Search

Sialophorin (CD43) of leukocytes and platelets is a surface sialoglycoprotein that is phenotypically defective on lymphocytes of patients with the X chromosome-linked immunodeficiency Wiskott-Aldrich syndrome. Previous studies with monoclonal antibodies indicate that sialophorin is a component of a T-lymphocyte activation pathway. Here we describe the cDNA cloning and derived amino acid sequence of human sialophorin. The sequence predicts an integral membrane polypeptide with an N-terminal hydrophobic signal region followed by a mucin-like 235-residue extracellular region with a uniform distribution of 46 serine, 47 threonine, and 24 proline residues. This is followed by a 23-residue transmembrane region and a 123-residue C-terminal intracellular region. These latter regions have been highly conserved during evolution; the intracellular region contains a number of potential phosphorylation sites that might mediate transduction of activation signals. The chromosomal location of the sialophorin gene was determined and the implications of this assignment for the pathogenesis of the Wiskott-Aldrich syndrome are discussed.

The T3 complex is composed of three polypeptide chains that are both structurally and functionally associated with the receptor for antigen on the surface of human T lymphocytes. In a series of experiments utilizing both somatic cell hybrids and chromosomal hybridization in situ, the genes encoding two members of the human T3 complex, T3-delta and T3-epsilon, were found to reside on the long arm of chromosome 11 in band q23. The murine T3-epsilon gene was localized to chromosome 9. The location of the T3-delta and T3-epsilon genes with respect to the Hu-ets-1 gene, which is also located in 11q23, is discussed. Recent assignments of several genes, preferentially expressed in human cells of hematopoietic and neuroectodermal origins, to band q23 of human chromosome 11 and the murine equivalents to murine chromosome 9 may define a conserved gene cluster important in cell proliferation and differentiation.

A cDNA that encodes the peptide core of the secretory granule proteoglycan of the human promyelocytic leukemic cell line, HL-60, has been isolated and analyzed. When human genomic DNA was digested and probed under conditions of low stringency with a rat cDNA that encodes a Mr = 18,600 serine/glycine-rich proteoglycan peptide core in L2 yolk sac tumor cells (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1321-1325) and basophilic leukemia-1 cells (Avraham, S., Stevens, R. L., Gartner, M. C., Austen, K. F., Lalley, P. A., and Weis, J. H. (1988) J. Biol. Chem. 263, 7292-7296), a number of DNA fragments were identified. A HL-60 cell-derived cDNA library was therefore screened under conditions of low stringency with the rat probe to identify and isolate a human homologue of this rat proteoglycan peptide core. Analysis of the resulting human cDNA clones indicated that the proteoglycan peptide core that is expressed in HL-60 cells is Mr = 17,600 and contains an 18-amino acid glycosaminoglycan attachment region that consists primarily of alternating serin and glycine. Northern blot analysis of total RNA probed with the human cDNA revealed that the major message for this proteoglycan peptide core in HL-60 cells is approximately 1.3 kilobase pairs in size. When a Southern blot of digested human genomic DNA was probed with the human cDNA, three bands of approximately 6, 9, and 12 kilobase pairs were detected. However, when the Southern blot was probed with the XmnI—-3' fragment of this human cDNA, one prominent band was detected, indicating that a single gene encodes this protein in the human. Analysis of the DNA from human/mouse and human/hamster somatic cell hybrids probed with the human cDNA demonstrated that the gene that encodes this molecule resides on human chromosome 10. Because the proteoglycans that are present in the secretory granules of different types of rat and mouse mast cells possess small peptide cores that are rich in serine and glycine, we propose that this HL-60 cell-3 derived cDNA encodes the peptide core of the proteoglycan that is expressed in the secretory granules of this human promyelocytic cell.

DNA amplification, manifested by homogeneously staining regions in chromosomes and by extrachromosomal, double minute bodies, is characteristic of many neuroblastoma cell lines. Sequences recruited from a specific domain on the short arm of chromosome 2 (2p) are amplified in advanced-stage primary neuroblastomas, whereas sequences from distinctly different regions of 2p are amplified in the neuroblastoma cell line IMR-32. Five different DNA segments, which include the oncogene N-myc, three other fragments derived from the homogeneously staining region of the neuroblastoma cell line IMR-32, and a fifth fragment, derived from the neuroblastoma cell line NB-9, showed differential and variable amplification in 24 advanced-stage neuroblastoma tumors out of 112 tested specimens. All five fragments were mapped within the chromosomal region 2p23-2p25 by three different approaches. However, eight other fragments cloned from the homogeneously staining region of IMR-32 cells, which were not amplified in the tumor tissues examined, were mapped to two more proximal domains of 2p, thousands of kilobases apart from each other and from the chromosomal domain that is amplified in the tumors. These results establish the amplification, to different degrees, of a variable-sized segment of one domain near the terminus of 2p in advanced neuroblastomas. These tumors might ultimately be distinguished according to the pattern of amplification of DNA segments within this domain. The data presented also indicate the existence of a new and complex amplification mechanism in at least one neuroblastoma cell line (IMR-32), which involves not only relocation of DNA from specific genomic domains but also the formation of novel units by splicing together very distant DNA segments.

