A highly polymorphic microsatellite marker located within the choroideremia gene

Although serotonergic dysregulation is a leading pathogenetic hypothesis for obsessive-compulsive disorder (OCD), some evidence also suggests a possible dysregulation of the dopaminergic system in this disorder. Therefore, individual differences in deoxyribonucleic acid (DNA) coding for dopamine receptor proteins might contribute to the genetic background of this disorder. Previously we reported a null mutation in exon 1 of the dopamine D4 receptor gene. The variant type is characterized by a 13 bp deletion and is predicted to code for a truncated, non-functional receptor. We assessed the frequency of this polymorphism in 157 OCD patients, 196 schizophrenics, 111 bipolars and 162 healthy controls of Italian descent. Our findings do not implicate a role for this mutation in conferring a susceptibility to OCD and confirm previous negative results regarding its involvement in schizophrenia and bipolar disorder.     

A highly polymorphic CA/GT repeat in intron 3 of the human urokinase receptor gene (PLAUR)

We describe the first highly polymorphic microsatellite marker for the human urokinase plasminogen activator receptor gene (PLAUR). The urokinase receptor (uPAR) has a central role in cancer invasion and metastasis, which may enable the development of new anti-metastatic therapies. Analysis of the marker genotypes in colorectal cancer cell lines revealed three alleles that were not detected in a series of healthy control individuals, which encourages further genetic study of the role of uPAR in cancer.     

A novel microsatellite polymorphism in the human OB gene: a highly polymorphic marker for linkage analysis

The mouse ob gene and its human homologue OB have recently been cloned. The mutations in the ob gene are known to be associated with extreme obesity. The relationship between the human OB gene and disease, however, is largely unknown due to the lack of suitable markers within or adjacent to the OB gene. To obtain informative markers, we searched for simple tandem repeat polymorphisms in the genomic sequence of the human OB gene and identified a novel tetranucleotide repeat in the 3' flanking region. Fifteen alleles were detected in this marker with a heterozygosity of 0.85 and polymorphism information content of 0.83, indicating a highly informative nature of this marker. Two-point linkage mapping in two Centre Etude Polymorphisme Humaine (CEPH) reference families suggested that this marker is located in the interval between D7S514 and D7S530, the same interval where the OB gene is located (recombination fractions with D7S514 and D7S530 were 0.026 and 0.034, respectively). Although allele frequency distributions of this marker did not differ between 84 control subjects and 69 NIDDM patients, there was a tendency to higher body weight in control subjects with class I/class I genotype than in those without this genotype (68.8 +/- 11.1 vs 60.8 +/- 10.3 kg, p = 0.05). The highly polymorphic nature of this marker and its location in the OB gene makes this marker useful for linkage studies of the OB gene with a number of phenotypes, such as obesity, non-insulin-dependent diabetes mellitus, hypertension and the insulin resistance syndrome.     

The alpha/beta subunit interaction in H(+)-ATPase (ATP synthase). An Escherichia coli alpha subunit mutation (Arg-alpha 296-->Cys) restores coupling efficiency to the deleterious beta subunit mutant (Ser-beta 174-->Phe)

The Ser-beta 174 residue of the Escherichia coli H(+)-ATPase beta subunit has been shown to be near the catalytic site together with Gly-beta 149, Gly-beta 172, Glu-beta 192, and Val-beta 198 (Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 3156-3160). In this study, we introduced various residues at position 174 and found that the larger the side chain volume of the residue introduced, the lower the enzyme activity became. The Phe-beta 174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-beta 174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (approximately 10% of the wild type). The defective energy coupling of the Phe-beta 174 mutant was suppressed by the second mutation (Arg-alpha 296-->Cys) in the alpha subunit. The Cys-alpha 296/Phe-beta 174 mutant had essentially the same membrane ATPase activity as the Phe-beta 174 single mutant when assayed under the conditions that stabilize the double mutant enzyme. These results indicate the importance of the alpha/beta interaction, especially that between the regions near Arg-alpha 296 and Ser-beta 174, for energy coupling in the H(+)-ATPase. The 2 residues (Ser-beta 174 and Arg-alpha 296) may be located nearby at the interface of the two subunits. About 1 mol of N-[14C]ethylmaleimide could bind to 1 mol of the alpha subunit of Cys-alpha 296/Phe-beta 174 or Cys-alpha 296 mutant ATPase, but could not inhibit the enzyme activity. This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.     

The vacuolar H(+)-ATPase inhibitor bafilomycin A1 differentially affects proteolytic processing of mutant and wild-type beta-amyloid precursor protein

We analyzed the effect of the vacuolar H(+)-ATPase inhibitor bafilomycin A1 (bafA1) on the processing of beta-amyloid precursor protein (beta APP). In kidney 293 cells stably transfected with the wild-type beta APP cDNA, bafA1 caused a stabilization of mature beta APP and its 10-kDa COOH-terminal fragment. Moreover, it caused a 2-3-fold increase in secretion of soluble APP and amyloid-beta protein (A beta). Interestingly, bafA1 treatment of cells transfected with a mutant beta APP isoform that occurs in a Swedish kindred with familial Alzheimer's disease resulted in a decrease of A beta production and no increase of soluble APP secretion. Identical results were obtained when the effect of bafA1 was analyzed on fibroblasts derived from affected versus unaffected members of the Swedish family. These data demonstrate a differential effect of bafA1 on the production of A beta derived from wild-type or Swedish mutant beta APP. Radiosequencing of A beta derived from bafA1-treated cells expressing wild-type beta APP revealed a marked increase of A beta peptides starting at amino acids phenylalanine 4 and valine -3 and a relative decrease of A beta molecules beginning at the typical NH2 terminus of aspartate 1. Cells transfected with the Swedish mutation and treated with bafA1 did not produce these alternative A beta peptides, so that bafA1 treatment resulted in a decrease of A beta starting at aspartate 1. Our data indicate that multiple proteases are able to cleave A beta at or near its NH2 terminus. Inhibition of the protease cleaving at aspartate 1 by bafA1 and perhaps other similar agents can result in an increase of alternatively cleaved peptides.     

A highly polymorphic microsatellite in the class II Eb gene allows tracing of major histocompatibility complex evolution in mouse

A hallmark of major histocompatibility complex (MHC) genes is their extraordinarily high level of polymorphism. Polymorphic residues on MHC molecules determine which peptide ligands they bind and present to effector T lymphocytes. Although the genetic mechanisms responsible for MHC polymorphism have been delineated, the timetable and the pathway of their diversification remain unclear. To trace MHC evolution, we have characterized a highly polymorphic microsatellite containing tandem repeats (TRs) of two tetranucleotide units, TGGA and GGCA, located at the 3' end of the second intron in the class II Eb gene of mouse. On the basis of length as well as sequence variations, 11 TR alleles were defined in 55 inbred mouse strains, which included MHC recombinant haplotypes and haplotypes derived from different subspecies of mouse. In this extensive sampling, a striking concordance was observed between the serologically identified class II proteins and the associated TR alleles. Examination of several strains carrying the same MHC haplotypes as well as strains carrying recombinant MHC haplotypes indicates that TR alleles are extremely stable. These observations suggest that TR polymorphism predates the separation of various subspecies of mouse. On the basis of sequence divergence, a genealogical tree has been constructed to depict evolution of the different TR alleles. Finally, evidence is presented that suggests this microsatellite polymorphism is generated by slipped-strand mispairing during DNA replication.     

A highly informative CA/GT repeat polymorphism in intron 38 of the human neurofibromatosis type 1 (NF1) gene

We describe a polymorphic microsatellite in intron 38 of the neurofibromatosis type 1 (NF1) gene. The microsatellite consists of a CA/GT dinucleotide repeat detecting 8 alleles; it has a heterozygosity of 82%.     

Site-directed mutagenesis of the yeast V-ATPase B subunit (Vma2p)

The B subunit of the vacuolar (H+)-ATPase (V-ATPase) has previously been shown to participate in nucleotide binding and to possess significant sequence homology with the alpha subunit of the mitochondrial F-ATPase, which forms the major portion of the noncatalytic nucleotide binding sites and contributes several residues to the catalytic sites of this complex. Based upon the recent x-ray structure of the mitochondrial F1 ATPase (Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker, J.E. (1994) Nature 370,621-628), site-directed mutagenesis of the yeast VMA2 gene has been carried out in a strain containing a deletion of this gene. VMA2 encodes the yeast V-ATPase B subunit (Vma2p). Mutations at two residues postulated to be contributed by Vma2p to the catalytic site (R381S and Y352S) resulted in a complete loss of ATPase activity and proton transport, with the former having a partial effect on V-ATPase assembly. Interestingly, substitution of Phe for Tyr-352 had only minor effects on activity (15-30% inhibition), suggesting the requirement for an aromatic ring at this position. Alteration of Tyr-370, which is postulated to be near the adenine binding pocket at the noncatalytic sites, to Arg, Phe, or Ser caused a 30-50% inhibition of proton transport and ATPase activity, suggesting that an aromatic ring is not essential at this position. Finally, mutagenesis of residues in the region corresponding to the P-loop of the alpha subunit (H180K, H180G, H180D, N181V) also inhibited proton transport and ATPase activity by approximately 30-50%. None of the mutations in either the putative adenine binding pocket nor the P-loop region had any effect on the ability of Vma2p to correctly fold nor on the V-ATPase to correctly assemble. The significance of these results for the structure and function of the nucleotide binding sites on the B subunit is discussed.     

Population variation at the polymorphic ApaLI restriction enzyme site in intron 5 of the WT1 gene

We examined 63 unrelated individuals from the United States for the Apa-LI polymorphism in intron 5 of the WT1 gene. Allele frequencies of 0.13 and 0.87 for the A and B alleles, respectively, and a heterozygosity index of 24% contrast sharply with previous data obtained in the Japanese population where allele frequencies of 0.55 and 0.45 for the A and B alleles and a heterozygosity index of 59% were reported. These data suggest genetic heterogeneity at the WT1 locus, which may contribute to the differences in the incidence of Wilms tumor between the two population groups.     

Mutations of G158 and their second-site revertants in the plasma membrane H(+)-ATPase gene (pma1) in Saccharomyces cerevisiae

A G158D mutation residing near the cytoplasmic end of transmembrane segment 2 of the H(+)-ATPase from Saccharomyces cerevisiae appears to alter electrogenic proton transport by the proton pump (Perlin et al. (1988) J. Biol. Chem. 263, 18118-18122.) The mutation confers upon whole cells a pronounced growth sensitivity to low pH and a resistance to the antibiotic hygromycin B. The isolated enzyme retains high activity (70% of wild type) but is inefficient at pumping protons in a reconstituted vesicle system, suggesting that this enzyme may be partially uncoupled (Perlin et al. (1989) J. Biol. Chem. 264, 21857-21864). In this study, the acid-sensitive growth phenotype of the pma1-D158 mutant was utilized to isolate second site suppressor mutations in an attempt to probe structural interactions involving amino acid 158. Site-directed mutagenesis of the G158 locus was also performed to explore its local environment. Nineteen independent revertants of pma1-G158D were selected as low pH-resistant colonies. Four were full phenotypic revertants showing both low pH resistance and hygromycin B sensitivity. Of three full revertants analyzed further, one restored the original glycine residue at position 158 while the other two carried compensatory mutations V336A or F830S, in transmembrane segments 4 and 7, respectively. Partial revertants, which could grow on low pH medium but still retained hygromycin B resistance, were identified in transmembrane segments 1 (V127A) and 2 (C148T, G156C), as well as in the cytoplasmic N-terminal domain (E110K) and in the cytoplasmic loop between transmembrane segments 2 and 3 (D170N, L275S). Relative to the G158D mutant, all revertants showed enhanced net proton transport in whole-cell medium acidification assays and/or improved ATP hydrolysis activity. Small polar amino acids (Asp and Ser) could be substituted for glycine at the 158 position to produce active, albeit somewhat defective, enzymes; larger hydrophobic residues (Leu and Val) produced more severe phenotypes. These results suggest that G158 is likely to reside in a tightly packed polar environment which interacts, either directly or indirectly, with transmembrane segments 1, 4 and 7. The revertant data are consistent with transmembrane segments 1 and 2 forming a conformationally sensitive helical hairpin structure.     

Numerous PCR-RFLPs within the porcine CYP21 (steroid 21-hydroxylase) gene

Mesenteric cysts are rare intra-abdominal tumors with an incidence around one case per 100,000 hospital admissions. The clinical presentation is variable; patients may be asymptomatic or present with either acute or chronic abdominal pain. Physical examination commonly demonstrates a smooth, round and mobile abdominal mass. Differential diagnosis includes any abdominal cyst or tumor. Laboratory tests are usually helpless. Ultrasonography and CT scans are the best diagnostic tools. The treatment of choice is the total resection of the cyst, which is regularly performed by open surgery. This paper reports a case of a mesenteric cyst successfully resected by laparoscopy, and addresses the possible uses of this approach.     

Structural and functional characterisation of the signal recognition particle-specific 54 kDa protein (SRP54) of tomato

The major oligosaccharide chains of bovine, frog and human rhodopsins are identical in structure. These molecules are unique in terms of their abridged size as compared to other asparagine-linked glycoproteins, a property which must be reflected in the activities and properties of the glycosyltransferases of the retina that are concerned with their biosynthesis. One of the unusual characteristics of the major oligosaccharide chain is its lack of branching. A key enzymatic step required for branching to occur would be the transfer of a residue of N-acetylglucosamine to the terminal unsubstituted mannose residue of the major rhodopsin oligosaccharide. We have investigated the kinetic properties of enzymes that would catalyse this process. The N-acetylglucosaminyl-transferases (GlcNAc-transferases) of Golgi-enriched fractions of bovine retina, human retina, rat liver Golgi and a partially purified GlcNAc-transferase II from rat liver were examined using as acceptors rhodopsin, opsin and the oligosaccharide isolated from rhodopsin. The identification of the substrates and products of the reactions was carried out by chromatographic means. From an evaluation of the Vmax/K(m) ratios it was observed that bovine and human retinas have very limited abilities compared to the rat liver enzymes to carry out the transfer of GlcNAc to these acceptors, showing from 100- to about 800-fold lower efficiency in this regard. A comparison of the activities obtained with intact rhodopsin or opsin and the rhodopsin oligosaccharide indicated that the activity of GlcNAc-transferase II was not appreciably influenced by the polypeptide portion of the molecule. It was also observed that prior galactosylation of rhodopsin blocked the addition of GlcNAc to rhodopsin. It is suggested that these properties contribute to the assembly of the abridged structures that have been observed in the oligosaccharides of rhodopsins of all species thus far examined.     

A highly conserved microsatellite in the dystrophin gene of diverse mammalian species

Corticobasal degeneration (C.B.D.) is a neurodegenerative disorder characterized mainly by an asymmetrical a kineto-rigid syndrome associated with fronto-parietal cortical signs, particularly apraxia. Conventional imaging even magnetic resonance imaging (M.R.I.) has often been considered as poorly contributive for the diagnosis of C.B.D. We retrospectively studied routinely performed M.R.I. scans of 15 patients presenting a clinical and metabolic (P.E.T/S.P.E.C.T.) syndrome characteristic of probable C.B.D. M.R.I. scans were assessed by 3 investigators, not aware of the clinically most affected side, taking into account M.R.I. technical parameters. We quantified, on each side, the cortical atrophy (frontal, parietal and temporal) and the white matter changes, by using the semi-quantified method of Victoroff et al. (1994). Abnormalities were considered if observed by at least 2 of the 3 investigators. Abnormalities were then correlated with the side initially and most severely affected. The most contributive findings were the asymmetric parietal atrophy (clinically correlated in 93 p. 100 of cases), asymmetric frontal atrophy (clinically correlated in 60 p. 100) and asymmetric dilatation of the lateral ventricles (clinically correlated in 60 p. 100). 80 p. 100 of affected subjects displayed at least 2 of these M.R.I. abnormalities. These results are in accordance with the metabolic and pathologic features of C.B.D. This study demonstrates that M.R.I. evaluation of the cortical atrophy asymmetry may contribute to the diagnosis of C.B.D.     

Estimation of pH and the number of lytic granules in a CD8+ CTL clone treated with an inhibitor of vacuolar type H(+)-ATPase concanamycin A

A vacuolar type H(+)-ATPase inhibitor, concanamycin A (CMA), raised the pH and induced morphologic changes such as vacuolation in lytic granules in the CD8+ cytotoxic T lymphocyte clone OE4. Here we measured the pH in lytic granules present in OE4 by immunoelectron microscopic observation using (3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) as a pH probe. In control OE4, only the peripheral region, but not cores, were heavily stained with DAMP. Exposure to 100 nM CMA for 60 min raised the pH in the lytic granules from 5.7 +/- 0.2 to 6.8 +/- 0.2. Furthermore, the number of lytic granules in OE4 was estimated under phase contrast microscopy after Wright staining. About 20 lytic granules in average were detected in control cells, while CMA induced vacuolation and decreased their number by 20 to 30%.     

A minisatellite sequence within the propeptide region of the vacuolar carboxypeptidase Y gene of Schizosaccharomyces pombe

We describe the presence of a minisatellite sequence that displays length polymorphisms in the fission yeast Schizosaccharomyces pombe. The minisatellite sequence was found to reside within the propeptide region of the vacuolar carboxypeptidase Y gene. The minisatellite sequence, which was found only at a single locus, was mitotically stable and displayed length polymorphisms between the two varieties of S. pombe (S. pombe var. pombe and S. pombe var. malidevorans). The minisatellite sequence, however, appeared to be species specific and was absent in other members of the Schizosaccharomyces genus. This report constitutes the first experimental demonstration of the presence of such sequences in yeasts.     

5-(5,6-Dichloro-2-indolyl)-2-methoxy-2,4-pentadienamides: novel and selective inhibitors of the vacuolar H+-ATPase of osteoclasts with bone antiresorptive activity

The vacuolar H+-ATPase (V-ATPase), located on the ruffled border of the osteoclast, is a proton pump which is responsible for secreting the massive amounts of protons that are required for the bone resorption process. With the aim to identify new agents which are able to prevent the excessive bone resorption associated with osteoporosis, we have designed a novel class of potent and selective inhibitors of the osteoclast proton pump, starting from the structure of the specific V-ATPase inhibitor bafilomycin A1. Compounds 3a-d potently inhibited the V-ATPase in chicken osteoclast membranes (IC50 = 60-180 nM) and were able to prevent bone resorption by human osteoclasts in vitro at low-nanomolar concentrations. Notably, the EC50 of compound 3c in this assay was 45-fold lower than the concentration required for half-maximal inhibition of the V-ATPase from human kidney cortex. These results support the validity of the osteoclast proton pump as a useful molecular target to produce novel inhibitors of bone resorption, potentially useful as antiosteporotic agents.     

Development of a highly polymorphic STR marker for identity testing purposes at the human androgen receptor gene (HUMARA)

We developed a non-isotopic method which improves the technical quality of the X-linked HUMARA locus typing process. The use of formamide and a low concentration of acrylamide increased resolution and sharpness of HUMARA alleles in silver-stained polyacrylamide gels. In addition, the construction of an allelic ladder containing amplified sequence of 9 alleles (even-numbered alleles) of the HUMARA locus, allows confident, rapid and precise assignment of discretely defined alleles. Allele and genotype frequencies for the HUMARA locus were determined in a French Canadian population sample. Observed genotype frequencies in females conformed to Hardy-Weinberg expectations. Furthermore, the HUMARA locus is highly polymorphic with 18 observed alleles and an heterozygosity value of 89.3%. Also, this locus has average powers of discrimination of 97.8% and 88.7% for testing samples of female and male origin, respectively. In the French Canadian population, the average probability of excluding a random man as the father in paternity analysis when both mother and daughter are tested for this locus is 88.0%. Together, the results indicate that the HUMARA locus provides a highly discriminatory system that is appropriate for the purposes of forensic identification and paternity testing involving a female child.     

Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1.