Structure and developmental regulation of the B-lymphoid tyrosine kinase gene blk.

The murine blk gene, which encodes a B-lymphoid-specific tyrosine kinase of the Src family (p55blk), contains 13 exons that span more than 30 kilobases of DNA on chromosome 14. In the first three exons, which encode the 5'-untranslated region and N-terminal amino acid sequence unique to p55blk, the blk gene differs from other members of the src family; in the last 10 exons, the organization of the blk gene is similar to that of other src genes. By primer extension and S1 nuclease protection analyses, we show that blk transcripts initiate from four major sites at the 5'-flank of blk; two sites predominate. The resulting transcripts differ only in the lengths of their 5'-untranslated sequences and encode identical proteins. None of the transcriptional start sites are preceded by consensus TATA elements, AT-rich elements, or extensive GC-rich regions. Expression of blk is regulated during B-cell development: blk RNA is expressed in all pro-B-, pre-B-, and mature B-cell lines examined, but is absent from plasma cell lines. Immunolocalization of p55blk in normal mouse spleen supports these observations: staining is restricted to lymphocytes and is concentrated in regions rich in B-cells; plasma cells and stromal cells are not stained with anti-Blk antibodies. Assays for RNA synthesis in isolated nuclei indicate that the lineage and developmental stage specificities of blk expression are regulated at least in part by changes in its rate of transcription.

The blk gene, which encodes the protein-tyrosine kinase p5fibLk, is the most recently identified member of a family that includes src, yes, fgr, lyn, fyn, hck, and lck (1). Several of these genes, notably k k , hck, fgr, and blk, exhibit restricted patterns of expression: lck is expressed predominantly in T-lymphoid cells (2), hck and fgr in cells of myeloid lineage (3)(4)(5)(6), and blk in B-lymphoid cells (7). Because blk is expressed specifically in cells of the B-lineage, it may function in a signal transduction pathway specific to B-lymphocytes. Several transmembrane proteins expressed on B-cells appear to signal through tyrosine phosphorylation yet lack intrinsic kinase activity. The best characterized include surface immunoglobulin (8-* This work was supported by the Howard Hughes Medical Institute and by Grant CA16519 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession number(s)
$ Trainee of the Medical Scientist Training Program of the National Institutes of Health. 10) and class I1 major histocompatibility complex (MHC)' antigens (11). Cross-linking of surface Ig appears to stimulate the enzymatic activities of Blk, Fyn, and Lyn (12). Ig and class I1 MHC antigens each have a distinctive pattern of expression during B-cell development, but both of them are expressed on mature B-cells and are absent from plasma cells (13,14). The evidence implicating ~5 5 "~ in signaling pathways involving one or both of these proteins led us to examine the structure and developmental regulation of the blk gene.
The earliest B-cell precursors in the mouse (pro-B-cells) are null with respect to expression of immunoglobulin heavy and light chains. These cells give rise to pre-B-cells, which are defined by the expression of immunoglobulin p-heavy chain in the absence of light chain. Both pro-B-and pre-Bcells express a polypeptide (X5) homologous to the constant region of an immunoglobulin X-light chain (15). In pre-Bcells, the X5 polypeptide is expressed at the cell surface in a complex with p and a third polypeptide, Vpre.~ (16)(17)(18). The transition from pre-B-to B-cell is marked by the expression of functional immunoglobulin light chain, the disappearance of X5 and Vpre.B, and the appearance of a complete immunoglobulin molecule at the cell surface. Expression of membrane immunoglobulin is lost when B-cells differentiate into antibody-secreting plasma cells (14). For each of these broad stages in B-cell development (pro-B-, pre-B-, and B-cells and plasma cell), there exist representative transformed analogues; such cell lines have facilitated the study of B-lymphoid-specific gene expression as a function of developmental stage (19).
The Src kinases are structurally homologous, as are the genes that encode them (20-22). The structures of the chicken c-src (23), human c-fgr (4,24), murine and human k k (25,26), and murine hck (27) genes have been examined in detail. The exons that encode the noncatalytic Src homology regions, the catalytic domain, and the C-terminal domain are homologous in sequence and organization, whereas the exons encoding the 5"untranslated regions and the N-terminal amino acids are dissimilar. The sites of transcriptional initiation have been determined for several members of the src family (24,25,(27)(28)(29). Multiple sites of transcriptional initiation are common, as is the absence of TATA or GC-rich sequences upstream of these sites. In the case of lck, two independent widely separated promoters are used differentially during Tcell ontogeny (30, 31), suggesting that distinct sets of transcriptional factors govern lck expression at different stages in development.
