Large multi-chromosomal duplications encompass many members of the olfactory receptor gene family in the human genome

The human genome contains thousands of genes that encode a diverse repertoire of odorant receptors (ORs). We report here on the identification and chromosomal localization of 74 OR-containing genomic clones. Using fluorescence in situ hybridization (FISH), we demonstrate a striking homology among a set of approximately 20 OR locations, illustrating a history of duplications that have distributed OR sequences across the genome. Half of the OR-containing BACs cloned from total genomic DNA and 86% of cosmids derived from chromosome 3 cross-hybridize to a subset of these locations, many to 17 of them. These paralogous regions are distributed on 13 chromosomes, and eight lie in terminal bands. By analyzing clones from an approximately 250 kb clone-walk across one of these sites (3p13), we show that the homology among these sites is extensive (>150 kb) and encompasses both OR genes and intergenic genomic sequences. The FISH signals appear significantly larger at some sites than at the native location, indicating that portions of some duplicons have undergone local amplification/attrition. More restricted duplications involving pairs of other genomic locations are detected with 12% of the OR-BACs. Only a small subset of OR locations is sufficiently diverged from the others that clones derived from them behave as single-copy FISH probes. We estimate that duplications encompassing members of the OR gene family account for >0.1% of the human genome. A comparison of FISH signals at orthologous locations in other primates indicates that a portion of this OR 'subgenome' has been in flux during the divergence of primates, possibly as a mechanism for evolving the repertoire of olfactory receptors.     

CTX, a Xenopus thymocyte receptor, defines a molecular family conserved throughout vertebrates

CTX, a cortical thymocyte marker in Xenopus, is an immunoglobulin superfamily (Igsf) member comprising one variable and one constant C2-type Igsf domain, a transmembrane segment and a cytoplasmic tail. Although resembling that of the TCR and immunoglobulins, the variable domain is not encoded by somatic rearrangement of the gene but by splicing of two half-domain exons. The C2 domain, also encoded by two exons, has an extra pair of cysteines. The transmembrane segment is free of charged residues, and the cytoplasmic tail (70 amino acids) contains one tyrosine and many glutamic acid residues. ChT1, a chicken homologue of CTX, has the same structural and genetic features, and both molecules are expressed on the thymocyte surface. We cloned new mouse (CTM) and human (CTH) cDNA and genes which are highly homologous to CTX/ChT1 but not lymphocyte specific. Similarity with recently described human cell surface molecules, A33 antigen and CAR (coxsackie and adenovirus 5 receptor), and a number of expressed sequence tags leads us to propose that CTX defines a novel subset of the Igsf, conserved throughout vertebrates and extending beyond the immune system. Strong homologies within vertebrate sequences suggest that the V and C2 CTX domains are scions of a very ancient lineage.     

Characterization of rice endo-beta-glucanase genes (Gns2-Gns14) defines a new subgroup within the gene family

Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled. Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes. However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling). We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times. We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one. Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration. The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway. Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one. Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control. Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients.     

Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain

We have characterized an Arabidopsis receptor-like serine/threonine kinase gene, Ath.lecRK1 (Arabidopsis thaliana lectin-receptor kinase), defining a new and putatively important class of plant receptor kinases. Structural features of the predicted polypeptide include an amino-terminal membrane-targeting signal sequence, a legume lectin-like extracellular domain, a single membrane-spanning domain, and a characteristic serine/threonine protein kinase domain. A recombinant protein containing the kinase domain can be autophosphorylated on a serine residue. Ath.lecRK1 is a member of a gene family of at least two closely related genes. Northern blot analysis indicates that the Ath.lecRK1 gene is weakly expressed in a variety of organs and is regulated in Arabidopsis cell suspension cultures according to the growth phase of cells. The role this new class of plant receptor kinase could play is discussed with regard to the transduction of oligosaccharide and plant hormone signals.     

Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana

Disseminated neuroblastoma frequently show a very poor prognosis. N-myc gene amplification, 1p deletion and lack of CD44 gene expression, are all genetic factors associated with the disease's dissemination. Human neuroblastoma xenografts in nude mice has permitted to characterize, in disseminated neuroblasts, oncogenes overexpression, inactivation of tumor suppressor genes as well as detoxifying genes activation which contributes to increase cellular resistance to chemotherapy. These genetic abnormalities permit to propose a nosology of this very aggressive pediatric solid tumor. Hopefully, this genetic classification could be of great value for new therapeutic approaches.     

The human LARGE gene from 22q12.3-q13.1 is a new, distinct member of the glycosyltransferase gene family

Meningioma, a tumor of the meninges covering the central nervous system, shows frequent loss of material from human chromosome 22. Homozygous and heterozygous deletions in meningiomas defined a candidate region of >1 Mbp in 22q12.3-q13.1 and directed us to gene cloning in this segment. We characterized a new member of the N-acetylglucosaminyltransferase gene family, the LARGE gene. It occupies >664 kilobases and is one of the largest human genes. The predicted 756-aa N-acetylglucosaminyltransferase encoded by LARGE displays features that are absent in other glycosyltransferases. The human like-acetylglucosaminyltransferase polypeptide is much longer and contains putative coiled-coil domains. We characterized the mouse LARGE ortholog, which encodes a protein 97.75% identical with the human counterpart. Both genes reveal ubiquitous expression as assessed by Northern blot analysis and in situ histochemistry. Chromosomal mapping of the mouse gene reveals that mouse chromosome 8C1 corresponds to human 22q12.3-q13.1. Abnormal glycosylation of proteins and glycosphingolipids has been shown as a mechanism behind an increased potential of tumor formation and/or progression. Human tumors overexpress ganglioside GD3 (NeuAcalpha2,8NeuAcalpha2, 3Galbeta1,4Glc-Cer), which in meningiomas correlates with deletions on chromosome 22. It is the first time that a glycosyltransferase gene is involved in tumor-specific genomic rearrangements. An abnormal function of the human like-acetylglucosaminyltransferase protein may be linked to the development/progression of meningioma by altering the composition of gangliosides and/or by effect(s) on other glycosylated molecules in tumor cells.     

Organization of the human natriuretic peptide receptor A gene

We have determined the structural organization of the human natriuretic peptide receptor A (hNPRA) gene without screening the genomic library. Based on the information for the cDNA of hNPRA, we amplified some fragments of the long polymerase chain reaction (PCR) products covering all genomic sequences of the gene and directly sequenced the products after extraction and purification. The hNPRA gene spans approximately 16 kb and contains 22 exons and 21 introns. All of the exon-intron junction sequences coincide with the GT/AG consensus sequence. The sequence encoding the transmembrane domain, one part of the hNPRA topological structure, exists in exon 7. The results of this first study, determination of the hNPRA gene structure, will facilitate further genetic analysis of the hNPRA gene and its related diseases.     

The DAM gene family encodes a new group of tumor-specific antigens recognized by human leukocyte antigen A2-restricted cytotoxic T lymphocytes

The DAM family of genes has a high degree of homology with MAGE, both in nucleotide sequence and in neoplastic tissue-specific expression. This study describes, for the first time, the identification of CTLs specific for a peptide epitope encoded by DAM genes. A human leukocyte antigen (HLA)-A2-restricted CTL clone was raised against a peptide, D10/6-271, encoded by codons 271-279 in the DAM cDNA. The corresponding peptide in the MAGE-3 sequence, M3-271, has been shown previously to be a natural T-cell epitope for HLA-A2-restricted CTLs recognizing the MAGE-3 protein. The D10/6-271-specific CTL clone required approximately 3 nM exogenous peptide for half-maximal lysis of target cells and was able to specifically recognize endogenous DAM antigen on HLA-A2+ melanoma cells infected with a vaccinia vector recombinant for gene DAM-6. These data suggest that DAM genes might encode a new group of tumor-specific antigens useful for the design of specific antitumor vaccines.     

Clinical validation of a new olfactory test

A new approach to clinical olfactometry is presented using nasal sprays. A special dosage valve was used to standardize an aerosol size to 40 microns. For evaluation a 6 x 6 matrix (substance/olfactory quality) with verbal associative clues was used according to test psychological findings. Validation took place in three steps after determining smell-associated thresholds in preliminary examinations. Recognition of different aromas administered either by spraying into the open mouth from a distance of 5 cm using nasal sprays or sniffing opened bottles was compared in 50 subjectively normosmic subjects. Findings showed that the correspondence between the two procedures was relatively high, with global identification of odorous substances as high as 98.4% in the spray test. The number of substances used was next reduced to those six substances providing the most reproducible results in an investigation with 56 normosmic and 55 hyposmic subjects. Verbal associative clues were also tested. In a last step 50 patients with hyposmia of various origin and 110 normosmic subjects were tested, allowing previous results to be reproduced regardless of the cause of hyposmia. The spray test was shown to be easily performed and was suitable as a screening test, with a sensitivity of 88% and a specificity of 100%.     

A new member of the MER2 repeat family is detected in the promoter region of the human X11 gene

AIMS: To evaluate the clinical utility of two new tests for serum trypsinogen 2 and trypsin 2-alpha 1 antitrypsin complex (trypsin 2-AAT) in diagnosing and assessing the severity of acute pancreatitis (AP) induced by endoscopic retrograde cholangiopancreatography (ERCP). PATIENTS: Three hundred and eight consecutive patients undergoing ERCP at Helsinki University Central Hospital in 1994 and 1995. METHODS: Patients were followed prospectively for pancreatitis and clinical outcome. They were tested for serum trypsinogen 2, trypsin 2-AAT, and amylase in samples obtained before and one, six, and 24 hours after ERCP. RESULTS: Pancreatitis developed in 31 patients (10%). Their median serum trypsinogen 2 increased 26-fold to 1401 micrograms/l at six hours after the procedure and trypsin 2-AAT showed an 11-fold increase to 88 micrograms/l at 24 hours. The increase in both markers was stronger in severe than in mild pancreatitis, and in patients without pancreatitis there was no significant increase. Baseline trypsinogen 2 and trypsin 2-AAT concentrations were elevated in 29% and 32% of patients, respectively. The diagnostic accuracy of a threefold elevation over the baseline value was therefore analysed. The sensitivity and specificity of these parameters in the diagnosis of post-ERCP pancreatitis was 93% and 91%, respectively, for serum trypsinogen 2 at six hours after the examination, and 93% and 90%, for trypsin 2-AAT at 24 hours. CONCLUSIONS: Serum trypsinogen 2 and trypsin 2-AAT reflect pancreatic injury after ERCP. High concentrations are associated with severe pancreatic damage. The delayed increase in trypsin 2-AAT compared with trypsinogen 2 appears to reflect the pathophysiology of AP. A greater than threefold increase in trypsinogen 2 six hours after ERCP is an accurate indicator of pancreatitis.     

Genomic organization of a human killer cell inhibitory receptor gene

We have cloned a region of human chromosome 19q13.4 which contains multiple killer cell inhibitory receptor (KIR) loci. By random and directed sequence analysis of these KIR-specific clones, we deduced the genomic structure of KIR genes. A locus encoding a member of the NKAT-2 family of KIRs is presented here. The structure of the gene is reminiscent of loci of the Fc receptor gene family, and the two sets of genes may derive from a common ancestor. The KIR gene contains potentially nine exons. The first two exons encode the leader sequence, as in Fc receptor genes. The third exon encodes an untranslated pseudo exon specifying an immunoglobulin domain with an in-frame stop codon. Expressed cDNAs do not contain this exon. This finding is consistent with the hypothesis that certain KIR genes may have been derived from the duplication of a primordial three immunoglobulin domain structure with subsequent skipping of one exon to derive genes with two expressed immunoglobulin domains. Variation in numbers of immunoglobulin domains in different KIR genes is facilitated by conservation of splicing frame in respect to the codon triplet for each immunoglobulin domain.     

Bgp2, a new member of the carcinoembryonic antigen-related gene family, encodes an alternative receptor for mouse hepatitis viruses

Murine coronaviruses such as mouse hepatitis virus (MHV) infect mouse cells via cellular receptors that are isoforms of biliary glycoprotein (Bgp) of the carcinoembryonic antigen gene family (G. S. Dveksler, C. W. Dieffenbach, C. B. Cardellichio, K. McCuaig, M. N. Pensiero, G.-S. Jiang, N. Beauchemin, and K. V. Holmes, J. Virol. 67:1-8, 1993). The Bgp isoforms are generated through alternative splicing of the mouse Bgp1 gene that has two allelic forms called MHVR (or mmCGM1), expressed in MHV-susceptible mouse strains, and mmCGM2, expressed in SJL/J mice, which are resistant to MHV. We here report the cloning and characterization of a new Bgp-related gene designated Bgp2. The Bgp2 cDNA allowed the prediction of a 271-amino-acid glycoprotein with two immunoglobulin domains, a transmembrane, and a putative cytoplasmic tail. There is considerable divergence in the amino acid sequences of the N-terminal domains of the proteins coded by the Bgp1 gene from that of the Bgp2-encoded protein. RNase protection assays and RNA PCR showed that Bgp2 was expressed in BALB/c kidney, colon, and brain tissue, in SJL/J colon and liver tissue, in BALB/c and CD1 spleen tissue, in C3H macrophages, and in mouse rectal carcinoma CMT-93 cells. When Bgp2-transfected hamster cells were challenged with MHV-A59, MHV-JHM, or MHV-3, the Bgp2-encoded protein served as a functional MHV receptor, although with a lower efficiency than that of the MHVR glycoprotein. The Bgp2-mediated virus infection could not be inhibited by monoclonal antibody CC1 that is specific for the N-terminal domain of MHVR. Although CMT-93 cells express both MHVR and Bgp2, infection with the three strains of MHV was blocked by pretreatment with monoclonal antibody CC1, suggesting that MHVR was the only functional receptor in these cells. Thus, a novel murine Bgp gene has been identified that can be coexpressed in inbred mice with the Bgp1 glycoproteins and that can serve as a receptor for MHV strains when expressed in transfected hamster cells.     

Androgen receptor gene mutations in human prostate cancer

To investigate the structural abnormality of the androgen receptor (AR) in human prostate cancers, exons B-H encoding DNA- and hormone-binding domains were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products using originally designed oligoprimers. Tissues from 7 cases of untreated stage B prostate cancer surgically removed and from 8 cases of endocrine therapy-resistant cancers obtained at autopsy were used in the study. Two different mutations were identified in exons D and H in the different cancer foci of the same cancer death patient. One mutation in exon D (at codon 701, Leu to His) was detected in the prostate, and the other in exon H (at codon 877, Thr to Ala) was found in metastatic tissues. In untreated cancer tissues and the other autopsy samples, no mutations were detected. The mutation in exon H was identical to that reported in LNCaP cells. These results indicate that AR gene mutations occur in relation to endocrine therapy-resistance, although the mutation was found in 1 out of 8 resistant cases (12.5%) at autopsy.     

A highly conserved major histocompatibility complex class I-related gene in mammals

We report here a cDNA sequence of a murine homolog of the human major histocompatibility complex (MHC) class I-related gene, MR1. The analyses revealed unprecedentedly high conservation of MR1 in the alpha1 and alpha2 domains (corresponding to the peptide-binding domains in the classical MHC class I molecules) between human and mouse (predicted amino acid identity: 90 and 89% for the alpha1 and alpha2 domain, respectively), compared to MHC class I and other class I molecules. On the other hand, conservation in the alpha3 domain (73%) is comparable to those of others, suggesting domain-specific conservation of MR1. The localization of the mouse MR1 gene was determined to be chromosome 1H1, which corresponds to the human chromosomal region where the human MR1 gene is located (chromosome 1q25). High conservation of MR1 among mammals suggests that MR1 may be involved in critical conserved biological function(s).