human Hematopoietic Stem Cell Antigen ANNE B. SATTERTHWAITE,*3t

TIMOTHY

C. BURN,* MICHELLE M. LE BEAU,+ AND DANIEL G. TENEN*

*Hematology/Oncology Division, Beth Israel Hospital, and Department of Medicine and tfrogram in Cell and Developmental Harvard Medical School, Boston, Massachusetts 02215; and *Section of Hematology/Oncology, University of Chicago, Chicago, Illinois 60637 Received

July 2, 1991;

revised

Press, Inc.

INTRODUCTION

CD34 is a 115kDa transmembrane protein of unknown function and is expressed in l-4% of human bone marrow cells; this population consists of pluripotential hematopoietic stem cells as well as committed progenitors of each hematopoietic lineage (Andrews et al., 1986; Beschorner et al., 1985; Civin et al., 1984; Katz et al., 1985; Strauss et al., 1986; Tindle et al., 1985; Watt et al., 1987). CD34” bone marrow cells can reconstitute the hematopoietic systems of lethally irradiated nonhuman primates (Berenson et al., 1988). Blasts from approximately 30% of patients with acute lymphocytic and myeloid leukemia are also CD34+, with less differentiated subtypes more likely to express the antigen (Civin et al., 1984; Tindle et al., 1985). Outside the hematopoietic system, CD34 is expressed in the vascular endothelium of a variety of organs (Watt et al., 1987; Fina et al., 1990). The mouse CD34 gene seemsto have a wider pattern of expression, with CD34 mRNA also found at high levels in brain and a number of fibroblast lines (Brown et al., 1991). A human CD34 cDNA clone has been isolated (Simmons et al., 1992) and used to map the CD34 locus to the long arm of chromosome 1 (Molgaard et al, 1989; Tenen oss&7543/92

$3.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

3 1, 1991

et al., 1990). This region contains a cluster of complement genes at band lq32 (Weis et al., 1987; Bora et al.,

CD34 is a cell surface antigen of unknown function expressed in humans in hematopoietic stem cells, vascular endothelium, and blasts from 30% of patients with acute myeloid and lymphocytic leukemia. To begin to investigate the cis-acting elements required for this tissue-specific expression, the human CD34 locus was isolated and its genomic structure and transcriptional start site were characterized. The human CD34 gene spans 26 kb and has 8 exons, a structure quite similar to that of the murine gene. The start site of CD34 transcription was determined to be 258 bp upstream of the translational start site using RNase protection. These experiments also indicated that the 5’ untranslated region has extensive secondary structure. In addition, fluorescence in situ hybridization was used to map the CD34 locus to band Iq32. o 1s~ Academic

October

Biology,

788

1989), the gene for CD45 (Bruns and Sherman, 1989), the proto-oncogenes SKI (Chaganti et al., 1986) and TRK (Miozzo et al., 1990), as well as a group of leukocyte/endothelial adhesion molecules at lq22-q25 (Tedder et al., 1989; Watson et al., 1990). Although CD34 is not homologous to any known protein (Simmons et al., in press), it has been hypothesized to be an adhesion or signaling molecule. Of further interest, cells from the CD34+ myeloblast line KG-l have a partial chromosome 1 trisomy; in addition to the two normal homologues, KG-l cells contain a third marker chromosome 1 with abnormalities in the short and long arms (lp34qll::?::lq21-q32) (Furley et al., 1986). To obtain the information necessary to identify cisacting DNA elements required for the tissue- and stagespecific expression of CD34 and to investigate the potential rearrangement of the CD34 gene in leukemic cells, we cloned and characterized the human CD34 locus. The gene spans 26 kb and includes 8 exons. We localized the gene with further resolution to band lq32 by fluorescence in situ hybridization. In addition, we identified the CD34 transcriptional start site. MATERIALS