Here we describe the genomic organization and developmental regulation of the murine blk gene. The blk gene contains 13 exons that span >30 kb of DNA on mouse chromo-' The abbreviations used are: MHC, major histocompatibility complex; kb, kilobase(s); bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid. 4815 some 14. In the first three exons, which encode the 5'untranslated region and N-terminal amino acid sequence unique to p55*lk, the blk gene differs from other members of the src family; in the last 10 exons, the organization of the blk gene is similar to that of other src genes. Expression of blk was found to be regulated during B-cell development: blk RNA was detected in all pro-B-, pre-B-, and mature B-cell lines tested, but was absent from plasma cell lines. Immunolocalization of p55*lk in spleen was consistent with these observations: staining was restricted to lymphocytes and was concentrated in regions rich in B-cells; plasma cells and stromal cells did not react with the antibody. Measurement of relative transcription rates in isolated nuclei revealed that the lineage and developmental stage specificities of blk expression reflect regulation at the level of transcriptional initiation.

MATERIALS AND METHODS
Cell Lines-All lymphoid and erythroid cell lines, except the B-cell hybridoma LK, were grown in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, 50 pg/ml streptomycin, and 50 p M 2-mercaptoethanol. The LK cell line was maintained in 1:l RPMI 1640 medium/Eagle's Hanks' amino acid medium supplemented as described above. The NIH 3T3 and L(tk-) cell lines were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and 50 pg/ml streptomycin.
Antibodies and Immunohistochemistry-Anti-Blk antibodies were directed against a synthetic peptide (SD3) corresponding to amino acids 27-47 of mouse ~55'" (7). This peptide was cross-linked to keyhole limpet hemocyanin through glutaraldehyde or carbodiimide as described (32). New Zealand White rabbits were immunized intradermally with 250 pg of coupled peptide (1:l mixture of glutaraldehyde-and carbodiimide-coupled material). Booster immunizations (250 pg of peptide) were administered every other week beginning 21 days after the initial immunization. Antiserum was obtained at 8 weeks and anti-Blk antibodies were affinity-purified by adsorption to the SD3 peptide coupled to CH-Sepharose 4B. Bound antibodies were eluted with 200 mM glycine HCl (pH 2.3), 100 mM NaCl, 0.1% Triton X-100 followed by 100 mM triethylamine (pH 11.5) and were used at a 1:2000 dilution.
Immunohistochemical localization of p55'" was performed as follows. Spleens were obtained from C57BL/6 mice, fixed in 10% formalin in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HP0,, 1.8 mM KH2P0, (pH 7.4)), and embedded in paraffin; 4 -~m sections were cut. Sections were deparaffinized, treated with 3% hydrogen peroxide in Tris-buffered saline (137 mM NaC1, 2.7 mM KCl, 25 mM Tris-C1 (pH 7.4)), and blocked with 2% bovine serum albumin in Tris-buffered saline. Sections were then reacted with affinity-purified anti-SD3 antibodies alone or in the presence of a 4000-fold molar excess of homologous (SD3) or heterologous (SD5; amino acids 454-466 of ~5 5 "~) competitor peptide. Alternatively, sections were reacted with normal rabbit serum at 1:2000 dilution. After overnight incubation with primary antibodies at 4 "C, sections were incubated with biotinylated anti-rabbit IgG for 30 min at room temperature. Sections were stained with peroxidase-conjugated streptavidin and diaminobenzidine and counterstained with methylene blue. Plasma cells were identified by staining additional sections with methyl green/pyronin. DNA Probes-Restriction fragments used as hybridization probes were purified by electrophoresis through agarose and were labeled with 32P by the random priming method (33) to specific activities of 108-109 Cerenkov cpm/pg. The blk-specific probes used in this study included the EcoRI fragment of blk cDNA clone 205, spanning base pairs 1-1292 of blk cDNA, and the EcoRI-Mae1 fragment of clone 205, spanning base pairs 1-275 of blk cDNA (7).
