All seven of a set of CD34 monoclonal antibodies that recognize epitopes on an approximately 110 Kd glycoprotein on human hemopoietic progenitor cells also bind to vascular endothelium. Capillaries of most tissues are CD34 positive, as are umbilical artery and, to a lesser extent, vein, but the endothelium of most large vessels and the endothelium of placental sinuses are not. Angioblastoma cells and parafollicular mesenchymal cells in fetal skin are also CD34 positive, as are some stromal elements. An approximately 110 Kd protein can be identified by Western blot analysis with CD34 antibodies in detergent extracts of freshly isolated umbilical vessel endothelial cells, and CD34 mRNA is present in cultured umbilical vein cells as well as other tissues rich in vascular endothelium (breast, placenta). These data indicate that the binding of CD34 antibodies to vascular endothelium is to the CD34 gene product, and not to crossreactive epitopes. Despite the presence of CD34 mRNA in cultured, proliferating endothelial cells, the latter do not bind CD34 antibodies. In addition, CD34 antigen cannot be upregulated by growth factors. We conclude that under these conditions, CD34 protein is downregulated or processed into another form that is unreactive with CD34 antibodies. Electron microscopy of umbilical artery, breast, and kidney capillary vessels reveals that in all three sites, CD34 molecules are concentrated on membrane processes, many of which interdigitate between adjacent endothelial cells. However, well-established endothelial cell contacts with tight junctions are CD34 negative. CD34 may function as an adhesion molecule on both endothelial cells and hematopoietic progenitors.

All seven of a set of CD34 monoclonal antibodies that recognize epitopes on an approximately 110 Kd glycoprotein on human hemopoietic progenitor cells also bind to vascular endothelium. Capillaries of most tissues are CD34 positive, as are umbilical artery and, to a lesser extent, vein, but the endothelium of most large vessels and the endothelium of placental sinuses are not. Angioblastoma cells and parafollicular mesenchymal cells in fetal skin are also CD34 positive, as are some stromal elements. An approximately 110 Kd protein can be identified by Western blot analysis with CD34 antibodies in detergent extracts of freshly isolated umbilical vessel endothelial cells, and CD34 mRNA is present in cultured umbilical vein cells as well as other tissues rich in vascular endothelium (breast, placenta). These data indicate that the binding of CD34 antibodies to vascular endothelium is to the CD34 gene product, and not to crossreactive epitopes. Despite the presence of CD34 mRNA in cultured, proliferating endothelial cells, the latter do not bind CD34 antibodies. In addition, CD34 antigen cannot be upregulated by growth factors. We conclude that under these conditions, CD34 protein is downregulated or processed into another form that is unreactive with CD34 antibodies. Electron microscopy of umbilical artery, breast, and kidney capillary vessels reveals that in all three sites, CD34 molecules are concentrated on membrane processes, many of which interdigitate between adjacent endothelial cells. However, well-established endothelial cell contacts with tight junctions are CD34 negative. CD34 may function as an adhesion molecule on both endothelial cells and hematopoietic progenitors.

The human haemopoietic cell surface antigen, CD34, is a 105 - 120 kd cell surface glycoprotein whose stage-specific expression by stem cells and lineage-specific progenitor cells suggests a role in regulating early events in blood cell differentiation. A murine gene and cDNA encoding a closely homologous protein have been isolated. The gene is organized in eight exons in 22 kb of DNA. The first exon lies in a GC- and CpG-rich island. The sequence of the gene and the cDNA predict a 382 amino acid-long protein containing an N-terminal signal peptide and one transmembrane region 73 amino acids from the C-terminus. The extracellular part of the protein contains: a 140 amino acid-long-N-terminal region, 40% of whose residues are serine or threonine potential attachment sites for O-linked carbohydrate, as well as five potential attachment sites for N-linked carbohydrate. Proximal to the extracellular membrane there is a 79 amino acid-long cysteine-rich region. The homology with the human sequence is highest in the intracellular domain (90% amino acid identity) and lowest in the N-terminal region (43% amino acid identity). The protein is not homologous with any other proteins currently in the databases. The expression of the murine gene by a number of haemopoietic progenitor cell lines suggests that the CD34 function in haemopoiesis may be conserved between man and mouse. The high level of expression in a number of embryonic fibroblast cell lines and in brain imply a function outside of haemopoiesis.