AND

METHODS

Isolation. ofgenomic clones. Recombinants (2 X 105) from a human genomic pWE15 cosmid library and a CBXB cosmid library (both provided by David Housman) as well as 5 X 1O’plaques from a Charon-4A human genomic library (a gift of Arthur Banks) were screened with a 1.5.kb CD34 cDNA fragment containing the entire coding region (Simmons et al., 1992). The probe was labeled with [Cy-32P]dCTP by the hexanucleotide random priming method (Feinberg and Vogelstein, 1983). Ch.wacterization of genomic clones. Restriction fragments of the genomic clones were subcloned into pGEM3zf(-) (Promega), pBR322, M13mp18, or M13mp19, and mapped further by single, double, and partial digestion with several restriction enzymes. DNA fragments were run on agarose gels, blotted to Zetaprobe membrane (BioRad), and hybridized to CD34 cDNA labeled as above or to CD34-specific oligonucleotides 3’ end-labeled with [a-32P]dCTP (Rosenberg et al., 1990) to localize exons within the gene. PCR was used as previously described (Saiki et al., 1988) to determine the boundaries of exon 8; cDNA and genomic DNA fragments amplified using primers from

STRUCTURE

A.

OF

THE

HUMAN

CD34

789

GENE

CD34-2A

‘I’ Lambda-FIB

CD34-8

”

I I I

’

-

Sm B

gene

Sa

H

EBK

B S EX HHE

H

X S XBMEH

XKEXBX

EHEH

BXB

HSBBXSE

I I II I 47I III I II IIIIIIrrllIIIII III IIIY I III IIIIII I q I

FIG. 1. Isolation and characterization genomic libraries using a 1.5-kb CD34 tively, and Lambda-RB from a Charon-4A portions of the clones. (B) Restriction BamHI; E, EcoRI; H, HindlII; K, KpnI; map. The 3.5-kb SalI-EcoRI fragment map, with filled regions corresponding

00

P

UP0 m

23

4

567

0

of human CD34 genomic clones. (A) Three overlapping clones were isolated from three human cDNA fragment as a probe: CD34-2A and CD348 from a pWE15 and a CZXB cosmid library, respeclibrary. Solid lines represent regions that have been mapped. Dotted lines represent uncharacterized map of the CD34 locus. CD34 genomic clones were mapped using seven restriction endonucleases: B, Sm, SmaI; S, SatI; and X, XbaI. Sal1 (Sa) was not used except as a reference point for the 5’ end of the at the 5’end of the gene was not mapped with all seven enzymes. Exons are depicted by boxes under the to untranslated regions and open boxes representing coding sequence.

the 5’ and 3’ ends of the region were compared by agarose gel electrophoresis. All intron/exon junctions, exons l-7, and the 5’ flanking region were sequenced using the Sanger dideoxy method (Sanger et al., 1977) with double- or single-stranded templates. RNase protection. A l.l-kb BamHI-SstI fragment containing the putative transcriptional start site was subcloned into pGEM3zf(-). The plasmid was digested with DdeI, and 321-bp transcripts were prepared using T7 RNA polymerase (Promega) in the presence of [a32P]CTP as previously described (Ausubel et al., 1989) with the following changes: 1 pg of template DNA was used, and 50 pCi of [o-32P]CTP and unlabeled CTP to a final concentration of 12 FM were used. Then, 5 X lo5 cpm of the probe were hybridized as described (Ausubel et al., 1989) to 10 pg of yeast tRNA or total RNA isolated from KG-l cells or HL-60 cells using guanidium isothiocyanate (Chirgwin et al., 1979). Samples were digested with RNase A and RNase Tl as described (Ausubel et al., 1989) and run on a 6% polyacrylamide, 8.3 M urea gel. M13mp18 sequence was used as size markers. Analysis translated (Devereux

HE XX Sm XE S K XB

lkb

of secondary structure. Secondary structure of the 5’ unregion was analyzed using the programs Fold and Stemloop et al, 1984).

Fluorescence in situ chromosomul hybridization. Human metaphase cells were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes. The CD34 probes (CD34-2A, CD34-8) were 30- and 34-kb genomic fragments cloned into the vectors pWE15 and CPXB, respectively. The procedure used for fluorescence in situ hybridization is a modification (Rowley et al., 1990) of the method described by Lichter et al. (1988). Biotin-labeled probes were prepared by nick-translation using Bio-11-dUTP (Enzo Diagnostics). Hybridization was detected with Auorescein-conjugated avidin, and chromosomes were identified by staining with 4,6-diamidino-2-phenylindoledihydrochloride (DAPI).