Molecular Cloning of Murine blk Gene-Molecular clones spanning exons 1'-12 of blk were isolated from a mouse genomic DNA library that was constructed by insertion of partial MboI restriction frag-ments into the BamHI site of bacteriophage EMBL-3 (34) (Clonetech). The library was screened as described (7) by hybridization to 32P-labeled blk cDNA clone 205. DNA spanning exon 1 of blk was isolated from a size-selected EcoRI mouse genomic library (average insert size of -6 kb) that was constructed in the bacteriophage vector XZap (Stratagene). The library was screened by hybridization to the 32P-labeled EcoRI-Mae1 restriction fragment from blk cDNA clone 205. Positive bacteriophage were plaque-purified and isolated by isopycnic centrifugation in CsC1. Mouse genomic DNA was subcloned into pBluescript (Stratagene).
Restriction maps of murine genomic DNA were determined by standard methods. Nucleotide sequences of blk exons were determined by the dideoxynucleotide termination method (35); sequencing primers included synthetic oligonucleotides directed against sense and antisense strands of blk cDNA. The genomic sequence and the published cDNA sequence were found to differ at two positions. In the genomic sequence, an insertion of a C residue was found following nucleotide 35 in the 5"untranslated region of blk cDNA, and nucleotide 1611 was found to read A, whereas the published cDNA sequence has T. Resequencing the cDNA in the region in question showed the genomic sequence to be correct. This correction leads to a change in the predicted amino acid sequence of ~5 5 "~: residue 421 is E, not V as originally stated (7). Intron-exon boundaries were determined by comparison of genomic blk sequence to that of blk cDNA. Positions of exons were determined directly, by nucleotide sequence, or indirectly, by hybridization of exon-specific synthetic oligonucleotides to genomic clones that had been digested with various restriction endonucleases, fractionated by electrophoresis, and transferred to nitrocellulose.
RNA Isolation and RNase Protection Assays-Total cellular RNA from cell lines was isolated by organic extraction (36) or by the guanidinium isothiocyanate/cesium chloride method (37). Polyadenylated RNA was isolated by oligo(dT)-cellulose chromatography (38).
Radiolabeled RNA probes were prepared as described (39). To prepare a blk-specific probe, the plasmid p102T, which contains base pairs 1394-1878 of blk cDNA, was linearized with BamHI and transcribed in vitro with T3 RNA polymerase in the presence of [cP~*P] CTP (800 Ci/mmol). A 8-tubulin-specific probe was similarly synthesized from the BamHI-linearized form of plasmid pp5, which carries a 306-bp BamHI-StuI 8-tubulin cDNA fragment from pm85 (40). To permit comparison of blk and @tubulin signals from the same autoradiographic exposure, the 8-tubulin probe was synthesized at onetenth the specific activity of the blk probe. After transcription, DNA was removed by digestion with 400 units/ml RNase-free DNase I.
RNase protection assays were performed as described (39) with modifications. For each cell line assayed, the blk probe (3.5 x 10' cpm) and the 8-tubulin probe (3.5 X lo4 cpm) were mixed and annealed to 20 pg of total cellular RNA for 16 h at 54 "C in a reaction volume of 30 pl. After digestion with RNase A (40 pg/ml) and RNase T1 (2 pg/ml) for 30 min at 15 "C, proteinase K and sodium dodecyl sulfate were added to 150 pg/ml and 1%, respectively; and the reactions were incubated for an additional 15 min at 37 'C. Products were extracted with phenol/chloroform, precipitated with ethanol in the presence of 10 pg of tRNA, and resuspended in sample buffer containing 80% formamide. Products were fractionated by electrophoresis through a 4% polyacrylamide gel containing 7 M urea and detected by autoradiography.
