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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3125-3132
HEMATOPOIESIS
From the Division of Bone Marrow Transplantation and Stem Cell
Biology, Washington University School of Medicine, St Louis, MO; and
Laboratory of Experimental Carcinogenesis, Molecular Cytogenetics
Section, National Cancer Institute, National Institutes of Health,
Bethesda, MD.
The Ly-6 family includes a number of highly homologous,
low molecular weight glycophosphatidylinositol-linked proteins
expressed on hematopoietic and lymphoid cells. The best characterized
family member is Sca-1 (Ly-6A/E), an antigen commonly used for
purification of murine pluripotent hematopoietic cells. We sought to
characterize the genomic locus surrounding the Sca-1 gene. We
identified several overlapping P1 artificial chromosomes containing the
Sca-1 gene and mapped one of these to mouse chromosome
15D3.1-3.3, the region previously shown to contain members of the
murine Ly-6 gene family. We then mapped this clone and found
that the Sca-2 gene lies 35.4 kilobase (kb) downstream of
Sca-1 in the opposite transcriptional orientation. This is the
first direct demonstration of physical linkage of Ly-6 genes. A
novel gene, highly homologous to Sca-1 was identified and
localized 13.4 kb downstream of Sca-1. This gene, which we
designated Ly-6M, shares several structural features conserved
among members of the Ly-6 family. Ly-6M messenger RNA (mRNA) is
easily detectable in hematopoietic tissue (bone marrow, spleen, thymus,
peritoneal macrophages) as well as kidney and lung. No mRNA expression
was detected in heart, stomach, liver, small intestine, brain, or skin.
Ly-6M protein is detectable on 10% to 15% of peripheral blood
leukocytes, including monocytes and a subpopulation of
B220+ cells. Ly-6M is broadly distributed in the bone
marrow, with prominent expression on monocytes and myeloid precursors.
The identification and characterization of Ly-6M adds a new
member to a complex family of homologous, tightly linked genes that
have proven extremely useful reagents for defining populations within the hematopoietic system.
(Blood. 2000;95:3125-3132)
The Ly-6 proteins are a group of highly homologous
molecules linked to the outer leaflet of cell membranes via a
glycophosphatidylinositol (GPI) anchor. The murine Ly-6 family includes
several proteins expressed during relatively discrete stages of
hematopoietic development, including Ly-6A/E (Sca-1), Sca-2, ThB,
Ly-6C, and Ly-6G (Gr-1). Sca-1 has proven to be an especially useful
cell surface marker, as essentially all hematopoietic stem cell
activity in commonly used mouse strains resides in the
Sca-1+ compartment.1 Consequently, selection
for Sca-1 expression is a well-established strategy for murine
hematopoietic stem cell enrichment.2,3
A variety of potential biologic functions have been proposed for Ly-6
proteins, ranging from signal transduction to intercellular adhesion.