Several phosphoglycerate kinase genes were previously detected in the human genome by blot hybridization with a phosphoglycerate kinase cDNA probe. Using subcloned fragments of the cDNA we estimate the presence of four independent phosphoglycerate kinase genes. These genes have been mapped to both the human X chromosome (band q13) and chromosome 6 (p12-21.1) using a panel of human-rodent somatic cell hybrids and by chromosomal in situ hybridization. The genomic distribution of phosphoglycerate kinase sequences is conserved in man and mouse, not only for the X chromosome, but also for linkage to the respective major histocompatibility complexes. Molecular cloning of X-linked phosphoglycerate kinase sequences led to the identification of a novel intronless phosphoglycerate kinase pseudogene which is localized proximal to the active gene on the X chromosome.

Using a mixture of synthetic 17-mer oligonucleotides encoding the 64 possible sequences for a peptide of adenosine deaminase as probe, we have isolated a clone for adenosine deaminase mRNA sequences from a collection of T-cell cDNA recombinants. This cDNA clone, phADA-1, contains an insert of 0.8 kilobase. In addition to the peptide chosen for synthesis of the oligonucleotide probe, the complete DNA sequence predicts 16 other experimentally determined peptides. Mapping of total cellular human DNAs with several restriction enzymes revealed relatively simple patterns of hybridization with phADA-1 as probe, including a polymorphism for PvuII cleavage. In agreement with previous studies, the adenosine deaminase gene was localized by blot hybridization to chromosome 20 in a hybrid cell mapping panel. Using the cDNA as probe, an 18-kilobase EcoRI fragment of human cellular DNA was also cloned in bacteriophage Charon 4A. These adenosine deaminase clones will prove valuable in the full characterization of the cellular gene, molecular analysis of inherited enzyme deficiency associated with immunodeficiency, and regional mapping of human chromosome 20.

cDNA clones encoding human apolipoprotein CII (apo CII) were identified by screening an adult human liver cDNA library with a mixed oligonucleotide probe corresponding to all possible codons for apo CII amino acid 6-10. One clone with an approximately equal to 500-base-pair (bp) insert, designated pCII -711, was selected for DNA sequence analysis. This clone contained a DNA sequence that corresponded with the previously reported amino acid sequence of apo CII with only minor differences. The DNA sequence specified a polypeptide of 79 amino acids, compared to the 78 amino acids previously reported. The pCII -711 clone contains a 36-bp DNA sequence upstream from that specifying the NH2-terminal threonine which, when read in frame, specifies the amino acid sequence Leu-Val-Leu-Leu-Val-Leu-Gly-Phe-Glu-Val-Gln-Gly and may be part of an apo CII signal peptide. The pCII -711 clone also contains a 144-bp region that corresponds to the 3' untranslated region of apo CII mRNA as well as a portion of the poly(A) tail. Clone pCII -711 was used to isolate and characterize by restriction endonuclease digestion the gene for apo CII from a human genomic library. In addition, through Southern blot analysis of DNA from human-rodent somatic cell hybrids, clone pCII -711 also was used to provisionally map the gene for apo CII to human chromosome 19.

The human apo-E gene has been isolated from a lambda phage library using as a probe the previously reported apo-E cDNA clone pE-301. Lambda apo-E was mapped and subcloned, and the apo-E gene was completely sequenced. The DNA sequence was compared with that of a near full length cDNA clone pE-368 and revealed three introns. The first intron was in the region that corresponds to the 5' untranslated region of apo-E mRNA. The second intron interrupted the codon specifying amino acid -4 of the apo-E signal peptide. The third intron interrupted the codon specifying amino acid 61 of the mature protein. Analysis of the DNA sequence revealed four Alu sequences. Two were in opposite orientations in the second intron, and one each occurred in the regions 5' and 3' to the apo-E gene. There were two base differences between the apo-E gene sequence and the sequence derived from the cDNA clones. At the codon for amino acid residue 112, the apo-E gene contained CGC, specifying Arg, whereas the cDNA contained TGC, specifying Cys. The other base difference was in the area corresponding to the 5' untranslated region of apo-E mRNA. Apo-E is commonly polymorphic in the population and the data suggest that the genomic clone was derived from the epsilon 4 apo-E allele, whereas the cDNA clones were derived from the epsilon 3 apo-E allele. S1 nuclease protection and primer extension experiments allowed the tentative assignment of the cap site of apo-E mRNA to the A approximately 44 base pairs upstream of the GT that begins the first intron. The sequence TATAATT was identified beginning 33 base pairs upstream of the proposed cap site and is presumably one element of the apo-E promoter. Finally, the apo-E gene was mapped in the human genome to chromosome 19 through the use of DNA probes and human-rodent somatic cell hybrids.