Primer Extension and SI Nuclease Protection Assays-The 5'-ends of blk mRNA species were mapped by a combination of primer extension and S1 nuclease protection assays. Extension assays were performed with an oligonucleotide primer (SD205) complementary to nucleotides +213 to +233 in Fig. 2 (corresponding to nucleotides 196-216 of blk cDNA clone 205). The oligonucleotide was labeled at its 5'-end with 32P by polynucleotide kinase (specific activity of lo9 cpm/ pg), and the labeled primer was hybridized in excess (2 X 1 0 ' cpm, 30 fmol) to 20 pg of total RNA or 2 pg of poly(A+) RNA in a reaction mixture containing 150 mM KCl, 10 mM Tris-C1 (pH 8.0), 1 mM EDTA for 6 h at 55 "C. The extension reaction was begun by adding 2.3 volumes of a solution containing 65 mM Tris-C1 (pH 81, 14 mM dithiothreitol, 5.5 mM MgC12, 71 pg/ml actinomycin D, 1.4 mM each dATP, dCTP, dGTP, and dTTP, and 200 units of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories). The extension reaction was allowed to proceed at 42 'C for 1.5 h. Extension products were fractionated by electrophoresis through a 6% polyacrylamide gel containing 7 M urea. Products of G-, A-, T-, and C-specific dideoxynucleotide chain termination reactions, primed with radiolabeled SD205, were fractionated alongside the extension products as a reference.
The single-stranded antisense DNA probe used in the S1 nuclease protection assays was synthesized as follows. Oligonucleotide SD205 (see above) was labeled at its 5'-end with 32P and annealed to genomic clone Ab1123 (see Fig. l), which had been denatured in alkali. The primer was extended in a 100-p1 reaction mixture containing 10 mM Tris-HC1 (pH 8), 10 mM MgCl,, 1 mM each dATP, dCTP, dGTP, and dTTP, and 14 units of DNA polymerase I large fragment at 37 "C for 4 h. After heating at 68 "C for 5 min, the DNA was digested with the restriction endonuclease AccI. DNA was precipitated in ethanol, dissolved in alkaline gel sample buffer (30 mM NaOH, 1 mM EDTA (pH 8), 10% Ficoll, and 0.025% bromcresol green), and fractionated by electrophoresis through a 1.2% low melting point agarose gel containing 30 mM NaOH and 1 mM EDTA. A 776-nucleotide singlestranded radiolabeled probe was isolated (see Fig. 2C). S1 nuclease assays of blk transcripts were performed as described (41) with modifications. Total cellular RNA (40 pg) was hybridized to 10 pmol of the antisense blk probe at 50 "C in a 2 0 4 reaction mixture containing 80% formamide, 40 mM PIPES (pH 6.4), 1 mM EDTA, and 400 mM NaC1. After 12 h, 280 pl of a solution containing 280 mM NaCl, 50 mM NaOAc (pH 4.5), 4.5 mM ZnS04, 36 ng/pl single-stranded salmon sperm DNA, and 1.0 unit/pl S1 nuclease were added; and the mixture was incubated for 1 h at 25 "C. Products were fractionated by electrophoresis through a 6% polyacrylamide gel containing 7 M urea; 32P was detected by autoradiography. Products of G -, A-, T-, and C-specific dideoxynucleotide chain termination reactions, primed with radiolabeled SD205, were fractionated alongside the nuclease products as a reference.
Measurement of Relative Transcription Rates in Isolated Nuclei-Relative rates of blk transcription in nuclei isolated from the pro-Bcell line FE2NC1, the pre-B-cell line PD31, the B-cell lines BF0.3 and M12.4.5, the plasmacytoma cell lines SP2/0 and S107, and the erythroleukemia cell line MEL were estimated as described (42,43) with modifications. Cells (10') were grown to a density of 106/ml, collected by centrifugation, washed three times with ice-cold phosphate-buffered saline, and pelleted at 500 X g for 5 min. The cell pellets were resuspended in 8 ml of lysis buffer (10 mM Tris-C1 (pH 7.4), 10 mM NaC1,3 mM MgC12, 0.5% Nonidet P-40), incubated for 5 min on ice, and centrifuged at 500 X g for 5 min. The resulting nuclear pellets were washed once with 8 ml of lysis buffer and centrifuged at 500 X g. The nuclei were resuspended in 400 p1 of a solution containing 50 mM Tris-C1 (pH 8.3), 40% glycerol, 5 mM MgC12, 0.1 mM EDTA and frozen in liquid nitrogen in 2 0 0 4 aliquots. Nuclei were thawed at room temperature; transcription assays were performed in the presence of [w3'P]UTP, and products were isolated by organic extraction as described (36). Labeled RNA was assayed for hybridization to immobilized probes, which were prepared as follows. Plasmids (20 pg) carrying blk cDNA (pBS-blk), X5 cDNA (pB11; a gift of Dr. Shiv Pillai, Massachusetts General Hospital, Charlestown, MA), p-heavy chain cDNA (pp) (44), mouse 8-tubulin (pm85) (40), and the pBluescript vector (pBS) were linearized by digestion with Sal1 and denatured by incubation with 0.2 M NaOH for 30 min at room temperature. After addition of 10 volumes of 6 X SSC (1 X SSC = 0.150 M NaCl, 0.015 M sodium citrate), plasmid DNA was applied to nitrocellulose under vacuum. Radiolabeled RNA that had hybridized to each cDNA probe was detected by autoradiography.