An increasing number of reports document instances in which individual leukemic cells coexpress markers normally believed to be restricted to a single lineage. This has been interpreted by McCulloch and colleagues as aberrant programming or lineage infidelity and contrasts with earlier suggestions that lineage fidelity of gene expression was usually maintained in leukemia. We argue that several examples of infidelity are suspect on technical grounds, whereas others are bona fide and require explanation, eg, partial rearrangements and expression of Ig heavy-chain and/or T cell receptor genes in inappropriate cells and terminal deoxynucleotidyl transferase in leukemic myeloblasts. Individual examples of truly aberrant gene expression may well occur in leukemia but with insufficient regularity to be of general significance. We suggest that verifiable and consistent examples of apparent lineage infidelity do not reflect genetic misprogramming but rather the existence of a transient phase of limited promiscuity of gene expression occurring in normal biopotential or multipotential progenitors and able to be preserved as a relic in leukemic blast cell populations that are in maturation arrest. This alternative explanation has interesting implications for mechanisms of hematopoietic differentiation and leads to some testable predictions.

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Messenger RNA has been isolated from cells of the human myeloma line 266BL which synthesizes IgE of the myeloma ND. A fraction enriched in mRNA for the e heavy chain was copied into DNA and the DNA was cloned in Escherichia coli A chemically synthesized oligonucleotide probe, based on the experimentally determined sequence of the specific message, was used to screen colonies. The largest E chain cDNA cloned, 2.0 kiobases, was characterized by restriction endonuclease mapping and DNA sequence analysis. It appears to encode the complete amino acid sequence ofthe E chain, including a signal peptide at the NH2 terminus as well as untranslated sequences at the 5' and 3' ends of the mRNA. The missing part ofthe previously published amino acid sequence.of the ND E chain was determined from the DNA sequence.The medical importance of IgE, which differs from other immunoglobulins in possessing an E heavy chain, stems from its central role in type 1 immediate hypersensitivity. IgE binds with high affinity to specific receptors on blood basophils, and its association with antigen then triggers an allergic reaction by causing degranulation of the cells (1). Because of the minute concentrations of IgE in normal serum (0.1 Ag/ml, compared with 13 mg/ml for IgG), studies on the purified protein have largely been confined to the secreted products of IgE myelomas. The incidence of IgE myelomas is low, reflecting the corresponding plasma cell population; only three cases (2-4) have been reported so far.For the study ofhuman e gene expression and the molecular and cellular bases of the pathogenicity of IgE, we wished to obtain a cloned DNA sequence that encoded a functional E chain. The usual path to such a clone is by way of isolation and enzymatic copying of mRNA from cells that synthesize the corresponding protein, followed by cDNA cloning. The usual source of immunoglobulin mRNA is an appropriate myeloma or myeloma cell line. In view of the rare occurrence of human IgE myelomas and the fact that human myelomas, in contrast to those of mouse and rat, are notoriously difficult to establish in culture, it was fortunate that Nilsson et aL (5, 6) were able to establish a cell line from the ND myeloma. Its recent adaptation to suspension culture greatly facilitated our endeavors in isolating E chain mRNA. The previously determined sequence of the ND e chain (7) enabled us then to prepare a genetic probe for selecting cDNA clones.The cloned DNA has been used to complete the protein sequence by analysis of the DNA, to correct the earlier variable region sequence and a part ofthe constant region sequence, and to determine the sequence ofthe NH2-terminal signal peptide.This new information has allowed us to identify the D and J elements expressed in the 266BL cell line. We have determined the DNA sequence encoding the 5' and 3' untranslated regions of the mRNA.