In vitro antisense and antibody cross-linking experiments have
suggested that Sca-1 is important for T-cell activation.4-6 Sca-2 has also been implicated in T-cell costimulation on the basis of
in vitro findings.7 These results appear to conflict with
the observation that Sca-1 deficient T cells have a modest hyperproliferative response to some mitogenic stimuli,8
rather than the predicted hypoproliferative response. Taken together, these data do not support the hypothesis that Sca-1 is a critical participant in T-cell activation. Furthermore, the T-cell costimulation model does not address the role of Ly-6 proteins in non-T-cell compartments. The possibility that these proteins may function as
adhesion molecules was suggested by the observation that an anti-Ly-6C
antibody induces aggregation of purified CD8+ T cells in
vitro and impairs homing of lymphocytes in vivo.9 Similarly, transgenic overexpression of Sca-1 induces homotypic aggregation of thymocytes.10 Finally, Sca-1 has been
implicated in the regulation of T-cell development, based on the
observation that thymocyte maturation is arrested at the double
negative (CD4 Ly-6 homologs have been identified in other rodents, as well as diverse
species from squid to primates (reviewed in Palfree12). This reinforces the notion that these proteins have important biologic
functions. Human Ly-6 homologs have only recently been identified. A
human homolog of mThB was identified in a squamous cell carcinoma line
by expression cloning.13 Despite high-sequence homology
between mThB and its human homolog (E48), it is not clear that they are
true orthologs because the human gene is apparently not expressed in
lymphocytes.13 The human Sca-2 gene was cloned by
several groups recently.14-17 Human Sca-2 is widely
expressed and is inducible in NB4 or HL60 cells on exposure to retinoic acid.15 Several other potential human Ly-6 family members
have been characterized to a limited extent (eg, GML,
Ly-6H),18,19 or exist only in the Genbank EST
database. A human Sca-1 ortholog has not yet been identified. The
larger Ly-6 superfamily includes proteins such as CD59 and
urokinase-type plasminogen activator receptor (uPAR), as well as snake
venom After the initial serologic characterization of several murine Ly-6
proteins, the genes encoding 6 of them were cloned.22-26 Chromosomal localization in all cases has mapped the genes to chromosome 15.22,25,26 Southern blot analysis of
overlapping cosmids with cross-reactive Ly-6 probes has
suggested that a large number of highly homologous genes (or
pseudogenes) may be clustered within a 630-kilobase (kb)
region.27 We sought to characterize this genomic region in
greater detail. We found that within a 40-kb region on chromosome
15D3.1-3.3, the Sca-1 and Sca-2 genes lie in opposite
transcriptional orientations and flank a novel Ly-6 family
member. This new gene, Ly-6M, is highly homologous to
Sca-1 and is expressed abundantly in hematopoietic cells.
P1 artificial chromosome library screening
Fluorescence in situ hybridization
P1 mapping Ten micrograms of P1 DNA was digested with rare cutting restriction endonucleases and the resulting fragments were separated by pulsed field gel electrophoresis. Southern blot analysis was performed under standard conditions using Sca-1, Sca-2, or ThB probes. The Sca-1 probe was generated by PCR amplification of 129/SvJ genomic DNA using the primers 5'-ATCTTTGCTTACCCATCTGC-3' (forward) and 5'-CCTCTTCACTGTGCTGGCTG-3' (reverse) spanning exon IV. The Sca-2 probe was generated using the primers 5'-TCTTCCTGCCTGTGCTGTTG-3' (forward) and 5'-CTGATCGGTACATGAGAAGC-3' (reverse) flanking intron II. The ThB probe was generated using the primers 5'-TGCTCGTCCTCCTTGTCTTG-3' (forward) and 5'-CACACGTGACATCGAAGTGC-3' (reverse) flanking intron II. The identity of all probes was confirmed by subcloning and automated sequencing. Subsequently, all BamHI and EcoRI fragments contained within the P1 clone were subcloned into a plasmid vector and used to create a high-resolution restriction map by sequential Southern blot hybridizations. The map assignments for Sca-1, Sca-2, and a novel Ly-6 gene were confirmed by multiple PCRs using "long and accurate" conditions.29 The primers used were specific for the P1 vector (pAd10SacBII)30 arms (5'-GGCCGCTAATACGACTCACTATAGGGAGAGGATC-3' and 5'-GATCCTTCTATAGTGTCACCTAAATGTCGA-3') or for sites within the 3 genes.Ly-6M cloning An 11.8-kb EcoRI fragment cross-reactive with a Sca-1 exon IV probe was subcloned from P1 clone 13561. From this plasmid, a 2.3-kb PstI fragment also cross-reactive with the Sca-1 probe was subcloned and sequenced. An additional 2.5 kb of sequence flanking the PstI fragment were determined using the 11.8 EcoRI plasmid as a template. After alignment of this sequence with previously characterized Ly-6 genes, nested primers predicted to lie in the 5' and 3' untranslated region of a novel Ly-6 gene were designed. The first set is 5'-CTGCAGCCAGGTCTGAGAGG-3' (forward) and 5'-ACAAGGGTGGGGACCATCAC-3' (reverse). The nested set is 5'-CAAGGATGGACACTTCTCACGCG-3' (forward) and 5'-GGTGGGGACCATCACATCAG-3' (reverse). These primers were used to amplify a full-length complementary DNA (cDNA) clone from a P388D1 (macrophage-like) cDNA library (Clontech, Palo Alto, CA). The sequence of this cDNA clone was used to interrogate the GenBank/EMBL/DDBJ nucleotide database using the NIH BLAST server. Potential regulatory elements in the 5' flanking sequences of the novel Ly-6 gene were identified using the algorithms at http://biomas.dcrt.nih.gov.Accession number The Ly-6M genomic sequence has been deposited in the GenBank/EMBL/DDBJ nucleotide database under the accession number AF118557.Reverse transcriptase-polymerase chain reaction The organs of healthy, young adult (age 6-10 weeks) wild-type C57BL/6 or 129/SvJ mice were harvested aseptically. Total cellular RNA was prepared, as previously described.31 RNA was also prepared from macrophages harvested from the peritoneal cavities of healthy, young mice 72 hours after instillation of thioglycollate (macrophage RNA kindly provided by Dr Steven Shapiro, Washington University, St Louis, MO). These macrophage preparations are routinely more than 99% pure, as assessed by flow cytometry. Contaminating genomic DNA was removed from 1-µg aliquots of the RNA samples by treatment with 1 unit DNAse (Gibco BRL, Gaithersburg, MD) at 23°C for 15 minutes, followed by inactivation with ethylenediamine tetraacetic acid (EDTA) (2.5 mmol/L Fc) at 65°C for 15 minutes. Half of each sample was used as a template in a random hexamer-primed reverse transcription reaction using 5 units of AMV RT (Promega, Madison, WI) and 20 units RNAsin (Promega) according to the manufacturer's instructions. The resulting cDNA pools were normalized for -actin content in a control PCR using the primers
5'-GCTGTATTCCCCTCCATCGTG-3' (forward) and
5'-CACGGTTGGCCTTAGGGTTCAG-3' (reverse). The cycling conditions for -actin amplification were initial denaturation at
94°C for 1 minute, then 24 cycles of denaturation at 94° for 30 seconds, annealing at 58° for 30 seconds, extension at 70° for
30 seconds. Aliquots were removed at 5 cycle intervals to ensure that
amplification remained in the linear range. Amplification of Ly-6M was
performed using nested primers in 2 rounds of PCR using the same
conditions described for -actin. The template was diluted 50-fold
between rounds. The primers for the first round of amplification
are 5'-CAAGGATGGACACTTCTCACGCG-3' (forward) and
5'-CAAAGGTAAAAAAGGGTGTTC-3' (reverse). The nested primers are: 5'-TTGCCCATCAATTACCTGCC-3' (forward) and
5'-TCATCTTGGTGTT- AGGATCC-3' (reverse).
32P-dATP was included in the PCR cocktail at a final
concentration of 10 nCi per sample. The products were resolved on 8%
polyacrylamide gels and visualized by autoradiography for 4 hours or
quantified by phosphorimaging (Molecular Dynamics, Sunnyvale, CA).
Ly-6M antisera production The cDNA encoding the predicted mature Ly-6M polypeptide (Leu27 to Asn105) was subcloned into the pTrcHis prokaryotic expression vector (Invitrogen, Carlsbad, CA). Recombinant protein expressed in Escherichia coli was purified on Ni-NTA Agarose (Qiagen, Valencia, CA) and used to immunize rabbits. After 3 boost injections, sera were collected and affinity purified using an immobilized synthetic Ly-6M oligopeptide (CDEIEKKFAADPNTKM) (Sulfolink, Pierce Chemical Co, Rockford, IL).Ly-6M overexpression The full-length Ly-6M cDNA was subcloned into the pcDNA3.1+ mammalian expression vector (Invitrogen, Carlsbad, CA) and transfected by electroporation into K562 cells. Stable G418R clones were isolated by limiting dilution. The cDNAs encoding full-length Ly-6A and Ly-6G were obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification from wild-type C57BL/6 bone marrow cells and used to generate stable K562 clones as controls for these experiments. The Ly-6C.2 cDNA (kindly provided by Dr Roger Palfree, McGill University, Montreal, Canada) was also used to generate stable K562 cell lines.Flow cytometry K562 cells were stained with affinity-purified anti-Ly-6M antisera or control polyclonal rabbit IgG (Sigma, St Louis, MO) at a concentration of 1.0 µg protein per 5 × 105 cells in FACS buffer (0.2% bovine serum albumin [BSA], 0.01% NaN3 in phosphate-buffered saline [PBS]) with 5% goat serum and 5% horse serum. Ly-6M expression was detected using a fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (Vector Laboratories, Burlingame, CA). For blocking experiments, 5 × 105 cells were incubated on ice for 15 minutes with 1 µg of purified recombinant Ly-6M protein before staining with the primary antibody. GPI anchor analysis was performed by incubating 5 × 105 cells with 1.0 unit recombinant phosphatidyl-specific phospholipase C (PI-PLC) (Oxford GlycoSciences, Wakefield, MA) in serum-free media at 37°C for 30 minutes before staining with the primary antibody. K562 clones were also analyzed using an antibody against mLy-6B (clone SK 38-86 kindly provided by Dr Ulrich Hammerling, Memorial Sloan Kettering Cancer Center, New York, NY), as well as antisera that detect hCD55 and hCD59 (kindly provided by Dr Douglas Lublin, Washington University, St Louis, MO). Bone marrow cells were obtained by flushing the femurs of young (8-10 weeks of age) 129/SvJ or C57BL/6 mice with 1× Hebs buffer. After washing and counting, the unlysed samples were preincubated in FACS buffer with 10% goat serum and 1.0 µg/106 cells Fc block (Pharmingen, San Diego, CA), followed by staining as above with anti-Ly-6M antisera or control IgG and the following directly conjugated lineage markers: B220, Gr-1, CD11b, NK1.1, CD3, CD34, CD44, Ter119 (Pharmingen), or F4/80 (Serotec). In other experiments, bone marrow cells were stained with an FITC-conjugated lineage cocktail (CD3, B220, Gr-1, CD11b), phycoerythrin-conjugated anti-Sca-1 (Pharmingen), and anti-Ly-6M antisera (or control IgG) detected by a biotinylated goat antirabbit antibody (Vector), followed by streptavidin-Cy5 (Dako Corp, Carapinteria, CA). Peripheral blood was harvested by cardiac puncture. Leukocytes were recovered after 1 round of red cell lysis and then stained, as above. Analytical flow cytometry was performed on a FACScan (Becton Dickinson, San Jose, CA). Sterile sorting was performed with an Epics Elite cytometer (Coulter Corp, Miami, FL). Sorted samples were cytospun and examined after May-Grunwald/ Giemsa staining.
Sca-1 and Sca-2 are closely linked on chromosome 15 To study the genomic region surrounding the Sca-1 gene, a 129/Ola (H-2b) P1 library was screened using PCR primers specific for the Sca-1 gene. Three P1 clones, averaging 90 kb in size, were obtained. All 3 clones contain the Sca-1 gene, as determined by Southern blot analysis (data not shown). Clone 13561 contains both the Sca-1 and Sca-2 genes, as well as 1 additional band that cross-hybridizes with a Sca-1 cDNA probe (Figure 1). This clone was chosen for further analysis. Southern blot analysis with a ThB probe was negative, suggesting that the ThB gene lies outside of the region studied. Probes for Ly-6C, Ly-6F, and Ly-6G were not informative because of numerous cross-hybridizing bands. FISH analysis revealed that clone 13561 localizes to murine chromosome 15D3.1-3.3 (Figure 2), the region previously reported to contain several Ly-6 family members. By long-range mapping using pulsed field gel electrophoresis, the distance from the Sca-1 gene to the Sca-2 gene appeared to be 35 to 40 kb (data not shown). Restriction mapping of the entire clone placed the Sca-2 gene just 35.4-kb downstream of the Sca-1 gene (Figure 3). Long-range PCR was used to confirm these map assignments and revealed that the Sca-1 and Sca-2 genes are in opposite transcriptional orientations.