RESULTS
Structure of blk Gene-Molecular clones of the murine blk gene were obtained from a mouse genomic DNA library in the bacteriophage vector EMBL-3 by hybridization to a radiolabeled blk cDNA probe. Except for the 5"untranslated region (nucleotides 1-349 of blk cDNA clone 205) (7), the entire blk cDNA sequence was represented on the overlapping inserts of recombinant bacteriophage Xblk25, Xblk28, and Xblk29 (Fig.  1). The remainder of the blk gene was molecularly cloned from a size-selected library of mouse genomic EcoRI fragments in the bacteriophage vector XZAP; two identical clones (Xblk3 and XblkB), each 5.6 kb in length, were independently isolated (Fig. 1).
The structure of the blk gene is shown in Fig. 1. The nucleotide sequence and location of each exon were determined as described under "Materials and Methods." Compar-ison with the cDNA sequence allowed the intron-exon boundaries of blk t o be defined, and these are summarized in Table   I. We previously mapped blk to mouse chromosome 14 (45); it is now seen that the gene contains 13 exons that span >30 kb. In conformity with the convention adopted for lck (26), the first exon is designated 1, the second l', and so forth. The first exon, 1, is untranslated and is separated from the second exon by >10 kb. The second exon, l', begins one nucleotide upstream of the initiator Met codon. At the predicted splice junctions, the intron sequences are in agreement with the consensus for splice donor and acceptor sites (46). Between residues 57 and 499, the amino acid sequence of p55*lk is similar to that of other Src kinases (58% identity between ~5 5~" and p60"", for example). In the corresponding genomic sequence, spanning exons 3-12, blk is remarkably similar to other src genes in that the exon boundaries are conserved (4,(23)(24)(25)(26). Since a polyadenylated cDNA for blk was not obtained, the 3'-end of the gene was not mapped directly. The nucleotide sequence of 580 bp downstream from the blk translational termination codon was determined. In this interval, a single potential polyadenylation signal (AATAAA) (47) was found 536-541 bp downstream from the blk termination codon. We suggest that this is likely to be the site at which blk mRNA is polyadenylated.
Transcription of blk Initiates at Four Major Sites-The sites at which blk transcription is initiated were mapped by S1 nuclease protection and primer extension with the probes depicted in Fig. 2C. T o generate an S1 nuclease probe, a synthetic 21-mer oligonucleotide (SD205), complementary to a portion of exon 1, was radiolabeled at its 5'-end, annealed t o blk genomic clone Xblk3, and extended. The product was cleaved with AccI at a site 776 bp upstream from the 5'-end of the primer and denatured to yield a single-stranded radiolabeled probe (Fig. 2C). RNA from the mature B-cell line BF0.3 protected four major radiolabeled fragments of 234, 180,154, and 144 nucleotides from S1 nuclease digestion (Fig.  2, A, lanes 3-9; C). When the same RNA was assayed by primer extension with radiolabeled oligonucleotide SD205, an extension product corresponding to each of the four S1 nuclease products was obtained (Fig. 2B, lanes 3-9). These S1 nuclease and primer extension products were not observed in reactions with tRNA (Fig. 2, A and B, lane 1) or with RNA from the erythroleukemia cell line MEL (Fig. 2, A  Taken together, these results map the major transcriptional initiation sites of blk to adenine residues that lie 275, 285, 311, and 365 bp upstream from the first intron-exon boundary (Fig. 3). Of these four nested transcripts, designated types I-IV, two predominate: a species containing a 365-nucleotide 5'-untranslated region (type I) and a species with a 285nucleotide 5"untranslated region (type 111). We used the primer extension assay to measure the relative abundance of type I and I11 transcripts at different stages of B-lymphoid development ( Fig. 3 and Table 11). Densitometric quantitation of extension products showed that type I11 RNA was roughly four times more abundant than type I RNA in most pro-Band pre-B-cell lines, but both were present at similar levels in mature B-cell lines (Table 11). In the case of the lck gene, transcription is driven by two distinct promoters: one upstream from exon 1, the other upstream from exon 1' (29- In conformity with the convention adopted for kk (26), the first exon is designated 1, the second l', and so forth.

