Human Bone Marrow cDNA Library Research Articles

Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic anemia.

The first and the rate-limiting enzyme of heme biosynthesis is delta-aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the beta subunit of human ATP-specific succinyl CoA synthetase (SCS-betaA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-betaA associates specifically with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-betaA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-betaA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria.

The Platelet Collagen Receptor Glycoprotein VI Is a Member of the Immunoglobulin Superfamily Closely Related to FcαR and the Natural Killer Receptors

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

Utilization of an 86 bp exon generates a novel adducin isoform (β4) lacking the MARCKS homology domain

A novel isoform of β-adducin has been amplified and characterized from a human bone marrow cDNA library (GenBank #U43959). This isoform arises from the insertion of an 86 bp alternatively spliced and previously unrecognized exon (now termed exon 15) within codon 581 of the human red blood cell β-adducin sequence. This results in an insertion of 28 novel amino acids. The remainder of the red cell β-adducin mRNA is then translated in a different reading frame, adding an additional 35 novel amino acids prior to the stop codon. This new isoform, thus, replaces β1-adducin sequence after residue 580 with a total of 63 new amino acids. Sequences from genomic clones of the human β-adducin gene show that this alternate exon is flanked by splice consensus sequences and is appropriately located in the genomic map between exons encoding up-stream and down-stream sequences, thus defining a new exon. The COOH-terminus of this new isoform, which we designate β4, lacks a 22 amino acid lysine-rich sequence common to both the human red cell α- and β-adducin subunits and homologous to a highly conserved region in MARCKS, a filamentous actin-cross linking protein regulated by protein kinase C and calcium /calmodulin. β4-adducin preserves a previously identified calmodulin binding domain. PCR analysis indicates that this new β-adducin isoform is expressed in fetal brain and liver, bone marrow, and NT-2 (neuroepithelial) cells, but is not detected in several other tissues. We anticipate that this new β4 isoform of β-adducin will display unique and tissue-specific functional properties.

Expression of Bomapin, a Novel Human Serpin, in Normal/Malignant Hematopoiesis and in the Monocytic Cell Lines THP-1 and AML-193

AbstractOur group recently cloned the cDNA-encoding bomapin, a member of the serine protease inhibitor (serpin) superfamily, from a human bone marrow cDNA library (J Biol Chem 270:2675, 1995). To understand its expression within the hematopoietic compartment, RNA extracted from bone marrow or peripheral blood from normal donors and patients with leukemia was reverse transcribed and analyzed by polymerase chain reaction (PCR). Bomapin PCR products were readily detected in normal bone marrow, which was designated as a medium mRNA level. In peripheral blood, bomapin expression was low or undetectable in normal donors (n = 6) and patients with chronic lymphocytic leukemia (n = 6). Blood from patients with chronic myeloid leukemia (n = 6), chronic myelomonocytic leukemia (n = 6), acute myeloid leukemia (n = 5), and acute lymphocytic leukemia (n = 5) exhibited low to medium levels of bomapin expression. Furthermore, a high level of bomapin expression was detected in one individual with acute monocytic leukemia. These data suggest that bomapin expression may be elevated in hematopoietic cells of monocytic lineage. Therefore, we analyzed the expression of bomapin within cell lines that exhibited characteristics of the monocytic lineage. Bomapin PCR products were detected in the monocytic THP-1 and AML-193 cell lines but not in CRL 7607, CRL 7541, KG-1, or K562 cells. Induction of bomapin transcripts was not detected in the latter series of cell lines following a 24-hour treatment with phorbol myristate acetate (PMA, 10−8mol/L) or tumor necrosis factor-α (TNF-α, 30 U/mL), whereas treatment of THP-1 or AML-193 cells with these agents reduced the intensity of the bomapin PCR products. Northern blotting confirmed these results and showed that the expression of bomapin in THP-1 cells was downregulated over a 4-day period by PMA and, to a lesser extent, TNF-α. Immunoblotting was used to show the presence of a 40-kD protein in THP-1 cytosol preparations. Bomapin antigen levels were correspondingly reduced after treatment with PMA. Because PMA and TNF-α induce monocytic differentiation in THP-1 and AML-193 cells, these data increase the possibility that bomapin may play a role in the regulation of protease activities specifically in early stages of cellular differentiation.