One in 10,000 children develops Wilms' tumour, an embryonal malignancy of the kidney1. Although most Wilms' tumours are sporadic, a genetic predisposition is associated with aniridia, genito-urinary malformations and mental retardation (the WAGR syndrome)2. Patients with this syndrome typically exhibit constitutional deletions involving band p13 of one chromosome 11 homologue3,4. It is likely that these deletions overlap a cluster of separate but closely linked genes that control the development of the kidney, iris and urogenital tract (the WAGR complex). A discrete aniridia locus, in particular, has been defined within this chromosomal segment by a reciprocal translocation, transmitted through three generations, which interrupts 11p13 (ref. 5). In addition, the specific loss of chromosome 11p alleles in sporadic Wilms' tumours has been demonstrated, suggesting that the WAGR complex includes a recessive oncogene6–9, analogous to the retinoblastoma locus on chromosome 13 (ref. 10). In WAGR patients, the inherited 11p deletion is thought to represent the first of two events required for the initiation of a Wilms' tumour, as suggested by Knudson from epidemiological data11. We have now isolated the deleted chromosomes 11 from four WAGR patients in hamster–human somatic cell hybrids, and have tested genomic DNA from the hybrids with chromosome 11-specific probes. We show that 4 of 31 markers are deleted in at least one patient, but that of these markers, only the gene encoding the β-subunit of follicle-stimulating hormone (FSHB) is deleted in all four patients. Our results demonstrate close physical linkage between FSHB and the WAGR locus, suggest a gene order for the four deleted markers and exclude other markers tested from this region. In hybrids prepared from a balanced translocation carrier with familial aniridia5, the four markers segregate into proximal and distal groups. The translocation breakpoint, which identifies the position of the aniridia gene on 11p, is immediately proximal to FSHB, in the interval between FSHB and the catalase gene.

C1 inhibitor is a heavily glycosylated plasma protein that regulates the activity of the first component of complement (C1) by inactivation of the serine protease subcomponents, C1r and C1s. C1 inhibitor cDNA clones have been isolated, and one of these (pC1INH1, 950 base pairs) has been partially sequenced. Sequence analysis demonstrates that the C1 inhibitor is a member of the serpin "superfamily" of protease inhibitors. In the region sequenced, C1 inhibitor has 22% identity with antithrombin III, 26% with alpha 1-antitrypsin and alpha 1-antichymotrypsin, and 18% with human angiotensinogen. C1 inhibitor has a larger amino-terminal extension than do the other plasma protease inhibitors. In addition, inspection of residues that are invariant among the other protease inhibitors shows that C1 inhibitor differs at 14 of 41 of these positions. Thus, it appears that C1 inhibitor diverged from the group relatively early in evolution, although probably after the divergence of angiotensinogen. Southern blot analysis of BamHI-digested DNA from normal individuals and from rodent-human somatic cell hybrid cell lines (that contain a limited but varied human chromosome complement) was used to localize the human C1 inhibitor gene to chromosome 11.

ApoB is a large glycoprotein with an apparent molecular mass of 550 kDa on NaDodSO4/PAGE. It is a major constituent of most lipoproteins and plays an important role in their metabolism. Recently, apoB cDNA clones have been isolated from an expression library made with mRNA from a human hepatoma cell line. These clones, which were all 1.5-1.6 kilobases (kb) long and corresponded to the 3' end of apoB mRNA, were used to demonstrate that hepatic apoB mRNA is approximately 22 kb long. In the current report, a probe derived from one of these cDNA clones, pB8, was used for in situ hybridization experiments to map the human gene for apoB, APOB, to the distal half of the short arm of chromosome 2. This probe was also used to analyze somatic cell hybrids and, in agreement with the in situ hybridization studies, concordancy was demonstrated with chromosome 2. In addition, two hybrids with chromosome 2 translocations that contain only the short arm reacted with the pB8 probe. A third hybrid with a complex rearrangement of chromosome 2, which deleted an interstitial region and the tip of the short arm of chromosome 2, did not react. These data indicate that APOB maps to either 2p21-p23 or 2p24-pter. In further studies, DNA from normal individuals, digested with the restriction endonuclease EcoRI and subjected to Southern blot analysis with the pB8 probe, revealed a two-allele restriction fragment length polymorphism (RFLP). The major allele was 11 kb, and the minor allele was 13 kb. The minor allele was present with a frequency of 20-25%. The inheritance of the two alleles was studied in an informative family, and they segregated in a typical autosomal Mendelian fashion. The mapping studies provide the means for understanding the relationship of the APOB locus to others in the human genome, whereas the demonstration of an APOB RFLP increases our ability to assess the role of this locus in determining plasma lipoprotein levels.