Ly-6M, a novel member of the Ly-6 family, lies between Sca-1 and Sca-2 We reasoned that the 11.8 kb EcoRI P1 fragment noted to cross-react with a Sca-1 exon IV probe (Figure 1) might contain a novel Ly-6 gene or pseudogene. We therefore subcloned and partially sequenced this fragment. Sequence alignment revealed that the 11.8-kb genomic fragment contains an open reading frame highly homologous to, but distinct from the Sca-1 gene. Primers predicted to lie in the 5' and 3' untranslated region of this candidate Ly-6 gene were used to amplify a 550-bp cDNA clone from a P388D1 (murine macrophage-like) cDNA library. There were no discrepancies between the sequence of this cDNA clone and the corresponding genomic sequence obtained from the subcloned P1 fragment, suggesting that the 11.8-kb EcoRI fragment contains a novel, expressed Ly-6 gene. The cDNA sequence was used to define the intron/exon borders of the genomic clone. We designated this new gene Ly-6M because we isolated its cDNA from a macrophage library. Ly-6M is located 13.4 kb downstream of Sca-1 (Figure 3), roughly equidistant between Sca-1 and Sca-2 in the same transcriptional orientation as Sca-1. The genomic structure of Ly-6M (Figure 4) shows typical Ly-6 features (the first one and a half of 4 exons noncoding, subsequent introns and exons of progressively larger size).
Ly-6M is expressed in hematopoietic organs
Ly-6M is a GPI-anchored protein on myelomonocytic cells
Characterization of the pattern of cell surface protein expression
has been a fundamental goal in the study of hematopoietic and lymphoid
cell biology. Antibodies specific for the Ly-6 family of GPI-linked
proteins have been particularly useful in these studies. The Ly-6
antigen Sca-1 (Ly-6A/E) is commonly used to identify pluripotent
hematopoietic stem cells in mice.2,3 Similarly, Sca-2 and
Gr-1 (Ly-6G) are well-established markers of immature thymocytes and
mature myeloid cells, respectively.35,36 The known
Ly-6 genes, as well as perhaps a dozen uncharacterized genes or
pseudogenes, appear to be clustered on mouse chromosome 15.27 In this study, we have provided the first direct
demonstration of physical linkage of 2 family members (Sca-1
and Sca-2) on chromosome 15, band D3.1-3.3. In addition, we
have identified a novel family member that maps between the
Sca-1 and Sca-2 genes. This novel gene, which we
designate Ly-6M, is a prototypical family member with conserved
Ly-6 structural features and a pattern of expression relatively
restricted to hematopoietic tissue.
We thank Ms Sally Meek for expert technical assistance and Jeff Haug
for performing flow cytometric sorting. We also acknowledge helpful
discussions with Dr Roger Palfree, Dr Dan Link, and Dr Timothy Ley.
Submitted September 20, 1999; accepted January 18, 2000.
Supported in part by National Heart, Lung, and Blood Institute grant
K08 HL03872-01 (T.A.G.).
Reprints: Timothy Graubert, Washington University School of
Medicine, Division of Bone Marrow Transplantation and Stem Cell
Biology, Campus Box 8007, 660 S Euclid Ave, St Louis, MO 63110;
e-mail: graubert{at}medicine.wustl.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
CD8
-neurotoxins that contain Ly-6-like domains, but map to
nonsyntenic chromosomal locations and diverge structurally from the
core Ly-6 family (reviewed in Gumley et al
new insights and new members: a C-terminal Ly-6 domain in sperm acrosomal protein SP-10.
Tissue Antigens.
1996;48:71-79