Two unique 5′ untranslated regions in mRNAs encoding human 14-3-3ζ: differential expression in hemopoietic cells

In this report, we describe the identification and characterization of a novel 14-3-3 cDNA using the polymerase chain reaction and the screening of a human bone marrow cDNA library. This cDNA encodes the ζ isoform of 14-3-3 and contains a novel 5′ untranslated region (UTR) that is G+C rich and only 50% identical to the 5′ UTR in the human placental 14-3-3ζ cDNA, suggesting that 14-3-3ζ is encoded by at least two mRNAs. Using specific probes to the 5′ UTRs of bone marrow and placental 14-3-3ζ cDNAs, we studied the expression of each transcript in human hemopoietic cells at various stages of differentiation in the myeloid and lymphoid lineages. Differences in the expression of the bone marrow and placental 14-3-3ζ transcripts were found, the most notable being the markedly decreased expression of both 14-3-3ζ transcripts in HL-60 myeloid leukemic cells. Western blot analysis of 14-3-3ζ levels in HL-60 cells revealed correspondingly decreased levels of 14-3-3ζ protein compared to Jurkat cells. The differences among cell types of relative expression of the two 14-3-3 transcripts may reflect normal regulatory patterns, while the strikingly decreased expression of both types in HL-60 are more likely to be reflective of its multiple genetic abnormalities which contribute to its transformed phenotype.

Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.

A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.

Human Monocyte Chemotactic Protein-2: cDNA Cloning and Regulated Expression of mRNA in Mesenchymal Cells

Stimulated MG-63 osteosarcoma cells have been used as a source to purify and identify the monocyte chemokines MCP-1, MCP-2 and MCP-3. In comparison with MCP-1, the production yields of MCP-2 and MCP-3 in these cells are rather low and variable. Although the protein sequence of human MCP-2 is identified, its DNA sequence remains elusive. A degenerate primer set was used to isolate an MCP-2 gene fragment from the chemokine YAC contig on human chromosome 17. Based on the gene sequence of MCP-2, a unique primer set was synthesized and used to screen cDNA libraries for the presence of MCP-2 transcripts by PCR. The complete MCP-2 cDNA was cloned from a human bone marrow cDNA library and sequenced. The cDNA-derived protein sequence was identical to that of purified natural MCP-2, except for Gln46which replaced Lys46. There seem thus to exist two MCP-2 allelic variants because at position 46 the codons of two residues (Lys46and Gln46) were detected in individual genomes. As shown by Northern hybridization, the MCP-2 steady-state mRNA levels in normal diploid fibroblasts were increased by IL-1β, IFN-γ and the double-stranded RNA poly rI:rC. RT-PCR analysis showed induction of MCP-2 mRNA in MG-63 cells by IFN-γ and IL-1β. The regulated production of MCP-2 by tumor cells and normal mesenchymal cells is indicative of a role in neoplasia and inflammatory host responses.

PR-39, a proline-rich peptide antibiotic from pig, and FALL-39, a tentative human counterpart

The peptide antibiotic PR-39 was originally isolated from the upper part of pig intestine. It has antibacterial activity against Gram negative bacteria at concentrations comparable with tetracycline. Studies of the mechanism of action showed that PR-39 inhibits both DNA and protein synthesis. Recently, PR-39 was found in wound fluid and was shown to have inductive activity on matrix components as part of the wound repair process. We have now sequenced the complete gene and possible mediators of its expression will be discussed. Our attempts to characterize the human counterpart of PR-39 by probing for the well conserved prepro-part led to a different peptide antibiotic. A clone containing the coding information for this new peptide was isolated from a human bone marrow cDNA library. The putative human peptide antibiotic was designated FALL-39 after the first four residues and the total number of residues. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family with three disulfide bridges, while FALL-39 lacks cysteine. The clone for the prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the peptide. In the presence of the basal medium E, synthetic FALL-39 was highly active against Escherichia coli D21 and Bacillus megaterium Bm11. Residues 13–34 in FALL-39 can be predicted to form a perfect amphipatic helix and CD spectra showed that medium E induced 30% helix formation in FALL-39. By Northern blot analyses the transcript was located in bone marrow and testis. The structure of the gene and the chromosomal location is under investigation.