RESULTS

Isolation and characterization of CD34 genomic clones. Three human genomic libraries were screened with a 1.5kb CD34 cDNA probe that contained the entire coding region (Simmons et al, 1992). One positive clone was isolated from each screen (Fig. 1A). CD34-2A, from a pWE15 cosmid library, contained the first three exons and 15 kb of 5’ flanking sequences. Lambda-RB, isolated from a Charon-4A library, began in the first intron of CD34 and extended 1.5 kb 3’ to the gene. A third

clone, CD34-8 from a CBXB cosmid library, contained exons 5-8 and over 20 kb of 3’ flanking sequence. The genomic clones were subcloned and restriction mapped by single, double, and partial digestion with seven restriction endonucleases (Fig. 1B). Oligonucleotide probes corresponding to various regions of the cDNA sequence were hybridized to Southern blots of these restriction digests to localize exons within the gene. Eight exons were identified. The region of genomic sequence designated exon 8 was shown to be colinear with the cDNA sequence by comparing cDNA and genomic DNA amplified by PCR using primers from the 5’ and 3’ ends of this region; there was no difference in size between PCR products generated from cDNA and those from genomic templates (data not shown). The junctions of exon 8 were confirmed by sequencing. The remaining seven exons and their junctions were sequenced in their entirety (Fig. 2). All splice donor and acceptor sequences matched the GT/AG consensus (Mount, 1982). The CD34 coding region can be divided into five “domains”: an N-terminal signal sequence, a region rich in N- and O-linked glycosylation sites, a membrane-proximal domain containing six regularly spaced cysteine residues, a transmembrane region, and a 73-amino-acid cytoplasmic tail (Simmons et al., 1992). The relationship of the CD34 exons to the cDNA is shown in Fig. 3. Exon 1 contains the 5’ untranslated region of CD34, which is 258 bp long and contains areas of high GC content and extensive secondary structure (see below), as well as most of the signal sequence. Exons 2 and 3, separated from the first exon by approximately 11 kb, contain the remainder of the signal peptide and the heavily glycosylated region. The cysteine-rich domain is encoded by exons 4 and 5, although an 8-kb intron separates these two exons. Exons 5-8 are clustered together at the 3’end of the gene. Exon 7 contains the transmembrane domain, and exon 8 comprises the majority of the cytoplasmic domain as well as the entire 1200-bp S’untranslated region. A polyadenylation signal, ATTAAA (Wilusz et

790

SATTERTHWAITE

ET AL.

aggatgatggtgatggggaactaaatggggaaatatggaaggtcacaggaaaagttaacacaagttagcaaaaagttaacataacacaaa

-451

aaggtcttgcaggaaaaaaaaaagaaaagaaaagaaagaaaaagtctccaagaatggtttggacagccaaaatgaatacttatagtcacg

-361

tatacctgctcactcctgacgcttcactcacacacagcacaggatctggtgaggctatcactaaatgtgccacattgtggttaagtttta

-271

cctgattaacgaaatgctcacacttctaaactgaggtccttacagtagattccttttgcaagattgttactggcttacaacttaaaaata

-181

aaggaaaatcacaaggaaagaaaagtggggaaaaaatcggaggaaacttgcccctgccctggccaccggcaaggctgccacaaaggggtt

-91

aaaagttaagtggaagtggagcttgaagaagtgggatggggcctctccaggaaagctgaacgaggcatctggagcccgaacaaacctcca exon 1 -> CCTTTTTTGGCCTCGACGGCGGCAACCCCAGCCTCCTCGCCCTCCGCCTTTGGGACCAACCAGGGGAGCTCAAGTTAGTAGCAGC

-1

CAAGGAGAGGCGCTGCCTTGCCAAGACTAAAAAGGGAGGGGAGAAGAGAGGAAAAAA

180

90

GCAAGAATCCCCCACCCCTCTCCCGGGCGGAGG

GGGCGGGAAGAGCGCGTCCTGGCCAAGCCGAGTAGTGTCTTCCACTCGGTGCGTCTCTCTAGGAGCCGCGCGG~GGATGCTGGTCCGC

270

1

mlvr AGGGGCGCGCGCGCAGGGCCCAGGATGCCGCGGGGCTGGACCGCGCTTTGCTTGCTGAGTTTGCTGCgtgagt.....-ll

kb.....c

337

5rgaragprmprgwtalcllsllp exon 2 -> tatagCTTCTGGGTTCATGAGTCTTGACAACAACGGTACTGCTACCCCAGAGTTACCTACCCAGGGAACATTTTCAAATGTTTCTACAAA

28

422

sgfmsldnngtatpelptqgtfsnvstn TGTATCCTACCAAGAAACTACAACACCTAGTACCCTTGGAAGTACCAGCCTGCACCCTGTGTCTCAACATGGCAATGAGGCCACAACAAA

56

512

vsyqetttpstlgstslhpvsqhgneattn CATCACAGgtaaaa.....