Physarum polycephalum nucleolar satellite DNA has been analysed by restriction enzyme digests and hybridisation to ribosomal RNA. The nucleolar DNA is isolated as molecules of molecular weight 39 x lo6. The positions of sites of endoR . EcoRI and endo R . Hind111 digestion have been determined accurately in the whole molecule. The endo R . EcoRI sites and one endo R . Hind111 site are within the 26-S rRNA complementary sequence and the other endo R . Hind111 site is within the 19-S rRNA complementary sequence. The sites are arranged symmetrically about the centre of the molecule as if it were a palindrome The Physarum polycephalum nucleolar satellite DNA is 1-2% of the nuclear DNA, contains the genes for 19-S and 26-S ribosomal RNA and is located in the nucleolus [I]. Hybridisation of ribosomal RNA to P. polycephalum nuclear DNA shows that there are about one hundred copies of these genes per nucleus [2-51. The occurrence of a high concentration of genes for rRNA in a satellite DNA that can be isolated in milligram quantities [6] together with the availability of a synchronous growth phase in P. polycephalum [7] makes these genes an important model system for study of control of eukaryote transcription and replication [8]. The existence of repeated gene-containing sequences in this satellite DNA suggested that the organisation of the genes in the repeat could be studied by analysis of the cleavage fragments produced by restriction enzymes and by hybridisation of ribosomal RNA to the fragments. EXPERIMENTAL PROCEDURE Isolation qf D N APhysarum polycephalum microplasmodia were grown in batches in two 5-1 fermenters with aeration on a rotary shaker using a growth medium essentially the same as that of Daniel and Baldwin [9]. Both strains axi and M~c were used. Nuclei were isolated by the method of Mohberg and Rusch [lo] in homogenising medium containing 1 mM CaC12. Nucleoli were isolated by sonicating the nuclei in the homogenising medium using an M.S.E. sonicator set at high power and amplitude 6 with the exponential probe tip diameter 0.125 in (0.318 cm). Sonication was carried out in 30-nil batches by 15-30-s bursts for 2min. The nucleoli and unbroken nuclei were pelleted by a 20-min spin at 1 500 x g and the sonication and pelleting repeated if necessary. The nucleoli were purified from unbroken nuclei and debris by spinning through a 5 -30 wjw sucrose gradient in an 'A' zonal rotor. The procedure is as described before [6] except that we now spin the zonal rotor for 30 min at 1600 rev./min. The DNA was isolated from the nucleoli by lysis in sodium dodecylsulphate, ribonuclease and pronase digestion, and chloroform deproteination, followed by purification of the satellite DNA from the main band by sedimentation to equilibrium in CsCl as described before [6]. Fig.1A shows the purity of the satellite DNA as checked by analytical CsCl gradient equilibrium centrifugation. Labelling and Isolation of Ribosomal R N AA 40-ml culture of P. polycephalum was grown overnight in the presence of 50 pCi/ml [32P]phosphate (...

We have analysed the organisation and expression of mu genes in the granulocytic phase and in the lymphoid and myeloid blast crises of Philadelphia chromosome (Ph1) chronic granulocytic leukaemia (CGL), a leukaemia which is known to arise in multipotential stem cells. We find that mu chain gene rearrangement occurs exclusively in lymphoid blast crisis leading in some, but not all, cases to the synthesis of small amounts of cytoplasmic mu chains characteristic of early pre‐B lymphocytes. In Southern blots, only one or two rearranged mu chain genes are seen, suggesting that a clonal event leading to blast crisis can occur in a committed B cell precursor rather than in the multipotential stem cell precursor, in which the Ph1 chromosome originated. The pattern of mu gene rearrangement observed in Ph1 CGL blast crisis is compared with that in normal B cells, other B lineage malignancies, myeloid leukaemias and T cell leukaemias.