Functional Expression of Soluble Human Interleukin-11 (IL-11) Receptor α and Stoichiometry of in Vitro IL-11 Receptor Complexes with gp130

The interleukin-6 (IL-6) family of cytokines activates signaling through the formation of either gp130 homodimers, as for IL-6, or gp130-leukemia inhibitory factor receptor (LIFR) heterodimers as for ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, oncostatinM, and cardiotrophin-1. Recent in vitro studies with IL-6 and CNTF have demonstrated that higher order hexameric receptor complexes are assembled in which signaling chain dimerization is accompanied by the dimerization of both the cytokine molecule and its specific receptor alpha subunits (IL-6Ralpha or CNTFRalpha, respectively). IL-11 is a member of the IL-6 family and known to require gp130 but not LIFR for signaling. In this study we investigate the functional and biochemical composition of the IL-11 receptor complex. The human IL-11 receptor alpha-chain was cloned from a human bone marrow cDNA library. IL-11Ralpha was shown to confer IL-11 responsiveness to human hepatoma cells either by cDNA transfection or by adding a soluble form of the receptor (sIL11Ralpha) expressed in the baculovirus system to the culture medium. In vitro immunoprecipitation experiments showed that sIL11Ralpha specifically binds IL-11 and that binding is enhanced by gp130. Similarly to IL-6 and CNTF, gp130 is able to induce dimerization of the IL-11.IL-11Ralpha subcomplex, the result of which is the formation of a pentameric receptor complex. However, in contrast to the other two cytokines, IL-11 was unable to induce either gp130 homodimerization or gp130/LIFR heterodimerization. These results strongly suggest that an as yet unidentified receptor beta-chain is involved in IL-11 signaling.

SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis-specific gene product and to a rodent sperm-coating glycoprotein

A novel 28 kDa glycoprotein was purified from exocytosed material from human neutrophils and its primary structure partially determined. Degenerate oligonucleotide primers were used to amplify cDNA clones from a human bone marrow cDNA library. The deduced 245 amino acid sequence of the 2124 bp full-length cDNA showed high degrees of similarity to the deduced sequences of the human gene TPX-1 and of sperm coating glycoprotein from rat and mouse. Subcellular fractionation of human neutrophils indicated that the protein is localized in specific granules. The protein was named SGP28 (specific granule protein of 28 kDa).

Molecular Cloning of Bomapin (Protease Inhibitor 10), a Novel Human Serpin That Is Expressed Specifically in the Bone Marrow

Serine proteinase inhibitors or serpins are a super-family of homologous proteins that are for the most part involved in the regulation of proteolytic processes in a variety of biological systems. Utilizing a polymerase chain reaction-based strategy we have cloned a novel member of the ovalbumin family of serpins from a human bone marrow cDNA library. The new gene encodes a 397-amino acid protein, designated bomapin, with a calculated molecular mass of 45 kDa and 48% amino acid identity with plasminogen activator inhibitor-2, human leukocyte elastase inhibitor, and cytoplasmic antiproteinase. A single 2.3-kilobase bomapin transcript is highly expressed in human bone marrow cells but was undetectable in all other analyzed human tissues. In vitro transcription and translation of the bomapin cDNA revealed the synthesis of an appropriately sized protein that was able to form SDS-stable complexes with thrombin and trypsin. The restricted expression of bomapin to the bone marrow raises the possibility that this serpin may play a role in the regulation of protease activities during hematopoiesis.

The LW blood group glycoprotein is homologous to intercellular adhesion molecules.

The LW blood group antigens reside on a 42-kDa erythrocyte membrane glycoprotein that was purified by immunoaffinity and partially sequenced. From this information, a specific PCR-amplified DNA fragment was used to screen a lambda gt11 human bone marrow cDNA library. Two forms of cDNA were isolated; the first encoded a single spanning transmembrane protein of 270 amino acids, including a 29-amino acid peptide signal and four potential N-glycosylation sites, and the second encoded a shortened protein form of 236 residues devoid of transmembrane and cytoplasm domains. A rabbit antibody raised against the 15 N-terminal amino acids of the predicted protein reacted on immunoblots with authentic LW glycoprotein and in indirect agglutination test with all human erythrocytes except those from LW(a-b-). This showed that the protein encoded by these clones was LW gene product and suggested that the N terminus of the LW protein is oriented extracellularly. Most interestingly, the LW protein was found to exhibit sequence similarities (with approximately 30% identity) with intercellular adhesion molecules ICAM-1, -2, and -3, which are the counter-receptors for the lymphocyte function-associated antigens LFA-1. The extracellular domain of LW consists, like that of ICAM-2, of two immunoglobulin-like domains, and the critical residues involved in the binding of LFA-1 to ICAMs were partially conserved in LW.

Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite.

cDNA clones encoding the major subunit of the Duffy blood group were isolated from a human bone marrow cDNA library using a PCR-amplified DNA fragment encoding an internal peptide sequence of glycoprotein D (gpD) protein. The open reading frame of the 1267-bp cDNA clone indicated that gpD protein was composed of 338 amino acids, predicting a M(r) of 35,733, which was the same as a deglycosylated gpD protein. Portions of the predicted amino acid sequence, matched with six CNBr/pepsin peptides obtained from affinity-purified gpD protein. In ELISA analysis, an anti-Duffy murine monoclonal antibody reacted with a synthetic peptide deduced from the cDNA clone. Hydropathy analysis suggested the presence of 9 membrane-spanning alpha-helices. In bone marrow RNA blot analysis, the gpD cDNA detected a 1.27-kb mRNA in Duffy-positive but not in Duffy-negative individuals. It also identified the same size mRNA in adult kidney, adult spleen, and fetal liver; in brain, it detected a prominent 8.5-kb and a minor 2.2-kb mRNA. In Southern blot analysis, gpD cDNA identified a single gene in Duffy-positive and -negative individuals. Duffy-negative individuals, therefore, have the gpD gene, but it is not expressed in bone marrow. The same or a similar gene is active in adult kidney, adult spleen, and fetal liver of Duffy-positive individuals. Whether this is true in Duffy-negative individuals remains to be demonstrated. A GenBank sequence search yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors.

Multiple Rh messenger RNA isoforms are produced by alternative splicing

Three Rh-related cDNAs have been isolated from a human bone marrow cDNA library and by polymerase chain reaction (PCR) amplification of human bone marrow and erythroblast mRNAs. They potentially encode a family of Rh protein isoforms that exhibit several unexpected structural properties as compared with the Rh polypeptide encoded by the cDNA clone identified previously. These modifications include several peptide deletions, the predicted alteration of Rh protein topology within the cell membrane, variations in the number and surface exposition of cysteine residues, and the generation of new C-terminal polypeptide segments caused by frameshift mutations. The four Rh mRNAs now described correspond to different splicing isoforms transcribed from the same Rh gene, and all exist in the same cell lineage (erythroid). Moreover, PCR experiments indicated that at least three of these RNA species exist in reticulocytes from donors with different commonly expressed Rh phenotypes. Although the translated proteins have not yet been characterized, these results suggest that the two genes at the RH locus may direct the synthesis of several protein species possibly corresponding to different Rh antigenic variants.

Multiple Rh Messenger RNA Isoforms Are Produced by Alternative Splicing

Abstract Three Rh-related cDNAs have been isolated from a human bone marrow cDNA library and by polymerase chain reaction (PCR) amplification of human bone marrow and erythroblast mRNAs. They potentially encode a family of Rh protein isoforms that exhibit several unexpected structural properties as compared with the Rh polypeptide encoded by the cDNA clone identified previously. These modifications include several peptide deletions, the predicted alteration of Rh protein topology within the cell membrane, variations in the number and surface exposition of cysteine residues, and the generation of new C-terminal polypeptide segments caused by frameshift mutations. The four Rh mRNAs now described correspond to different splicing isoforms transcribed from the same Rh gene, and all exist in the same cell lineage (erythroid). Moreover, PCR experiments indicated that at least three of these RNA species exist in reticulocytes from donors with different commonly expressed Rh phenotypes. Although the translated proteins have not yet been characterized, these results suggest that the two genes at the RH locus may direct the synthesis of several protein species possibly corresponding to different Rh antigenic variants.

CDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III

cDNA encoding the human homologue of mouse APEX nuclease was isolated from a human bone-marrow cDNA library by screening with cDNA for mouse APEX nuclease. The mouse enzyme has been shown to possess four enzymatic activities, i.e., apurinic/ apyrimidinic endonuclease, 3′–5′ exonuclease, DNA 3′-phosphatase and DNA 3′ repair diesterase activities. The cDNA for human APEX nuclease was 1420 nucleotides long, consisting of a 5′ terminal untranslated region of 205 nucleotide long, a coding region of 954 nucleotide long encoding 318 amino acid residues, a 3′ terminal untranslated region of 261 nucleotide long, and a poly(A) tail. Determination of the N-terminal amino acid sequence of APEX nuclease purified from HeLa cells showed that the mature enzyme lacks the N-terminal methionine. The amino acid sequence of human APEX nuclease has 94% sequence identity with that of mouse APEX nuclease, and shows significant homologies to those of Escherichia coli exonuclease III and Streptococcus pneumoniae ExoA protein. The coding sequence of human APEX nuclease was cloned into the pUC18 SmaI site in the control frame of the lacZ promoter. The construct was introduced into BW2001 ( xth-11, nfo-2) strain and BW9109 (Δ xth) strain cells of E. coli. The transformed cells expressed a 36.4 kDa polypeptide (the 317 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide derived from the part of pUC18 sequence), and were less sensitive to methylmethanesulfonate and tert-butyl-hydroperoxide than the parent cells. The N-terminal regions of the constructed protein and APEX nuclease were cleaved frequently during the extraction and purification processes of protein to produce the 31, 33 and 35 kDa C-terminal fragments showing priming activities for DNA polymerase on acid-depurinated DNA and bleomycin-damaged DNA. Formation of such enzymatically active fragments of APEX nuclease may be a cause of heterogeneity of purified preparations of mammalian AP endonucleases. Based on analyses of the deduced amino acid sequence and the active fragments of APEX nuclease, it is suggested that the enzyme is organized into two domains, a 6 kDa N-terminal domain having nuclear location signals and 29 kDa C-terminal, catalytic domain.

Characterization of a new subgroup of human Ig V λ cDNA clone and its expression

From a human bone marrow cDNA library, we have cloned and sequenced a gene which cross-hybridized with murine pre-B cell-specific gene 8HS-20 cDNA under the low-stringent condition. Sequence analysis predicted that this gene (YM-1) encoded 240 amino acids which had the basic structure of immunoglobulin λ light chain. The 3' half of the YM-1 sequence was identical to the J λ2 C λ2 region except for four nucleotides. The 5' part of the gene had 87.6% sequence homology with the reported V λ gene called T1. Comparison of the deduced amino acid sequences with representative members of the seven other V λ subgroups showed considerable structural homology, but the maximum homology with these chains was 44%. Therefore, we conclude that YM-1 belongs to a new V λ subgroup. Interestingly YM-1 showed higher homology with VpreB1 (56%) than with any of the other V λ subgroups. By Southern blot analysis four to six cross-hybridizing V λ bands were detected at high stringency. Expression of the V λ gene was observed in immature as well as mature B cell lines without accompanying V-JC gene joining, suggesting that V λ of the YM-1 locus is activated at the early stage of maturation.

Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family.

CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and approximately 2 kilobases of 3' untranslated sequence containing a polyadenylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.

Molecular cloning and primary structure of Kell blood group protein.

The Kell blood group is a major antigenic system in human erythrocytes. Kell antigens reside on a 93-kDa membrane glycoprotein that is surface-exposed and associated with the underlying cytoskeleton. We isolated tryptic peptides and, based on the amino acid sequence of one of the peptides and by using the PCR, prepared a specific oligonucleotide to screen a lambda gt10 human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. All known Kell amino acid sequences were present in the deduced sequence; moreover, rabbit antibody to a 30-amino acid peptide, prepared from this sequence, reacted on an immunoblot with authentic Kell protein. The Kell cDNA sequence predicts a 732-amino acid protein. Hydropathy analysis indicates a single membrane-spanning region, suggesting that Kell protein is oriented with 47 of its N-terminal amino acids in the cell cytoplasm, and a 665-amino acid segment, which contains six possible N-glycosylation sites, is located extracellularly. Computer-based search showed that Kell has structural and sequence homology to a family of zinc metalloglycoproteins with neutral endopeptidase activity.

Molecular cloning and protein structure of a human blood group Rh polypeptide.

cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO4/polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. In RNA blot-hybridization (Northern) analysis, the Rh cDNA probe detects a major 1.7-kilobase and a minor 3.5-kilobase mRNA species in adult erythroblasts, fetal liver, and erythroid (K562, HEL) and megakaryocytic (MEG01) leukemic cell lines, but not in adult liver and kidney tissues or lymphoid (Jurkat) and promyelocytic (HL60) cell lines. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters.