Parathyroid hormone (hPTH 1-38) stimulates the expression of UBP41, an ubiquitin-specific protease, in bone.

Parathyroid hormone (PTH) stimulates bone formation in both animals and humans, and the expression of a number of genes has been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon, we used differential display reverse transcription polymerase chain reaction (DDRT-PCR) and screened for genes, which are differentially expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single subcutaneous (s.c.) injection of hPTH (1-38) (8 microg/100 g). We found and cloned one full-length cDNA, which encodes a putative 348 amino acid protein. Sequence analysis of this protein demonstrates a 98, 93.7, and 82.5% identity with mouse, human, and chicken ubiquitin-specific protease UBP41, respectively. Northern blot analysis confirmed that a 3.8-4 kb UBP41 mRNA transcript was rapidly increased 1 h after acute hPTH (1-38) exposure in both metaphyseal (6- to 8-fold) and diaphyseal (3-fold) bone, but returned to control levels by 24 h after exposure. In contrast, continuous exposure to hPTH (1-38), resulted in a rapid and sustained elevation of UBP41 mRNA. PTH (1-31), which stimulates intracellular cAMP, and PTHrP (1-34) both induced UBP41 mRNA expression; whereas PTH analogs (3-34) and (7-34), that do not stimulate cAMP, had no effect on UBP41 expression. UBP41 mRNA expression was also rapidly induced 1 h after injection of PGE2, but returned to the control level by 6 to 24 h. In vitro, UBP41 mRNA is expressed in primary osteoblasts (metaphyseal and diaphyseal derived) and in the osteoblast-like cell lines UMR106, ROS17/2.8, and BALC. PTH (1-38) treatment induced UPB41 expression (3.6- to 13-fold) in both primary cultures of osteoblasts and in UMR106 cells. Further analysis in UMR 106 cells demonstrated that PGE2, forskolin and dibutyryl cAMP increased UBP41 mRNA expression 4-, 4.5-, and 2.4-fold, respectively. Tissue distribution analysis of UBP41 mRNA detected transcripts in brain, heart, skeletal muscle, kidney, liver, and testis. Together, these results demonstrate that UBP41, an ubiquitin-specific protease, is selectively upregulated in bone by the osteotropic agents PTH, PTHrP, and PGE2, possibly via the PKA/cAMP pathway. We speculate that the rapid induction of UBP41 in response to these physiological regulators contributes to the mechanism by which either the structure, activity, half-life or localization of essential proteins are modified to maintain bone homeostasis.

Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients.

Velo-cardio-facial syndrome (VCFS) is a relatively common developmental disorder characterized by craniofacial anomalies and conotruncal heart defects. Many VCFS patients have hemizygous deletions for a part of 22q11, suggesting that haploinsufficiency in this region is responsible for its etiology. Because most cases of VCFS are sporadic, portions of 22q11 may be prone to rearrangement. To understand the molecular basis for chromosomal deletions, we defined the extent of the deletion, by genotyping 151 VCFS patients and performing haplotype analysis on 105, using 15 consecutive polymorphic markers in 22q11. We found that 83% had a deletion and >90% of these had a similar approximately 3 Mb deletion, suggesting that sequences flanking the common breakpoints are susceptible to rearrangement. We found no correlation between the presence or size of the deletion and the phenotype. To further define the chromosomal breakpoints among the VCFS patients, we developed somatic hybrid cell lines from a set of VCFS patients. An 11-kb resolution physical map of a 1,080-kb region that includes deletion breakpoints was constructed, incorporating genes and expressed sequence tags (ESTs) isolated by the hybridization selection method. The ordered markers were used to examine the two separated copies of chromosome 22 in the somatic hybrid cell lines. In some cases, we were able to map the chromosome breakpoints within a single cosmid. A 480-kb critical region for VCFS has been delineated, including the genes for GSCL, CTP, CLTD, HIRA, and TMVCF, as well as a number of novel ordered ESTs.

Identification, characterization, and precise mapping of a human gene encoding a novel membrane-spanning protein from the 22q11 region deleted in velo-cardio-facial syndrome.

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of phenotypes including cleft palate, conotruncal heart defects, and facial dysmorphology. Hemizygosity for a portion of chromosome 22q11 has been detected in 80-85% of VCFS/DGS patients. Using a cDNA selection protocol, we have identified a new gene, TMVCF (transmembrane protein deleted in VCFS), which maps to the deleted interval. The genomic locus is positioned between polymorphic markers D22S944 and D22S941. TMVCF encodes a small protein of 219 amino acids that is predicted to contain two membrane-spanning domains. TMVCF is expressed abundantly in human adult lung, heart, and skeletal muscle, and transcripts can be detected at least as early as Day 9 of mouse development.