TABLE I Nucleotide and amino acid sequences at the predicted splice junctions of blk
Numbers in parentheses indicate the length of each exon in base pairs. GT and AG dinucleotides bounding putative introns are underlined. Gaps are indicated by dashes. Codons preceding splice donor and acceptor sites are translated below the nucleotide seauence.
). In contrast, using a polymerase chain reaction-based assay, we failed to detect blk transcripts initiating upstream from exon 1' (data not shown). In Fig. 4, the transcriptional initiation sites are shown in the context of the nucleotide sequence at the 5"flank of the blk gene. None of the transcriptional start sites are accompanied by consensus TATA elements; the consensus TATA element nearest the blk gene is found at base pairs -423 to -419 relative to start site I. Notably, several sequence motifs known to function in the regulation of class I1 MHC gene expression are found within 546 bp of start site I. These include five sites conforming to a consensus y-interferon response element (CTKKANNY) (48)

FIG. 2. Transcription of blk initiates at multiple sites. A, S1
nuclease mapping of blk transcripts in BF0.3 cells. A 776-nucleotide single-stranded DNA probe specific for the 5'-flank of blk was generated by primer extension using a 32P-labeled oligonucleotide (SD205) specific for the blk 5'-untranslated region as the primer and Xblk3 as the DNA template (see "Materials and Methods" and C). This probe was annealed to 40 pg of total cellular RNA or tRNA, and S1 nuclease-resistant products were fractionated by electrophoresis through a urea-polyacrylamide gel. The 32P-labeled oligonucleotide SD205 was annealed to 40 pg of total cellular RNA or tRNA, and extended products were fractionated by electrophoresis through a urea-polyacrylamide gel. Lanes 1-9 were the same as described for A. The four major BF0.3-specific radiolabeled extension products are indicated by Roman numerals. Sizes of markers, in nucleotides, are indicated to the left. C, diagram of S1 nuclease probe and the products of S1 nuclease protection and primer extension. nt, nucleotide. three pre-B-, and nine mature B-cell lines and five plasmacytoma cell lines (Fig. 5). Total RNA was annealed, in the same reaction mixture, to probes specific for blk and &tubulin; protection of these probes was expected to yield radiolabeled species of 460 and 302 nucleotides, respectively. The 460nucleotide blk-specific species was detected in all pro-B-, pre-B-, and mature B-cell lines assayed with the exception of the earliest putative pro-B-cell line, BaF3 (Fig. 5, compare lanes  4-22 with lane 3). No blk RNA was found in any of the five plasmacytoma lines assayed (lanes [23][24][25][26][27] or in the myeloid lineage cell line RAW286 ( l a n e 28). Among the cell lines tested, little variation was seen in the yield of the 302nucleotide 0-tubulin-specific species (lanes . These data suggest that expression of blk begins very early in pro-B-cell development, continues in pre-B-and mature B-cells, and ceases upon differentiation of B-cells to plasma cells. To verify that the differential expression of blk RNA seen     1 1 1 2 1 3 1 4 nantly of primary and memory B-cells. In the red pulp, which is separated from the white pulp by the marginal sinus, erythrocytes predominate, although many plasma cells and some Tand B-cells are found there (54).

+I75 J&ASC_rSTST_ ST_gTSCTSG-S A S~A~G _ C~A -~A~G _ C~A C T _ C -A J c _ c l~A g T S -A~n _ r~A S~A G +235 _ALTCC_nlC&_CLG&TSrGGGI\T_ ~G S~T l T _ r~T -~A~C _ r~C~~C -C _ A~A S c _ r l c _ r -G S~T~C~& C~ IRE
Immunohistochemistry was carried out with an antibody directed against amino acids 27-47 of ~5 5 "~; the sequence of this peptide is not found in any other member of the Src family. Anti-Blk antibody stained a subset of small mononuclear cells concentrated in the follicular mantles of germinal centers (Fig. 6A) and scattered within the red pulp. In individual cells, the pattern of staining was consistent with the presence of ~55"' in the cytoplasm or plasma membrane, rather than in the nucleus. No peroxidase staining was seen when normal rabbit serum was used in the immune reactions (Fig. 6B). As a further control for specificity, approximate serial sections were reacted with anti-Blk antibody alone, in excess heterologous peptide, or in excess homologous peptide (see "Materials and Methods"); staining was specifically abolished in the presence of the homologous competitor (data not shown). We have previously shown that blk transcripts are quantitatively removed from the splenic mononuclear cell population upon selective depletion of surface Ig+,B220+ cells; this indicates that in spleen, blk is preferentially expressed in B cells (7). The pattern of staining with anti-Blk antibody reveals that cells expressing ~55'" are concentrated in the Bcell-rich follicular mantle and are scattered through the red pulp of mouse spleen. Plasma cells are readily identified in spleen sections by their distinctive morphology and histochemistry. A cluster of plasma cells is identified in Fig. 7.
These are large ameboid cells with pale eccentrically placed nuclei and prominent nucleoli (Fig. 7A); the cytoplasm is extensive and stains bright red with methyl greenlpyronin, reflecting a relative abundance of ribosomes (Fig. 7, B and C) (55). Such cells failed to react with anti-Blk antibody and were easily distinguished from reactive cells, which were small and spherical, with heterochromatic nuclei surrounded by a thin rim of cytoplasm (Fig. 7 , the plasmacytoma cell lines SP2/0 and S107, and the erythroleukemia cell line MEL; and radiolabeled nuclear transcripts were prepared as described under "Materials and Methods.'' Each preparation was hybridized to a filter containing a panel of immobilized DNA probes (Fig. 8). Radiolabeled transcripts were detected in nuclei from FE2NC1, PD31, and BF0.3 cells, but not in nuclei from MEL, M12.4.5, SP2/0, and S107 cells (Fig. 8). As expected (15), transcription of the X5 gene was observed only in nuclei from the immature B-lymphoid cell lines FE2NCl and PD31. Transcription through the immunoglobulin p locus was observed in all B-lymphoid cell lines assayed (M12.4.5 was not tested for p transcription) and in the erythroleukemia cell line MEL. The p-tubulin gene was transcribed at similar rates in nuclei from all cell lines tested (Fig. 8). We conclude that the lineage and developmental stage specificities of blk expression reflect, at least in part, differences at the level of transcriptional initiation.

DISCUSSION
Organization of blk Gene-The murine blk gene consists of 13 exons that span >30 kb of DNA on chromosome 14. The overall organization of blk is similar to that of murine and human lck, murine hck, chicken c-src, and human c-fgr. In its first three exons, blk is not homologous in nucleotide sequence to other src family members. Nonetheless, the configuration of these three exons resembles that of the T-lymphocyte kinase gene lck. In both blk and lck (26,31), exon 1 is untranslated and is separated from exon 1' by >10 kb of DNA; exons 1' and 2 encode N-terminal amino acid residues that show little similarity among Src kinases. In the case of ~5 6~~~ the N-terminal sequence has been shown to associate specifically with the T-cell-surface glycoproteins CD4 and CD8 (56-62); a comparable association may exist between the corresponding N-terminal sequence of ~5 5 "~ and a B-cellspecific transmembrane protein. A more general similarity is evident in exons 3-12; in blk, lck, hck, c-src, and c-fgr, these exons, which encode the conserved Src homology regions SH3 and SH2, the catalytic region, and a C-terminal regulatory region, are homologous with respect to nucleotide sequence and organization.
Regulation of blk Expression-We previously showed that blk is expressed specifically in cells of the B-lineage (7). In the present study, we have used a more sensitive assay to determine the pattern of blk expression in B-lymphoid cells.
A survey of cell lines representative of distinct stages in Bcell development suggests that blk RNA 1) first appears in pro-B-cells, prior to assembly and functional expression of immunoglobulin heavy chain genes; 2) is maintained in pre-B-and B-cells; and 3) ceases or is greatly diminished upon differentiation of B-cells to plasma cells. The developmental pattern of blk expression overlaps, but does not coincide with, the patterns of surface Ig and class I1 MHC expression. In contrast, the gene mb-1, which encodes a 34-kDa glycoprotein associated with surface Ig (63, 64), exhibits a pattern of expression similar to that of blk (data not shown) (65), suggesting that these genes may share common elements of control.
Imrnunolocalization of p55*lk in normal mouse spleen was consistent with these observations: expression of the protein is restricted to lymphocytes and is concentrated in regions rich in primary and memory B-cells; the antibody does not stain plasma cells, stromal cells, or regions rich in T-cells. By measuring the relative rates of blk transcription in isolated nuclei, we demonstrated that the lineage and developmental stage specificities of blk expression reflect differences at the level of transcriptional initiation.
Transcription of blk initiates at four major sites nested within a span of 91 bp. Of the resulting four transcripts, two predominate: a species containing a 365-nucleotide 5'-untranslated region (type I) and a species with a 285-nucleotide 5'-untranslated region (type 111). All four transcripts differ only in the lengths of their 5"untranslated sequences and encode identical ~55'" products. In pro-B-and pre-B-cell lines, type I11 RNA was approximately four times more abundant than type I RNA; both transcripts were present at similar levels in mature B-cell limes. Differences between the 5'untranslated regions of type I and 111 transcripts may result in altered translational efficiency. Transcripts of most mammalian genes do not contain Met codons upstream from the initiator codon, but 5'-AUG codons are common among members of the src family; in the case of Zck, removal of these codons greatly increases translational efficiency (66). The 5'untranslated region of blk type I RNA carries six AUG codons. Type 111 blk transcripts lack two of the AUG codons that are present in the 5'-untranslated region of type I transcripts, suggesting that type 111 transcripts may be translated more efficiently.
None of the blk transcriptional s t a r t sites are preceded by consensus TATA elements, AT-rich elements, or extensive GC-rich regions; a single consensus Sp-1-binding site is present. Many cellular genes do not contain TATA boxes; the promoters of such genes can be divided into two classes: those that are GC-rich, found primarily upstream from housekeeping genes, and those that are not GC-rich. The latter typically are not constitutively active, appear to be regulated during development, and initiate transcription at multiple clustered sites. A number of genes that are regulated during lymphocyte differentiation fall into this latter class, including Zck (25), c-fgr (24), X5 (15), (17), the terminal deoxynucleotidyltransferase gene (67), and the V, segments of the Tcell receptor gene (68).
In addition to blk, at least one other src family member, lyn, has been reported to be expressed preferentially in Blymphoid cells (69); the distribution of Zyn RNA, however, differs markedly from that of bllz. First, in murine tissues, Zyn RNA is found in thymus, kidney, liver, heart, and brain, albeit at lower levels than in spleen (70,71). Second, Z j n is expressed in platelets, macrophages, and monocytes as well as in Blineage cells (70). Third, lyn expression does not appear to be diminished upon differentiation of B-cells to plasma cells (70). It therefore seems reasonable that the transductory pathway(s) in which lyn functions are more broadly distributed than those involving blk. By sequence, bZk is most closely related to the src family members Zck and hck; this similarity is reflected in the genomic organization of the three genes. The homology found among src family members suggests that they arose through duplication of an ancestral gene. A phylogenetic map of tyrosine kinases, based on the sequences of their catalytic domains, places Zck on a branch distinct from that which includes c-src and c-fgr (1). The apparent phylogenetic clustering of blk, hck, and kk suggests that they share a common progenitor distinct from that of c-src and c-fgr. It is unclear whether the upstream regulatory sequences of these genes and the exons encoding the distinctive N termini of their products have arisen by divergence of common progenitor sequences or whether they were acquired as a result of exon shuffling. Regardless of the mechanisms by which they arose, these differences in gene structure have important consequences in that they define, at least in part, the tissues in which particular Src kinases are expressed and the transmembrane mol-ecules with which they associate.