The gene encoding the 20-kDa glycoprotein of the T3-T-cell receptor complex (T3-delta chain) has been mapped to human chromosome 11 by hybridization of a T3-delta cDNA clone (pPGBC#9) to DNA from a panel of human-rodent somatic cell hybrids. In Southern blotting experiments with DNAs of somatic cell hybrids that contained segments of chromosome 11, we were able to assign the T3-delta gene to the distal portion of the long arm of human chromosome 11 (11q23-11qter). By use of a newly developed cDNA clone (pPEM-T3 delta) that codes for the murine T3-delta chain, the mouse T3-delta gene was mapped on chromosome 9. The importance of the T3-delta map position and its relationship to the other genes on the long arm of human chromosome 11 and to those on mouse chromosome 9 is discussed.

A cDNA clone encoding the alpha chain of the human T cell receptor was used in connection with somatic cell human-rodent hybrids to determine that the genes coding for the alpha chain are located on chromosome 14 in humans. In situ hybridization confirms this result and further localizes these genes to 14q11-14q12 on this chromosome. Since this region of chromosome has been shown to be nonrandomly involved in a number of T cell neoplasias, this assignment raises a number of interesting questions as to the possible involvement of the T cell receptor alpha chain genes in tumorigenesis.

The human mannose-binding protein (MBP) plays a role in first line host defense against certain pathogens. It is an acute phase protein that exists in serum as a multimer of a 32-kD subunit. The NH2 terminus is rich in cysteines that mediate interchain disulphide bonds and stabilize the second collagen-like region. This is followed by a short intervening region, and the carbohydrate recognition domain is found in the COOH-terminal region. Analysis of the human MBP gene reveals that the coding region is interrupted by three introns, and all four exons appear to encode a distinct domain of the protein. It appears that the human MBP gene has evolved by recombination of an ancestral nonfibrillar collagen gene with a gene that encodes carbohydrate recognition, and is therefore similar to the human surfactant SP-A gene and the rat MBP gene. The gene for MBP is located on the long arm of chromosome 10 at 10q11.2-q21, a region that is included in the assignment for the gene for multiple endocrine neoplasia type 2A.

Complementary DNA clones corresponding to the human serum amyloid P component (SAP) were isolated, and the complete nucleotide and derived amino acid sequence of preSAP was determined. PreSAP biosynthesis is directed by a 1.1-kilobase mRNA. Synthesis and postsynthetic processing of preSAP in Xenopus oocytes result in secretion of a protein with mobility similar to native purified SAP when analyzed by sodium dodecyl sulfate gel electrophoresis. The human SAP gene is on chromosome 1, probably closely linked to the gene for C-reactive protein which encodes the related acute phase reactant found in human plasma.

Profilin is an ubiquitous 12-15-kDa actin monomer-binding protein, the amino acid sequence of which was previously reported for the cow and Acanthamoeba. In the latter species, two isoforms of profilin have been identified. We have isolated full-length profilin cDNA clones from a human HepG2 library. All clones have the same nucleotide sequence, and Northern blot and RNase protection analyses of human tissues indicate that all tissues have the same approximately 850 base message, and provide no evidence of alternative message splicing. This result strongly implies a single profilin isoform in human cells, although differential post-translational modifications have not been excluded. Northern blot analysis extends the tissue distribution of profilin to include epithelial, muscle, and renal tissues. Comparison of the predicted human profilin amino acid sequence with that of published bovine profilin indicates 90% identity with a single 3-residue deletion in the human sequence. Southern blot analysis of somatic cell hybrid DNA indicates at least four dispersed genetic loci in the human genome hybridize with the profilin cDNA as well as untranslated region fragments, suggesting several of these loci represent pseudogenes of recent evolutionary origin. In addition, 5‘ and 3‘ untranslated regions are conserved between humans and rodents, implying a functional role for these regions of the profilin gene.