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Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%-37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88-6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27. 1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15-20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

A novel chromosomal translocation, t(2;11)(q31;p15), was identified in a patient with therapy-related acute myelogenous leukemia (t-AML). Fluorescence in situ hybridization experiments mapped the breakpoint near NUP98; Southern blot analysis demonstrated that the nucleoporin gene NUP98 was disrupted by this translocation. We used rapid amplification of cDNA ends to identify a chimeric mRNA. An in-frame, chimeric mRNA that fused NUP98 sequences to the homeobox gene HOXD13 was cloned; the predicted fusion protein contains both the GLFG repeats from NUP98 as well as the homeodomain from HOXD13. The NUP98-HOXD13 fusion is structurally similar to the NUP98-HOXA9 fusion previously identified in patients with AML, leading to the speculation that NUP98-homeobox gene fusions may be oncogenic. Moreover, this report, along with a recent study that demonstrated NUP98-DDX10 fusions in patients with t-AML, raises the possibility that NUP98 may be a previously unsuspected target for chromosomal translocations in patients with t-AML.

Fertilin alpha/beta (PH30 alpha/beta) is a heterodimeric sperm surface protein containing binding and fusion domains with potential for interaction with integrin receptors on the oocyte. We report the cDNA cloning, deduced amino acid sequence, tissue specificity, and chromosomal mapping of human fertilin beta. Encoded by a 2205 nucleotide open reading frame, the deduced amino acid sequence of human fertilin beta contains pro-, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like (EGF) repeat, transmembrane, and cytoplasmic domains. Due to this domain organization, human fertilin beta has been identified as a member of the ADAM family, which is composed of membrane-anchored proteins having A Disintegrin And Metalloprotease domain. The amino acid sequence of human fertilin beta shares 90%, 56%, and 55% identity, respectively, to monkey, guinea pig, and mouse fertilin beta homologs. A phenylalanine-glutamate-glutamate (FEE) binding tripeptide within the disintegrin-like domain of human fertilin beta, homologous to other fertilin beta RGD-like (arginine-glycine-aspartic acid) tripeptides, could compete for recognition by integrins and other receptors. Northern analysis from 16 human tissues revealed human fertilin beta's 2.9 kb message only in testis, which raises interest in possible clinical applications of this molecule as a contraceptive vaccinogen. Human fertilin beta maps to chromosome 8, band p11.2, by fluorescence in situ hybridization and mouse/human somatic cell hybrid Southern hybridization.

Type I procollagen COOH-terminal proteinase (C-proteinase) enhancer, a glycoprotein that binds to the COOH-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity, was purified from mouse fibroblast culture media. Partial amino acid sequences obtained from proteolytic fragments were found to have identity with the deduced amino acid sequence of a cDNA clone of unknown function, previously isolated from a mouse astrocyte library. Sequences of mouse enhancer cDNA, obtained in the present study, predict a approximately 50-kDa, 468-amino acid protein that differs from the 43-kDa, 402-amino acid protein predicted by the previously reported astrocyte-derived clone. Human cDNAs encode an enhancer of 449 amino acids. Previous biochemical studies have found the mouse enhancer as a 55-kDa form, which is readily processed to 36- and 34-kDa forms, retaining full C-proteinase enhancing activity and the ability to bind the COOH-terminal propeptide. Data presented here show the 36-kDa form to correspond to the amino-terminal portion of the 55-kDa protein. This is the most conserved region between mouse and human enhancers, comprising two domains with homology to domains found in a number of proteases and proteins with developmental functions. Such domains are thought to mediate interactions between proteins. Mouse enhancer RNA is shown to be at highest levels in collagen-rich tissues, especially tendon. The human enhancer gene, PCOLCE, is localized to 7q21.3--q22, the same chromosomal region containing the type I collagen alpha 2 chain gene, COL1A2.

Retrovirus infection is initiated by binding of the viral envelope glycoprotein to a cell-surface receptor. The envelope proteins of type C retroviruses of mammals demonstrate similarities in structural organization and protein sequence. These similarities suggest the possibility that retroviruses from different interference groups might use related proteins as receptors, despite the absence of any relationship between retrovirus receptors isolated to date. To investigate this possibility, we have identified a human cDNA clone encoding a protein closely related to the receptor for gibbon ape leukemia virus and have found that it functions as the receptor for the amphotropic group of murine retroviruses. Expression of this protein (GLVR-2) is likely to be a requirement for infection of human cells by amphotropic retroviral vectors for purposes of gene therapy.

The primary structure of the human laminin M chain was determined from cDNA clones isolated from human placental libraries. The clones covered a total of 6,942 bp, with 49-bp encoding a 5' end untranslated region and 6,893-bp coding for a translated sequence. The complete human laminin M chain contains a 22-residue signal peptide and 3,088 residues of the mature M chain. The M chain has a domain structure similar to that of the human and mouse A chains. The homology between the two human laminin heavy chains is highest in the short arm region and lowest in the long arm helical domain I + II. Northern blot analysis of human fetal tissues showed that the M chain was expressed in most tissues such as cardiac muscle, pancreas, lung, spleen, kidney, adrenal gland, skin, testis, meninges, choroid plexus, and some other regions of the brain, but not in liver, thymus, and bone. In situ hybridization localized the expression of the M chain gene to cells of mesenchymal origin. In contrast, expression of the A chain was observed only in kidney, testis, neuroretina and some region of brain as determined by Northern analyses. Epithelial and endothelial cells were negative for both M and A chain gene transcripts. The gene for the human M chain (LAMM) was localized to chromosome 6q22-->23.

The human beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1) (SiaT-1) gene is localized to human chromosome 3 (q21-q28) by Southern analysis of somatic cell hybrids and by in situ hybridization of metaphase chromosomes. Comparative analysis between the human and the previously reported rat SiaT-1 genomic sequences demonstrates precise conservation of the intron/exon boundaries throughout the coding domains. Furthermore, there is extensive inter-species sequence similarity in some of the exons that contain information only for the 5'-leader regions. Human genomic sequences were also analyzed to reconcile reported differences in the 5'-untranslated region in SiaT-1 mRNAs. In cultured cell lines of the B-lineage, Reh, Nalm-6, Jok-1, Ball-1, Daudi, and Louckes, the study demonstrates that three upstream exons, Exons(Y+Z) and Exon(X), are mutually exclusively utilized, resulting in at least two distinct populations of SiaT-1 mRNA being synthesized. None of these exons is present in the SiaT-1 mRNA isotype expressed in HepG2 human hepatoma cells. In all B-lymphoblastoid cell lines examined, the basal level SiaT-1 mRNA is maintained by the expression of an isotype containing the Exons(Y+Z) sequence. The slightly smaller SiaT-1 mRNA, which contains the Exon(X) sequence but not Exons(Y+Z) sequence, is synthesized at a high level and found only in Jok-1, Daudi, and Louckes, the cell lines with mature B-cell phenotype. The study also provides further evidence that induced SiaT-1 expression accompanies the appearance of CDw75, a putatively sialylated cell surface epitope and a marker of human mature B-lymphocytes. The SiaT-1 induction is the result of the appearance of a novel form of SiaT-1 mRNA isotype.

Lysyl oxidase catalyzes the oxidation of lysine residues to alpha-aminoadipic-delta-semialdehyde. This is the first step in the covalent cross-linking of collagen and tropoelastin and results in the formation of insoluble collagen and elastic fibers in the extracellular matrix. We have characterized the complete nucleotide sequence of human lysyl oxidase (EC 1.4.3.13) and compared the derived amino acid sequence (417-amino acids) to rat lysyl oxidase and the mouse ras recision gene (rrg). 88% of amino acids and 83% of nucleotides were conserved between human and rat lysyl oxidase. The mouse ras recision gene demonstrated 89% conservation of amino acids with human lysyl oxidase. The sequence conservation was not evenly distributed along the molecule. The carboxy terminus of the protein, which contains the putative copper binding sites and is likely to be the catalytically active domain, was more highly conserved than the amino terminus. The 89% amino acid sequence similarity between the murine ras recision gene and human lysyl oxidase suggests that they are the same gene product. Therefore, in addition to cross linking of extracellular matrix proteins, lysyl oxidase may have a direct role in tumor suppression. Northern blot analysis of poly A+RNA from cultured skin fibroblasts revealed at least three-distinct transcripts, sized 4.8 kb, 3.8 kb and 2.0 kb. In addition, using a panel of human mouse cell hybrids, the lysyl oxidase gene was assigned to human chromosome 5.

TNF is a proinflammatory cytokine that has pleiotropic effects on cells and tissues, mediated in large part by alterations in target tissue gene expression. We have used the technique of differential hybridization to identify several primary response genes induced by TNF in human umbilical vein endothelial (HUVE) cells, a cell type that is profoundly activated by cytokine treatment. One of these cDNA, designated B94, detects a rapidly and transiently induced 4-kb transcript in TNF-treated HUVE cells, and this transcript is superinduced in the concomitant presence of cycloheximide. Other proinflammatory stimuli including IL-1 beta and LPS are also able to induce B94 mRNA expression. Nuclear run-on experiments demonstrate that TNF induction of B94 transcript occurs primarily at the level of transcriptional activation. Further, B94 is shown to be a single copy gene that is evolutionarily conserved. The gene is localized to the q32 region of chromosome 14, a region that is often rearranged in lymphoid neoplasms. B94 transcript expression is also found to be regulated during mouse development and in an in vitro model of endothelial capillary tube formation. Developmental regulation occurs most prominently in mouse embryonic liver and kidney, and a second smaller form of B94 transcript is detected in the placenta and testes. B94 and other TNF-responsive transcripts are also induced during capillary tube formation suggesting overlap between genes induced by TNF and those induced during angiogenesis. Sequence analysis of the B94 cDNA reveals an open reading frame encoding a 73-kDa polypeptide that has no homology to any known protein. Polyclonal antisera directed against the carboxyl-terminal portion of the B94 protein immunoprecipitates a protein of the predicted molecular mass both from COS cells transfected with a B94 expression vector and from TNF-treated HUVE cells.

The response of endothelial cells to the cytokine tumor necrosis factor-alpha (TNF) is complex, involving the induction and suppression of multiple genes and gene products. Differential screening of a TNF-stimulated, cycloheximide-treated human umbilical vein endothelial cell library has resulted in the cloning of several novel cDNAs whose protein products are involved in the primary response of the endothelium to TNF. One of these cDNAs, designated B12, is further characterized here. B12 is encoded by a 3.5-kilobase transcript and is induced rapidly and transiently by TNF. Transcript expression is found to be developmentally regulated in a tissue-specific manner, with B12 message being differentially expressed in the heart and liver during mouse embryogenesis. The open reading frame of B12 predicts a 316-amino acid sequence rich in charged residues, particularly at the carboxyl terminus, and has neither significant homology to other known proteins nor to any extent sequence motifs. B12 is found to be a highly conserved single-copy gene which is located in the q22----q23 region of human chromosome 17. Polyclonal antibodies raised against a large portion of the B12 open reading frame immunoprecipitate a 36-kilodalton polypeptide from wheat germ lysates programmed to translate in vitro transcribed B12 mRNA. The B12 protein is further shown to be induced in human umbilical vein endothelial cells by TNF, and the protein is shown to be rapidly degraded.

We describe the identification of a novel laminin chain. Overlapping clones were isolated from a human fibrosarcoma HT1080 cell cDNA library spanning a total of 5,200 bp. A second set of clones contained an alternative 3' end sequence giving a total of 4,316 bp. The longer sequence contained an open reading frame for a 1,193-residue-long polypeptide. The alternative sequence was shortened at the carboxyl-terminal end coding for a 1,111-residue-long polypeptide. The amino acid sequence contained 21 amino acids of a putative signal peptide and 1,172 residues or alternatively 1,090 residues of a sequence with five distinct domains homologous to domains I-V in laminin chains. Comparison of the amino acid sequences showed that the novel laminin chain is homologous to the laminin B2 chain. However, the structure of the novel laminin chain isolated here differs significantly from that of the B2 chain in that it has no domain VI and domains V, IV, and III are shorter, resulting in a truncated laminin chain. The alternative sequence had a shortened domain I/II. In accordance with the current nomenclature, the chain characterized here is termed B2t. Calculation of possible chain interactions of laminin chains with the B2t chain domain I/II indicated that the B2t chain can replace the B2 chain in some laminin molecules. The gene for the laminin B2t chain (LAMB2T) was localized to chromosome 1q25-q31 in close proximity to the laminin B2 chain gene. Northern analysis showed that the B2t chain is expressed in several human fetal tissues but differently from the laminin B1 and B2 chains. By in situ hybridization expression of the B2t chain was localized to specific epithelial cells in skin, lung, and kidney as opposed to a general epithelial and endothelial cell expression of the laminin B2 chain in the same tissues.

The human sperm antigen SP-10 is a testis-specific, intra-acrosomal protein associated with the membranes of the acrosomal vesicle. The molecule has been designated a "primary vaccine candidate" by a World Health Organization (WHO) Taskforce on Contraceptive Vaccines. cDNA cloning and sequencing have indicated that SP-10 is encoded by a 795-base-pair (bp) reading frame that predicts a 265-amino acid protein of 28.3 kd. In this study, we used a 634-bp fragment (bp 68 through 700, amino acids 3 through 222) of the SP-10 sequence to probe, by Southern blotting, EcoRI-digested DNA from 33 mouse/human somatic cell hybrids involving 16 unrelated human cell lines and 4 mouse cell lines. The hybrids were characterized by karyotypic analysis and by mapped enzyme markers. The presence or absence of positive human bands was scored on the blots and the percent of concordance and discordance with a specific human chromosome was determined. The DNA probe for SP-10 showed a concordance of 31 and a discordancy of 0 for human chromosome 11, mapping SP-10 unequivocally to this chromosome. The hybrid XER-7 with the 11/X translocation: 11p12 or 11p11----11qter:: Xq11----Xqter and the hybrid EXR-5CSAZ with the X/11 translocation: Xpter----Xq22::11q13----11qter localized the SP-10 gene to the p12----q13 region. The SP-10 locus has been assigned the gene symbol ACRV1 (acrosomal vesicle protein-1).

The expression of a major cellular substrate for protein kinase C, the MARCKS protein, is regulated in a cell-, tissue-, and developmental stage-specific fashion; in addition, this expression can be stimulated acutely by various cytokines in certain cell types. We have begun to characterize the human gene in order to elucidate the genetic elements responsible for this highly regulated expression. We first cloned a human MARCKS cDNA, which encoded a predicted protein of 332 amino acids (Mr 31,600) that was approximately 89, 74, and 59% identical to the bovine, mouse, and chicken proteins, respectively. Regions conserved at the amino acid level included the amino-terminal myristoylation consensus sequence, the site of intron splicing, and the phosphorylation site domain. The human cDNA was used to demonstrate that tumor necrosis factor-alpha could rapidly stimulate MARCKS gene transcription in the human promyelocytic leukemia cell line HL60. Genomic clones were then isolated; sequence analysis identified a putative promoter region that had no TATA box and contained multiple transcription initiation sites in a region spanning 57 base pairs (bp). This was followed by a 5'-untranslated region of approximately 400 bp, which displayed a complex predicted secondary structure with a delta G of -73.4 kcal/mol. Plasmid constructions containing between 52 and 1453 bp of the human MARCKS promoter linked to the human growth hormone gene were then used in transient expression experiments. Constructions containing 52 and 110 bp of the MARCKS promoter did not exhibit promoter function while the larger constructions all exhibited promotor function; the 248-bp fragment of the MARCKS promoter was 80% as effective as the human ferritin promoter in stimulating expression of human growth hormone in intact cells. Using an insert from the human genomic clone as a probe, we identified human chromosome 6, q21-qter, as the location of the MARCKS gene; this has been assigned the gene symbol MACS.

We have previously described two cellular immediate-early genes, Egr-1 (mouse) and EGR2 (human) that encode zinc finger proteins. Here we report the characterization of a new member of the Egr family referred to as EGR3 (human). This cDNA clone was isolated using low stringency hybridization with the zinc finger domain of Egr-1. The EGR3 cDNA sequence predicts a 387 amino acid (a.a.) protein containing three Cys2-His2 zinc fingers nearly identical to those of Egr-1 and EGR2. This similarity has a functional consequence: EGR3 can activate transcription of a CAT gene linked to the sequence CGCCCCCGC, a cis element which is a target for Egr-1 and EGR2. We show that EGR3 is an immediate-early growth response gene induced by mitogenic stimulation of rodent and human fibroblasts and a monkey kidney epithelial cell line. The EGR3 gene has a single intron and maps to chromosome 8 at bands p21-23.

Lipoprotein-associated coagulation inhibitor (LACI) is a multivalent, Kunitz-type proteinase inhibitor which appears to play an important role in the regulation of hemostasis. LACI directly inhibits factor Xa, and, in a Xa-dependent fashion, also inhibits the factor VIIa-tissue factor catalytic complex. Hybridization of a LACI cDNA probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes localized the human LACI gene to chromosome 2. In situ hybridization to metaphase chromosomes further mapped the gene to the region 2q31----2q32.1. Exons of the human LACI gene were cloned from genomic or chromosome 2-specific phage libraries and sequenced, including approximately 500 base pairs of 5' upstream DNA. The 5' DNA did not contain a prototypical TATAA box or CCAAT sequence, and attempts to identify a unique site for the initiation of transcription were unsuccessful in that primer extension and S1 nuclease protection analysis indicate multiple transcription initiation sites for LACI messages. Comparing the gene sequence with LACI cDNA sequences indicates that the gene contains nine exons and that alternative splicing can occur, resulting in the absence of exon 2 in the 5' untranslated region of some messages. The three Kunitz domains in LACI are encoded on separate exons. Introns which interrupt coding sequences all occur in the same codon phase interrupting the first and second bases of the codon triplets. The data are consistent with LACI evolving by a combination of gene segment duplications and exon shuffling.

Neurokinin A (substance K) is a peptide neurotransmitter of the tachykinin family with potential as a major mediator in human airway and gastrointestinal tissues. Neurokinin A acts via a receptor (the NK-2 receptor) believed to be localized on smooth muscle cells and pharmacologically coupled to a GTP-binding protein. To characterize the human NK-2 receptor, we prepared a partial cDNA from human tracheal RNA using the polymerase chain reaction with oligonucleotide primers derived from the bovine NK-2 receptor cDNA sequence (Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., Nakanishi, S. (1987) Nature 329, 836-838). This partial human NK-2 receptor cDNA was used to screen a human genomic DNA library and yielded a clone, NGNK-2, of approximately 25 kilobases. Analysis of NGNK-2 indicates that it contains the entire coding sequence of the NK-2 receptor as well as 5'- and 3'-flanking sequences. The gene is organized with five exons interrupted by four introns. The complete sequence of the exons and the intron-exon junctions was determined, as were the transcription initiation site and the 3'-polyadenylation signal. Analysis of EcoRI digests of genomic DNA from human-mouse cell hybrids indicates a single gene for the human NK-2 receptor localized to chromosome 10. Sequence analysis of exons 1 and 5, where major differences occur between the human and animal species, provided information for polymerase chain reaction primers which allowed us to prepare full-length cDNA for the human NK-2 receptor. The protein predicted from the gene sequence is extended by 14 amino acids at the COOH terminus compared to the bovine and 9 residues compared to the rat molecules. The seven membrane-spanning regions are encoded by exons 1-4 and none is interrupted by introns. These regions are highly conserved among the species studied, suggesting stringent evolutionary control over these molecules.

Cartilage matrix protein (CMP) is a major component of the extracellular matrix of nonarticular cartilage. The structure and chromosomal location of the human gene encoding CMP was determined by molecular cloning analysis. We used a partial chicken CMP cDNA probe to isolate three overlapping human genomic clones. From one of these clones, a probe containing 2 human CMP exons was isolated and used to map the gene to chromosome 1p35 and to screen a human retina cDNA library. Two overlapping cDNA clones were isolated. The predicted protein sequence of 496 amino acids includes a 22-residue signal peptide and a 474-residue mature protein of Mr 51,344. The human CMP gene and polypeptide are strikingly similar to the chicken CMP gene and polypeptide. Human CMP is 79% identical to chicken CMP and contains two homologous domains separated by an epidermal growth factor-like domain. One potential N-glycosylation site is conserved between the two species. The human CMP gene spans 12 kilobase pairs with 8 exons and 7 introns which are similar in size to those of the chicken CMP gene. Both RNA splice junctions of intron G in the human and chicken CMP genes are nonconforming to the consensus splice sequences. This suggests that the CMP gene utilizes a new RNA splicing mechanism.

The polyamines are known to be essential for cellular proliferation. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the synthesis of these amines, and activity is elevated in colorectal tumors and polyps. Two ODC genes (designated ODC1 and ODC2) were localized by somatic cell hybridization and in situ techniques to 2p25 and 7q31-qter, respectively. Investigation of the expression of ODC in colorectal neoplasia reveals a consistent increase in mRNA expression compared with normal adjacent mucosa and control mucosa, ranging from 1.3- to 12.2-fold. No amplification of the loci was seen. Comparison of ODC mRNA expression with ODC activity from the same samples revealed no direct correlation, suggesting that regulation of ODC in this system occurs at the posttranscriptional level.

AMP deaminase is an ubiquitous enzyme in eukaryotic cells, and tissue-specific isoforms are produced in mammals by differential expression of the two genes which encode this enzyme activity as well as by alternative splicing of the primary transcript of one of these genes. Deficiency of this enzyme activity is one of the most common causes of metabolic myopathy in man. To provide a framework for understanding the molecular basis of this inherited disorder and the mechanisms responsible for regulating the expression of this enzyme activity, both the human and rat muscle-specific genes for AMP deaminase have been cloned and partially sequenced. Comparison of the two genes shows a high degree of conservation of sequence and structural organization. The two genes share the following characteristics: 1) both are approximately 20 kilobases in size, have identical exon/intron boundaries, and exhibit similar intron/exon structural organization; 2) the transcription start site is located at the same position in both genes, and comparison of 5'-flanking sequences reveals four highly conserved domains that together contain the information necessary for muscle-specific expression of a receptor cDNA; 3) coding sequences are 88% identical and the 5'-untranslated regions are 67% identical; 4) both genes have extremely short 3'-untranslated regions (13-17 nucleotides); 5) highly conserved intervening sequences of several hundred nucleotides surround most exon/intron boundaries. In situ hybridization and analysis of human-mouse somatic cell hybrids have localized the human gene (designated AMPD1) to chromosome 1 in the region p13-p21. The implications of these structural properties for identifying functional domains in the AMP deaminase peptide, regulation of expression of this gene, and inheritance of AMP deaminase deficiency are discussed.

Arylsulfatase C is a microsomal membrane-bound enzyme previously thought to be the same as steroid sulfatase, the only X-linked enzyme known to escape from X inactivation in man. We had shown that arylsulfatase C actually consists of two biochemically distinct isozymes, s and f. Only the s form has steroid sulfatase activity. The f and s forms were thought to be related through posttranslational or posttranscriptional modification of the same gene product. In part consistent with this hypothesis, we now report that in a panel of 28 rodent-human somatic cell hybrids, expression of both s and f was concordant only with the human X chromosome, thus showing that the f form is also X linked. In three separate somatic hybrids containing human X chromosomes in an inactive state, the f form was still expressed. Thus, similar to the s form, the f form also escapes from X inactivation. However, contrary to expectations, the s and f forms were not related by posttranslational modification of the same gene product. A full-length cDNA for the s form failed to hybridize to transcripts produced from an f-expressing cell line, showing that there is little sequence identity between the two. They are also not related by differential splicing of a common primary transcript, since fibroblasts from some patients with steroid sulfatase deficiency due to gene deletion of the s form continue to express the f form. Therefore, although the f and s isozymes of arylsulfatase C are X linked and escape from X inactivation, they are products from separate genes, thus providing a unique isoenzyme system to study possible gene duplication and regulation in the part of the human X chromosome that escapes inactivation.

To identify novel protein tyrosine kinase (PTK) genes expressed in human lymphoid cells, we have screened B- and T-cell cDNA libraries at low stringency using a c-fms tyrosine kinase domain probe. Three new PTK genes were identified, based on the presence of conserved amino acid sequence motifs characteristic of the catalytic domain of tyrosine kinases. Of these three genes, one (tyk1) appears to be the human homologue of a previously cloned murine gene (ltk), which has been reported to encode a tyrosine kinase with a unique structure; while the second gene, tyk2 cannot be clearly assigned to any of the known PTK subfamilies, and therefore may be the prototype of a new PTK gene subfamily. The third gene (tyk3/fer) has been very recently cloned by others; we present additional characterization in this report. We have performed Northern blots to establish the size of the mRNA encoded for by these genes, and to confirm their expression in lymphoid cells. Finally, we have determined the chromosomal location of all three genes by analyzing human-mouse somatic cell hybrids.

Human genes coding for hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49; D-glucose-6-phosphate:NADP+ 1-oxidoreductase), and phosphoglycerate kinase (PGK, EC 2.7.2.3; ATP:3-phospho-D-glycerate 1-phosphotransferase) have been assigned to specific regions on the long arm of the X chromosome by somatic cell gentic techniques. Gene assignment and linear order were determined by employing human somatic cells possessing an X/9 translocation or an X/22 translocation in man-mouse cell hybridization studies. The X/9 translocation involved the majority of the X long arm translocated to chromosome 9 and the X/22 translocation involved the distal half of the X long arm translocated to 22. In each case these rearrangements appeared to be reciprocal. Concordant segregation of X-linked enzymes and segments of the X chromosome generated by the translocations indicated assignment of the PGK gene to a proximal long arm region (q12-q22) and the HPRT and G6PD genes to the distal half (q22-qter) of the X long arm. Further evidence suggests a gene order on the X long arm of centromere-PGK-HPRT-G6PD.

Polymeric immunoglobulin receptor (PIGR) is a transmembrane glycoprotein which is expressed by epithelial cells and is involved in the transcellular transport of polymeric immunoglobulins into secretions. We cloned the human gene for PIGR and used the clone to obtain probes to determine the chromosomal localization of PIGR. Analysis of somatic cell hybrids and in situ chromosomal hybridization localized the human PIGR gene locus to 1q31→q41.

Overlapping cDNA clones, totaling 3.3 kilobases (kb) in length, which encode over 50% of the human erythrocyte beta-spectrin subunit, were isolated by antibody screening of a lambda gt11 expression library constructed from human fetal liver mRNA. The amino acid sequence of the C-terminus of beta-spectrin was derived. The size of beta-spectrin mRNA in human erythroleukemia cells was found to be 7.5 kb. Erythrocyte beta- spectrin is encoded by a gene located on human chromosome 14, as determined by cDNA hybridization to human X mouse somatic cell hybrids.

Two different mapping approaches were used to determine the human chromosomal location of the gene for protein S. A human protein S cDNA was used as a hybridization probe to analyze a panel of somatic cell hybrids containing different human chromosomes. Cosegregation of protein S-specific DNA restriction fragments with human chromosome 3 was observed. Three cell hybrids containing only a portion of chromosome 3 were analyzed in order to further localize protein S. Based on the somatic cell hybrid analysis, protein S is assigned to a region of chromosome 3 that contains a small part of the long arm and short arm of the chromosome including the centromere (3p21----3q21). In situ hybridization of the protein S cDNA probe to human metaphase chromosomes permitted a precise localization of protein S to the region of chromosome 3 immediately surrounding the centromere (3p11.1----3q11.2). Protein S is the first protein involved in blood coagulation that has been mapped to human chromosome 3.

The vasoactive peptide endothelin 1 (ET1) is encoded by a well characterized gene located on human chromosome 6. Recently, two human genomic fragments were isolated which potentially encode related vasoconstrictor peptides, endothelin 2 (ET2) and endothelin 3 (ET3) (Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K., and Masaki, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2863-2867). Inoue et al. were unable to detect transcripts of the ET2 and ET3 genes and observed ET1 gene expression exclusively in endothelial cells. In this study, we document transcription of the ET3 gene by isolating from a hypothalamic cDNA library DNA clones complementary to human ET3 mRNA. ET3 mRNA encodes a 238-amino acid precursor that includes ET3 and a 15-amino acid homologous segment, the ET3-like sequence. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, we assigned the ET3 gene to human chromosome 20. The ET3 and ET1 genes are, therefore, not genetically linked. RNA blot hybridization with restriction fragments derived from cDNAs revealed that the ET3 and ET1 genes are both expressed in lung, pancreas, and spleen. Cultured endothelial cells and cardiac tissues express the ET1 but not the ET3 gene. Observations that genes encoding endothelin-related peptides are expressed in a variety of human tissues suggest that these peptides may participate in complex vasoregulatory mechanisms.

Human alpha-mannosidase activity (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) from tissues and cultured skin fibroblasts was separated by gel electrophoresis into a neutral, cytoplasmic form (alpha-mannosidase A) and two closely related acidic, lysosomal components (alpha-mannosidase B). Human mannosidosis, an inherited glycoprotein storage disorder, has been associated with severe deficiency of both lysosomal alpha-mannosidase B molecular forms. Chromosome assignment of the gene coding for human alpha-mannosidase B (MANB) has been determined in human-mouse and human-Chinese hamster somatic cell hybrids. The human alpha-mannosidase B phenotype showed concordant segregation with the human enzyme glucosephosphate isomerase (GPI) (D-glucose-6-phosphate ketolisomerase, EC 5.3.1.9) but discordant segregation with 30 other enzyme markers representing 20 linkage groups. The glucose-phosphate isomerase gene has been assigned to chromosome 19 in man. This MANB-GPI linkage and confirming chromosome studies demonstrate assignment of the alpha-mannosidase B structural gene to chromosome 19 in man. Since mannosidosis is believed to result from a structural defect in alpha-mannosidase B, these findings suggest that the mannosidosis mutation is located on chromosome 19 in man.

The assignment of the human gene for uroporphyrinogen decarboxylase (UROD) to chromosome 1 is confirmed and further localized to the pter----p21 region through the use of human-mouse somatic cell hybrids. Human and mouse UROD were separated by electrophoresis and identified with antibodies to the human enzyme after electrophoretic transfer to nitrocellulose membranes.

A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage lambda gt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16+ natural killer cells and CD3+, CD16- T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The predicted protein has a 22-amino acid presegment, a 6-amino acid prosegment, and an active enzyme of 234 amino acids with a calculated unglycosylated molecular weight of 25,820. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. We propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells.

The structural gene (beta GALA) coding for lysosomal beta-galactosidase-A (EC 3.2.1.23) has been assigned to human chromosome 3 using man--mouse somatic cell hybrids. Human beta-galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver beta-galactosidase-A. The antiserum precipitates beta-galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with GM1 gangliosidosis. We have analyzed 90 primary man--mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for beta GALA assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of beta GALA to chromosome 3; all other chromosomes were excluded. The evidence suggests that GM1 gangliosidosis is a consequence of mutation at this beta GALA locus on chromosome 3.

The adenylate kinase 1 (AK1), adenylate kinase3 (AK3), and aconitaseS (ACONS) genes have been assigned to chromosome 9 in man by employing an X/9 translocation segregating in man-mouse somatic cell hybrids. Segregation was controlled by taking advantage of the HAT/8-azaguanine selection-counterselection strategy directed at the X-linked HPRT locus. Assignment of AK1 to chromosome 9 has suggested the assignment of the ABO blood-group locus and the nail-patella (Np) locus to 9, since both loci are linked to AK1 by family studies.

A complementary DNA (cDNA) probe for the predominant (pI 7) form of human monocyte-derived interleukin-1 (IL1) and a collection of 30 human-mouse somatic cell hybrids were used to assign the IL1 gene to human chromosome 2 by Southern blot analysis of hybrid cell DNA digested with the restriction endonuclease BglII. In situ hybridization to human metaphase chromosomes localized the IL1 gene to the long arm of chromosome 2 at position 2q13-2q21 between two fragile sites.

A cDNA probe (HOAT1) for ornithine aminotransferase (OAT) has recently been used to map (1) the structural gene for this enzyme to chromosome 10 and (2) several related DNA sequences to the X chromosome. We have defined six RFLPs for OAT, to explore its possible role in gyrate atrophy (GA) of the choroid and retina, an autosomal recessive genetic disorder associated with a deficiency of OAT activity. The RFLPs, which are detected by noncoding single-copy probes from the OAT gene and by subclones of the HOAT1 cDNA, all map on human chromosome 10, producing an overall level of heterozygosity for the OAT locus of 83%. Using the RFLPs, we have determined that the OAT locus segregates concordantly with GA in one available pedigree. Furthermore, the RFLPs display significant disequilibrium with GA, providing genetic evidence implicating a defect in the OAT structural gene as the cause of this disorder. The RFLPs for OAT are potentially applicable to prenatal diagnosis and carrier detection in families with a previous history of GA. They will also allow identification of specific haplotypes associated with GA chromosomes, as a guide for more detailed molecular-genetic investigations of the mutations underlying the disorder.