-0.6

exon 3 -> . . . . .ttccagAAACGACAGTCATTCACATCTACCTCTGTGAT~CCTCAGTTTATGG~

kb

ite

86

t

571

vkftstsvitsvygn

t

ACACAAACTCTTCTGTCCAGTCACAGACCTCTGTAATCAGCACAGTGTTCACCACCCCAGCC~CGTTTC~CTCCAGAGAC~CCTTGA

tn

106

s

s

661

vqsqtsvistvfttpanvstpettlk

AGCCTAGCCTGTCACCTGGAAATGTTTCAGACCTTTCAACCACTAGCACTAGCCTTGCAACATCTCCCACTAAACCCTATACATCATCTT

136

p

s

1

sp

g

n

v

s

d

1

CTCCTATCCTAAGTGACATCAAGgtgggg.....

p

166

i

1

s

di

s

ttsts

-1.5

kb

1

1

t

q

g

i

s

s

exon 4 -> . . . . .atacagGCAGAAATCAAATGTTCAGGCATCAGAGAAGTGAAA

k

a

e

TTGACTCAGGGCATCTGCCTGGAGCAAAATAAGACCTCCAGCTGTgtaagt.....-8 185

751

atsptkpyts i

810

kcsgirevk exon 5 -> . . . . .tcccagGCGGAGTTTAAGAAGGA

kb

cleqnktssc

a

e

fk

872

k

d

CAGGGGAGAGGGCCTGGCCCGAGTGCTGTGTGGGGAGGAGCAGGCTGATGCTGATGCTGGGGCCCAGGTATGCTCCCTGCTCCTTGCCCA

206 236

962

rgeglarvlcgeeqadadagaqvcslllaq GTCTGAGGTGAGGCCTCAGTGTCTACTGCTGGTCTTGGCC~CAG~CAGgtaagg..... se vrp qc 111 vl an rte

-0.2

GCAAACTCCAACTTATGAAAAAGCACCAATCTGACCTGAAAAAGgtaagt.....

k

256

1

q

1

m k

k

h

qs

d

1

k

-0.3

kb

exon 6 -> . . . ..ttctagAAATTTCCA iss exon 7 -> . . . . .ttcaagCTGGGGATCCTAGAT kb

1

k

gi

1

TTCACTGAGCAAGATGTTGCAAGCCACCAGAGCTATTCCCAAAAGACCCTGATTGCACTGGTCACCTCGGGAGCCCTGCTGGCTGTCTTG

275

fte

q

d

va

s

h

qs

y

s

qktlia

1

vts

g

GGCATCACTGGCTATTTCCTGATGAATCGCCGCCGCAGCTGGAGCCCCACAGGAG~GGCTGgtcagt.....

305 315

g i tgyflmnrrswsp exon 8 ->

tg

e

gGGCGAAGACCCTTATTACACGGAAAACGGTGGAGGCCAGGGCTATAGCTCAGGACCTGGGACCTCCCCTGAGGCTCAGGG~GGCCAG s P a gedpyytengggqgys sgpgt

v

n

a kb

. ..

vl ..gtCta

1230

aqgkas

1319 1409

rgaqkngtgqatsrnghsarqhvvadte

ATTGTGACTCGGCTAGGTGGGGCAAGGCTGGGCAGTGTCCGAGAGAGCACCCCTCTCTGCATCTGACCACGTGCTACCCCCATGCTGGAG 385

11

1

TGTGAACCGAGGGGCTCAGACGGGACCGGCCAGGCCACCTCCAG~CGGCCATTCAGC~GAC~CACGTGGTGGCTGATACCGA 355

1080

d 1170

a -0.8

r

1021

1499

1

FIG. 2. Sequence of the CD34 exons, intron/exon junctions, and 5’ flanking sequence. The complete sequence of all eight CD34 exons is presented in capital letters. The numbers to the right correspond to the nucleotides of the cDNA and 5’ flanking sequence. Intronic sequences at the splice junctions are depicted by lowercase letters, and the approximate size of each intron is written between the junction sequences. The GC-rich region in the 5’ untranslated region is underlined and the polyadenylation signal is underlined in bold. The translated amino acid sequence is presented in italics below the nucleic acid sequence. Numbers to the left correspond to the amino acid sequence of the precursor protein. The dot following amino acid 385 represents the stop codon. The cDNA sequence is available from GenBank under Accession NO. MS1104 and the genomic sequences under Accession Nos. M81937-M81945.

al., 1989), is present 22 bp 5’ of the point of poly(A) addition to the cDNA (Fig. 2, bp 2955-2601). No AUUUA sequences implicated in regulating mRNA stability

(Shaw and Kamen, 1986) are found in the 3’ untranslated region despite the involvement of mRNA degradation as a mechanism of CD34 gene regulation in some

STRUCTURE

OF

THE

CD34

791

GENE

GTGACATCTCTTACGCCCAACCCTTCCCCACTGCACACACCTCAGAGGCTGTTCTTGGGGCCCTACACCTTGAGGAGGGGGCAGGTAAAC

1589

TCCTGTCCTTTACACATTCGGCTCCCTGGAGCCAGACTCTGGTCTTCTTTGGGTAAACGTGTGACGGGGGAAAGCCAAGGTCTGGAGAG

1679

CTCCCAGGAACAATCGATGGCCTTGCAGCACTCACACAGGACCCCCTTCCCCTACCCCCTCCTCTCTGCCGCAATACAGGAACCCCCAGG

1769

GGAAAGATGAGCTTTTCTAGGCTACAATTTTCTCCCAGGAAGCTTTGATTTTTACCGTTTCTTCCCTGTATTTTCTTTCTCTACTTTGAG

1859

GAAACCAAAGTAACCTTTTGCACCTGCTCTCTTGTAATGATATAGCCAG~CGTGTTGCCTTG~CCACTTCCCTCATCTCTCCTCC

1949

AAGACACTGTGGACTTGGTCACCAGCTCCTCCCTTGTTCTCT~GTTCCACTGAGCTCCATGTGCCCCCTCTACCATTTGCAGAGTCCTG

2039

CACAGTTTTCTGGCTGGAGCCTAGAACAGGCCTCCCAAGTTTTAGGAC~CAGCTCAGTTCTAGTCTCTCTGGGGCCACACAG~CTC

2129

TTTTTGGGCTCCTTTTTCTCCCTCTGGATCAAAGTAGGCAGGACCATGGGACCAGGTCTTGGAGCTGAGCCTCTCACCTGTACTCTTCCG

2219

AAAAATCCTCTTCCTCTGAGGCTGGATCCTAGCCTTATCCTCTGATCTCCATGGCTTCCTCCTCCCTCCTGCCGACTCCTGGGTTGAGCT

2309

GTTGCCTCAGTCCCCCAACAGATGCTTTTCTGTCTCTGCCTCCCTCACCCTGAGCCCCTTCCTTGCTCTGCACCCCCATATGGTCATAGC

2399

CCAGATCAGCTCCTAACCCTTATCACCAGCTGCCTCTTCTGTGGGTGACCCAGGTCCTTGTTTGCTGTTGATTTCTTTCCAGAGGGGTTG

2489

AGCAGGGATCCTGGTTTCAATGACGGTTGGAAATAGAAATTTCCAGAGAAGAGAGTATTGGGTAGATATTTTTTCTGAATACAAAGTGAT

2579

GTGTTTAAATACTGCAATTAAAGTGATACTGAAACAC

2616

FIG.

Z-Continued

systems (Satterthwaite et aZ., 1990). The sequence AUUUUUA is found at bp 1817 (Fig. 2), but whether this can serve as a destabilizing element is not known. Mapping the transcriptional start site. The 5’ end of the CD34 cDNA was previously determined by amplifying CD34 mRNA from total RNA isolated from KG-1 cells and AML-1 cells (Simmons et al., 1992). The longest product extended 258 bp upstream of the translational start site. If transcription of CD34 indeed begins at this point, a 76-bp band should be protected in an RNase protection assay using the 321-bp probe depicted in Fig. 4B. A band comigrating with a 77-bp DNA marker was protected in KG-l RNA but not in HL-60 RNA or yeast tRNA control samples (Fig. 4A), consistent with the cDNA clone being full length and CD34 N- and

l-337

FIG. below.

HUMAN

transcription beginning 258 bp 5’ to the translational start site. We have determined that equal-sized RNA and DNA fragments in this size range run within 1 or 2 bp of each other in the gel system utilized here (data not shown). The sequence of the 540 bp 5’ to the transcriptional start site is presented in Fig. 2. A consensus CAP site, CANYYY (Bucher and Trifonov, 1986), starts 2 bp upstream of the 5’ end of the longest cDNA clone, although no TATA box is found immediately 5’ to this point. The promoter region contains a GATA site at -306 (Martin et al., 1989) as well as numerous purine-rich areas which may provide binding sites for the ets family of transcription factors (Karim et al., 1990). Experiments to define cis elements required for transcription of the CD34 gene

O-

338-

521-

775-

856-

1013-

1066-

520

774

855

1012

IO65

I230

3. Relationship of exons to cDNA. The basepairs spanned by each exon

A schematic are indicated

diagram of the CD34 under each box.

cDNA

1231-2616

is depicted

at the top.

Exons

are represented

by boxes

792

SATTERTHWAITE

A

ET

AL.

Mi3mp18 ACGT

B

Dde I

+I of cDNA I

Sst 1

321

bp probe

76 bp, expected 77 bp, observed FIG. digested primer specific structure was the length.

4. RNase protection. (A) 10 pg of total KG-1 and HL-60 RNA and yeast tRNA were hybridized to the riboprobe depicted with RNase A and RNase Tl. The samples were run on a 6% denaturing polyacrylamide gel. M13mp18 DNA was sequenced (USB) and is shown as size marker. An arrow indicates the KG-1 specific band, which comigrates with a 77-bp DNA marker. 77-bp band is a band present in all three samples and likely represents nondigested probe, presumably due to extensive and G-C pairing in this region (see text). Other areas of the gel (not shown) also contained bands in all three lanes. The only band present in a single sample. (B) The riboprobe is shown. A protected band of 76 bp is expected if the longest cDNA

are currently in progress. Preliminary results indicate that this upstream region functions as an active promoter in CD34+ cells (T. C. Burn, A. B. Satterthwaite, and D. G. Tenen, unpublished observations). Chromosomal localization of CD34. We and others have previously reported that the CD34 locus is on the long arm of chromosome 1 (Molgaard et al, 1989; Tenen et al., 1990). To sublocalize the CD34 gene, we performed fluorescence in situ hybridization of a biotin-labeled CD34 probe to normal human metaphase chromosomes. Fluorescent signals from biotin-labeled probes are visualized as discrete green-yellow dots on unstained chromosomes; specific signal is frequently observed on all four chromatids. Hybridization of the CD34-2A probe resulted in specific labeling only of chromosome 1 (Fig. 5). Specific labeling of lq32 was observed on two (5 cells), three (8 cells), or all four (12 cells) chromatids of the chromosome 1 homologues in 25 cells examined. Similar results were obtained in a second hybridization ex-

in B and with a -40 Below this secondary 77-bp band clone is full

periment using this probe and in hybridizations using the CD34-8 probe. Thus, the CD34 gene is localized to chromosome 1, band q32. DISCUSSION We have cloned and determined the structure of the human CD34 locus. The gene has eight exons, spans 26 kb, and is very similar in overall structure to the murine gene (Brown et al., 1992). There are five “domains” of the CD34 protein which roughly correspond to individual or pairs of exons, although these domains have not been shown to have specific functions. RNase protection confirmed that the full-length CD34 cDNA clone previously isolated (Simmons et al, 1991) begins at the transcriptional start site. This clone has a 258-bp 5’ untranslated region. We are currently analyzing the 15 kb of 5’ flanking sequence in cosmid CD34-2A to define cis-acting DNA elements required for CD34 transcription in

FIG. 5. In situ hybridization of a biotin-labeled CD34 probe to human metaphase cells from phytohemagglutinin-stimulated blood lymphocytes. (A) Counterstained with DAPI. (B) Detection of the probe with FITC-conjugated avidin. The chromosome are identified by arrows; specific labeling was observed at lq32. (C) Partial karyotype of a chromosome 1 homologue illustrating at lq32 (arrowhead).