Identification of amino groups in the carbohydrate binding activity of winged bean acidic agglutinin.

Chemical modification studies reveal that the modification of amino groups in WBA II leads to a complete loss in the hemagglutinating and saccharide binding activities. Since WBA II is a dimeric molecule and contains two binding sites, one amino group in each of the binding sites is inferred to be essential for its activity. The presence of amino group which has a potential to form hydrogen bonded interactions with the ligand, substantiates our observation regarding the forces involved in WBA II-receptor and WBA II-simple sugar interactions.

Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardio-facial syndrome.

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.

Chromosomal localization and expressed sequence tag generation of clones from a normalized human adult thymus cDNA library.

Expressed sequence tags (ESTs) from 298 clones have been generated from a randomly primed, normalized human adult thymus cDNA library. We describe the chromosomal localization of 136 of these ESTs by PCR-based mapping to a human monochromosomal somatic cell hybrid panel. Data base similarities to known genes are also described. A subset (n = 18) of these randomly primed ESTs extended the sequence of ESTs from other tissues currently in dbEST. Of the nonrepetitive human adult thymus ESTs generated in this study, 237 (79.5%) have no similarity to current data base entries. This would suggest that our collection contains approximately 100 new coding regions from thymus tissue, a large proportion of which likely will represent the middle regions of genes. The mapped ESTs should prove useful as new gene-based markers for mapping and candidate gene hunting, particularly when anchored to a well-developed physical map of the human genome.

Molecular cloning and expression pattern of genes from a 470 Kb region near BRCA1 locus on chromosome 17q21.

We have used direct cDNA selection to isolate expressed sequences from a set of four overlapping YACs, spanning approximately 470 kb of the chromosomal interval 17q21 centromeric to BRCA1 gene. Thirty-eight nonoverlapping unique cDNA fragments were identified in this region. Twenty-two of the selected cDNAs showed complete identity with known genes and expressed sequence tags. Two of these cDNAs shared sequence homology with genes known to encode potential DNA binding motifs and hence could function in transcriptional regulation. The remaining 16 unique cDNA fragments showed no significant similarity to sequences in the databases and could potentially encode novel genes. Northern analysis of the novel cDNAs showed differential expression in various tissues, supporting the transcribed nature of these sequences. Our results place the gene for a G-protein coupled receptor (GPR2) previously mapped to 17q within a 400 kb region on 17q21.

A single ataxia telangiectasia gene with a product similar to PI-3 kinase.

A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.

Isolation and refined regional mapping of expressed sequences from human chromosome 21.

To increase candidate genes from human chromosome 21 for the analysis of Down syndrome and other genetic diseases localized on this chromosome, we have isolated and studied 9 cDNA clones encoded by chromosome 21. For isolating cDNAs, single-copy microclones from a chromosome 21 microdissection library were used in direct screening of various cDNA libraries. Seven of the cDNA clones have been regionally mapped on chromosome 21 using a comprehensive hybrid mapping panel comprising 24 cell hybrids that divide the chromosome into 33 subregions. These cDNA clones with refined mapping positions should be useful for identification and cloning of genes responsible for the specific component phenotypes of Down syndrome and other diseases on chromosome 21, including progressive myoclonus epilepsy in 21q22.3.

Mechanism of lectin-cell interactions: thermodynamic and kinetic analysis of the binding of winged bean acidic lectin (WBA II) to human erythrocytes.

In order to identify the forces involved in the binding and to understand the mechanism involved, equilibrium and kinetic studies were performed on the binding of the winged bean acidic lectin to human erythrocytes. The magnitudes of delta S and delta H were positive and negative respectively, an observation differing markedly from the lectin-simple sugar interactions where delta S and delta H are generally negative. Analysis of the sign and magnitudes of these values indicate that ionic and hydrogen bonded interactions prevail over hydrophobic interactions resulting in net -ve delta H (-37.12 kJ.mol-1) and +ve delta S (14.4 J.mole-1 K-1 at 20 degrees C), thereby suggesting that this entropy driven reaction also reflects conformational changes in the lectin and/or the receptor. Presence of two kinds of receptors for WBA II on erythrocytes, as observed by equilibrium studies, is consistent with the biexponential dissociation rate constants (at 20 degrees C K1 = 1.67 x 10(-3) M-1 sec-1 and K2 = 11.1 x 10(-3) M-1 sec-1). These two rate constants differed by an order of magnitude accounting for the difference in the association constants of the two receptors of WBA II. However, the association process remains monoexponential suggesting no observable difference in the association rates of the lectin molecule with both the receptors, under the experimental conditions studied. The thermodynamic parameters calculated from kinetic data correlate well with those observed by equilibrium. A two-step binding mechanism is proposed based on the kinetic parameters for WBA II-receptor interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments.

Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus.

Construction of a uniform-abundance (normalized) cDNA library.

We have used a kinetic approach to construct cDNA libraries containing approximately equal representations of all sequences in a preparation of poly(A)+ RNA. Randomly primed cDNA fragments of a selected size range were cloned in lambda phage vector. Inserts were amplified by the polymerase chain reaction (PCR), denatured, and self-annealed under optimized conditions. After extensive but incomplete reannealing, the single-stranded fraction was relatively depleted of more abundant species of cDNA. Libraries of these fragments are suitable for cDNA subtraction, screening, or selection by hybridization and make it possible to detect and analyze cDNA corresponding to species of mRNA present at a low level in a small fraction of the cells in a complex tissue.

Thermodynamic analysis of ligand binding to winged bean (Psophocarpus tetragonolobus) acidic agglutinin reveals its specificity for terminally monofucosylated H-reactive sugars.

The sugar-specific binding of N-dansylgalactosamine to WBA II (n = 2; Ka = 5.6 x 10(3) M-1; delta H = -21 kJ.mol-1; delta S = -21.3 J.mol-1.K-1) was utilized in substitution titrations for evaluating the association constants for the interaction of sugars with the lectin. An axial hydroxyl at C-4 and equatorial hydroxyls at C-3 and C-6 as in D-galacto configuration are crucial for binding. Both axial and equatorial hydroxyls are tolerated at C-2. Conformationally akin disaccharides such as lactose, N-acetyllactosamine, Gal beta 1-3GlcNAc, and Gal beta 1-3GalNAc show similar affinities. 2'-Fucosyllactose and H-disaccharide display 146 and 13 times stronger affinity over lactose and galactose, yet fucose by itself is devoid of activity. An interesting feature, noted for the first time, in protein-sugar interactions is the positive entropy change for the binding of 2'-fucosyllactose, suggesting that nonpolar interactions play an important role in stabilization of the lectin-sugar complex. 3-Fucosyllactose, lactodifucotetraose, lacto-N-fucopentaose II and III are inactive, whereas lacto-N-fucopentaose I has 14-fold lower affinity as compared with 2'-fucosyllactose. Conformational analysis indicates that the substitution at subterminal glucose or GlcNAc by L-fucose in either alpha 1-3 or alpha 1-4 linkage leads to its projection so as to sterically hinder the access of 3'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose II and III to the binding site of winged bean agglutinin II. Similarly the projection of alpha 1-3 linked Gal/GalNAc also leads to steric hindrance and hence prevents the binding of blood group A and B reactive sugars. Considering its unique specificity winged bean agglutinin II should be useful in the isolation and characterization of terminally monofucosylated H-reactive oligosaccharides from those that are difucosylated or internally fucosylated.

Erythrocyte-binding studies on an acidic lectin from winged bean (Psophocarpus tetragonolobus).

An acidic lectin (WBA II) was isolated to homogeneity from the crude seed extract of the winged bean (Psophocarpus tetragonolobus) by affinity chromatography on lactosylaminoethyl-Bio-Gel. Binding of WBA II to human erythrocytes of type-A, -B and -O blood groups showed the presence of 10(5) receptors/cell, with high association constants (10(6)-10(8) M-1). Competitive binding studies with blood-group-specific lectins reveal that WBA II binds to H- and T-antigenic determinants on human erythrocytes. Affinity-chromatographic studies using A-, B-, H- and T-antigenic determinants coupled to an insoluble matrix confirm the specificity of WBA II towards H- and T-antigenic determinants. Inhibition of the binding of WBA II by various sugars show that N-acetylgalactosamine and T-antigenic disaccharide (Thomsen-Friedenreich antigen, Gal beta 1-3GalNAc) are the most potent mono- and di-saccharide inhibitors respectively. In addition, inhibition of the binding of WBA II to erythrocytes by dog intestine H-fucolipid prove that the lectin binds to H-antigenic determinant.

Studies on tryptophan residues of Abrus agglutinin. Stopped-flow kinetics of modification and fluorescence-quenching studies.

The presence of two essential tryptophan residues/molecule was implicated in the binding site of Abrus agglutinin [Patanjali, Swamy, Anantharam, Khan & Surolia (1984) Biochem. J. 217, 773-781]. A detailed study of the stopped-flow kinetics of the oxidation of tryptophan residues revealed three classes of tryptophan residues in the native protein. A discrete reorganization of tryptophan residues revealed three classes of tryptophan residues in the native protein. A discrete reorganization of tryptophan residues into two phases was observed upon ligand binding. The heterogeneity of tryptophan exposure was substantiated by quenching studies with acrylamide, succinimide and Cs+. Our study revealed the microenvironment of tryptophan residues to be hydrophobic, and also the presence of acidic amino acid residues in the vicinity of surface-localized tryptophan residues.

Isolation, macromolecular properties, and combining site of a chito-oligosaccharide-specific lectin from the exudate of ridge gourd (Luffa acutangula).

A lectin specific for chito-oligosaccharides from the exudate of ridge gourd (Luffa acutangula) fruits has been purified to homogeneity by affinity chromatography. The lectin has a molecular weight of 48,000, an S(0)20,w of 4.06 S and a Stokes radius of 2.9 nm. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single band corresponding to Mr of 24,000 was observed both in the presence and absence of beta-mercaptoethanol. The subunits in this dimeric lectin are, therefore, held together solely by noncovalent interactions. The lectin is not a glycoprotein, and secondary structure analysis by CD measurements showed 31% alpha-helix. The hemagglutinating activity of L. acutangula agglutinin was not inhibited by any of the monosaccharides tested. Among the disaccharides only di-N-acetylchitobiose was inhibitory. The inhibitory potency of chito-oligosaccharides increased dramatically with their size up to penta-N-acetylchitopentaose. The lectin has two binding sites for saccharides. The affinity of chito-oligosaccharides for L. acutangula lectin, as monitored by titrating the changes in the near UV-CD spectra and intrinsic fluorescence, increased strikingly with the number of GlcNAc units in them. The values of delta G, delta H, and delta S for the binding process showed a pronounced dependence on the size of the chito-oligosaccharides, indicating that the binding of higher oligomers is progressively more favored thermodynamically than di-N-acetylchitobiose. The thermodynamic data are consistent with an extended binding site in this lectin, which accommodates a tetrasaccharide.

Analysis of saccharide binding to Artocarpus integrifolia lectin reveals specific recognition of T-antigen (beta-D-Gal(1----3)D-GalNAc).

The binding of Artocarpus integrifolia lectin to N-dansylgalactosamine (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) leads to a 100% increase in dansyl fluorescence with a concomitant blue shift in the emission maximum by 10 nm. This binding is carbohydrate-specific and has an association constant of 1.74 X 10(4) M-1 at 20 degrees C. The lectin has two binding sites for N-dansylgalactosamine. The values of -delta H and -delta S for the binding of N-dansylgalactosamine are in the range of values reported for several lectin-monosaccharide interactions, indicating an absence of nonpolar interaction of the dansyl moiety of the sugar with the combining region of the protein. Dissociation of the bound N-dansylgalactosamine from its complex with the lectin and the consequent change in its fluorescence on addition of nonfluorescent sugars allowed evaluation of the association constant for competing ligands. The thermodynamic parameters for the binding of monosaccharides suggest that the OH groups at C-2, C-3, C-4, and C-6 in the D-galactose configuration are important loci for interaction with the lectin. The acetamido group at C-2 of 2-acetamido-2-deoxygalactopyranose and a methoxyl group at C-1 of methyl-alpha-D-galactopyranoside are presumably also involved in binding through nonpolar and van der Waals' interactions. The T-antigenic disaccharide Gal beta 1----3GalNAc binds very strongly to the lectin when compared with methyl-beta-D-galactopyranoside, the beta(1----3)-linked disaccharides such as Gal beta 1----3GlcNAc, and the beta(1----4)-linked disaccharides, N-acetyllactosamine and lactose. The major stabilizing force for the avid binding of T-antigenic disaccharide appears to be a favorable enthalpic contribution. The combining site of the lectin is, therefore, extended. These data taken together suggest that the Artocarpus lectin is specific toward the Thomsen-Friedenreich (T) antigen. There are subtle differences in the overall topography of its combining site when compared with that of peanut (Arachis hypogaea) agglutinin. The results of stopped flow spectrometry for the binding of N-dansylgalactosamine tot he Artocarpus lectin are consistent with a simple single-step bimolecular association and unimolecular dissociation rate processes. The value of K+1 and K-1 at 21 degrees C are 8.1 X 10(5) M-1 s-1 and 50 s-1, respectively. The activation parameters indicate an enthalpy-controlled association process.