peripheral 1 homologues specific labeling

STRUCTURE

OF THE

hematopoietic stem cells and endothelial cells. Preliminary experiments suggest that this region contains a functional promoter (Burn et al, unpublished observations). Analysis of the transcriptional start site was hampered by apparent secondary structure in the 5’ untranslated region of CD34. RNA probes from this region were relatively insensitive to RNase A and RNase Tl, giving

reproducible

cleavage patterns

in the absence of any

complementary RNA or DNA (Fig. 4A and data not shown). Traditional primer extension experiments failed, requiring amplification with PCR to visualize full-length extension products (Simmons et a& 1992). There is a 161-bp stretch in exon 1 that is 75% GC rich (Fig. 2, bp 156-316) and has the potential to form a stem-loop structure (Devereux et al., 1984) which would include the translational start site. There appears to be similar secondary structure in the 5’ untranslated region of the murine CD34 gene (Brown et al, 1991), suggesting that it plays a functional role. Hairpin structures in the 5’ untranslated region have been postulated to repress translation of a number of eukaryotic genes, including ferritin (Hentze et al., 1987; Wang et al., 1990), rat ornithine decarboxylase (Manzella and Blackshear, 1990), rat aldehyde dehydrogenase (Guan and Weiner, 1989), porcine pro-opiomelanocortin (Chevrier et al., 1988), and c-sis (Rao et al., 1988). It is possible that there is translational regulation of CD34; human umbilical vein endothelial cells maintain expression of CD34 mRNA for at least lo-15 passagesbut lose surface expression of the protein much earlier after isolation (Fina et al., 1990). Other human tissues that appear negative for CD34 by antibody screening may still express CD34 mRNA. To address this question, Northern blots, quantitative PCR, or in situ hybridization using samples from various tissues could be performed. The CD34 locus has previously been mapped to lq12qter using somatic cell hybrids (Molgaard et al., 1989; Tenen et al., 1990). We have sublocalized the gene to band lq32 using fluorescence in situ hybridization. The regulator of complement activation (RCA) cluster, which includes genes encoding membrane cofactor protein, CRl, CR2, decay accelerating factor, C4 binding protein, and factor H, is also located at lq32 (Weis et al., 1987; Bora et al., 1989). The gene for CD45, a transmembrane tyrosine phosphatase that is expressed in almost all hematopoietic cells except mature erythrocytes and is involved in regulating T and B cell activation, is located at lq31-q32 (Bruns and Sherman, 1989). The transmembrane tyrosine kinase TRK is expressed in some human tumor lines derived from hematopoietic and mesenchymal origin (Martin-Zanca et al., 1986) and has been mapped to lq32-q41 (Miozzo et cd., 1990). Although CD34 is not homologous to any of these genes (Simmons et al., in press) and its function is unknown, it is interesting that its gene is located near a cluster of genes encoding hematopoietic regulatory and signaling molecules.

HUMAN

793

CD34 GENE

The CD34+ myeloblast

line KG-l

expresses higher lev-

els of CD34 mRNA than other CD34+ cells (A. B. Sat-

terthwaite and D. G. Tenen, unpublished In addition to two normal chromosome

observations). 1 homologues,

KG-l cells have a third marker chromosome 1 resulting from several breakpoints, one of which is at lq32 (Furley et aZ., 1986). Perhaps the abnormally high level of CD34 expression in these cells is due to a rearrangement of the CD34 gene on the marker chromosome. If so, this hypothesis could be extended to other CD34+ blast cells from leukemic patients. Rearrangement of the CD34 gene in a number of such samples may suggest a causal role for abnormal CD34 expression in leukemogenesis. The genomic clones isolated here will provide useful probes for investigating this question. ACKNOWLEDGMENTS The authors thank David Simmons and Brian Seed for providing the CD34 cDNA and Rachel Borson for isolating the clones LambdaRB and CD348 We also acknowledge Rafael Espinosa III and Yogesh Pate1 for technical assistance, Helene F. Rosenberg, Steven J. Ackerman, and Linda K. Clayton for criticism of the manuscript, and Judith E. Coffin for helpful discussions. This work was supported by NIH Grant CA34183. D.G.T. is a scholar of the Leukemia Society of America.

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