Discovery of molecular and catalytic diversity among human diphosphoinositol-polyphosphate phosphohydrolases. An expanding Nudt family

The turnover of the "high energy" diphosphoinositol polyphosphates by Ca(2+)- and cyclic nucleotide-modulated enzymes is considered a regulatory, molecular switching activity. Target processes may include intracellular trafficking. Following our earlier identification of a prototype human diphosphoinositol-polyphosphate phosphohydrolase (hDIPP1), we now describe new 21-kDa human isoforms, hDIPP2alpha and hDIPP2beta, distinguished from each other solely by hDIPP2beta possessing one additional amino acid (Gln(86)). Candidate DIPP2alpha and DIPP2beta homologues in rat and mouse were also identified. The rank order for catalytic activity is hDIPP1 > hDIPP2alpha > hDIPP2beta. Differential expression of hDIPP isoforms may provide flexibility in response times of the molecular switches. The 76% identity between hDIPP1 and the hDIPP2s includes conservation of an emerging signature sequence, namely, a Nudt (MutT) motif with a GX(2)GX(6)G carboxy extension. Northern and Western analyses indicate expression of hDIPP2s is broad but atypically controlled; these proteins are translated from multiple mRNAs that differ in the length of the 3'-untranslated region because of utilization of an array of alternative (canonical and noncanonical) polyadenylation signals. Thus, cells can recruit sophisticated molecular processes to regulate diphosphoinositol polyphosphate turnover.     

Molecular cloning and characterization of the human topoisomerase IIalpha and IIbeta genes: evidence for isoform evolution through gene duplication

Human DNA topoisomerase II is essential for chromosome segregation and is the target for several clinically important anticancer agents. It is expressed as genetically distinct alpha and beta isoforms encoded by the TOP2alpha and TOP2beta genes that map to chromosomes 17q21-22 and 3p24, respectively. The genes display different patterns of cell cycle- and tissue-specific expression, with the alpha isoform markedly upregulated in proliferating cells. In addition to the fundamental role of TOP2alpha and TOP2beta genes in cell growth and development, altered expression and rearrangement of both genes are implicated in anticancer drug resistance. Here, we report the complete structure of the human topoisomerase IIalpha gene, which consists of 35 exons spanning 27.5 kb. Sequence data for the exon-intron boundaries were determined and examined in the context of topoisomerase IIalpha protein structure comprising three functional domains associated with energy transduction, DNA breakage-reunion activity and nuclear localization. The organization of the 3' half of human TOP2beta, including sequence specifying the C-terminal nuclear localization domain, was also elucidated. Of the 15 introns identified in this 20 kb region of TOP2beta, the first nine and the last intron align in identical positions and display the same phases as introns in TOP2alpha. Though their extreme 3' ends differ, the striking conservation suggests the two genes diverged recently in evolutionary terms consistent with a gene duplication event. Access to TOP2alpha and TOP2beta gene structures should aid studies of mutations and gene rearrangements associated with anticancer drug resistance.     

A Cluster of Keratin-Associated Proteins on Mouse Chromosome 10 in the Region of Conserved Linkage with Human Chromosome 21

A gene cluster of three to five high-cysteine keratin-associated proteins (KAPs) has been identified on mouse Chromosome 10 (MMU10) in the region of conserved linkage with human chromosome 21 (HSA21). One of these genes,Krtap12-1,has been sequenced in its entirety and shown to be an intronless gene encoding a predicted 130-amino-acid protein.Krtap12-1is most closely related to two previously identified KAP4 genes, but variation in sequence and cysteine content suggests that it represents a new KAP family.Krtap12-1is expressed in the skin of a 3-day-old mouse. The corresponding region of HSA21, betweenITGB2(integrin β2) andPFKL(the liver isoform of phosphofructokinase), has proven refractory to cloning, and thus mapping of this region at high resolution has been problematic. Based on the KAP gene cluster position in mouse, evidence has been found for an orthologous human KAP cluster on HSA21q22.3, reinforcing the observation that comparative genomics can play an essential and practical role in determining mammalian genome organization.     

Cytosolic glutamine synthetase in soybean is encoded by a multigene family, and the members are regulated in an organ-specific and developmental manner.

Gln synthetase (GS) is the key enzyme in N metabolism and it catalyzes the synthesis of Gln from glutamic acid, ATP, and NH4+. There are two major isoforms of GS in plants, a cytosolic form (GS1) and a chloroplastic form (GS2). In leaves, GS2 functions to assimilate ammonia produced by nitrate reduction and photorespiration, and GS1 is the major isoform assimilating NH3 produced by all other metabolic processes, including symbiotic N2 fixation in the nodules. GS1 is encoded by a small multigene family in soybean (Glycine max), and cDNA clones for the different members have been isolated. Based on sequence divergence in the 3'-untranslated region, three distinct classes of GS1 genes have been identified (alpha, beta, and gamma). Genomic Southern analysis and analysis of hybrid-select translation products suggest that each class has two distinct members. The alpha forms are the major isoforms in the cotyledons and young roots. The beta forms, although constitutive in their expression pattern, are ammonia inducible and show high expression in N2-fixing nodules. The gamma1 gene appears to be more nodule specific, whereas the gamma2 gene member, although nodule enhanced, is also expressed in the cotyledons and flowers. The two members of the alpha and beta class of GS1 genes show subtle differences in the expression pattern. Analysis of the promoter regions of the gamma1 and gamma2 genes show sequence conservation around the TATA box but complete divergence in the rest of the promoter region. We postulate that each member of the three GS1 gene classes may be derived from the two ancestral genomes from which the allotetraploid soybean was derived.     

Chemokine PARC gene (SCYA18) generated by fusion of two MIP-1alpha/LD78alpha-like genes

Two loci in the human genome, chromosomes 4q12-q21 and 17q11.2, contain clusters of CXC and CC chemokine subfamily genes, respectively. Since mice appear to contain fewer chemokine genes than humans, numerous gene duplications might have occurred in each locus of the human genome. Here we describe the genomic organization of the human pulmonary and activation-regulated CC chemokine (PARC), also known as DC-CK1 and AMAC-1. Despite high sequence similarity to a CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha)/LD78alpha, PARC is chemotactic for lymphocytes and not for monocytes and does not share its receptor with MIP-1alpha. Analyses of the BAC clones containing the human PARC gene indicated that the gene is located most closely to MIP-1alpha (HGMW-approved symbol SCYA3) and MIP-1beta (HGMW-approved symbol SCYA4) on chromosome 17q11.2. Dot-plot comparison suggested that the PARC gene had been generated by fusion of two MIP-1alpha-like genes with deletion and selective usage of exons. Base changes accumulated before and after the fusion might have adapted the gene to a new function. Since there are variably duplicated copies of the MIP-1alpha gene called LD78beta (HGMW-approved symbol SCYA3L) in the vicinity of the MIP-1alpha gene, the locus surrounding the MIP-1alpha gene seems to be a "hot spring" that continuously produces new family genes. This evidence provides a new model, duplication and fusion, of the molecular basis for diversity within a gene family.     

Chromosomal location of murine and human IL-1 receptor genes.

The gene for the type I interleukin-1 (IL-1) receptor has been mapped in both mouse and human. In the human genome, a combination of segregation analysis of rodent-human hybrid cells and chromosomal in situ hybridization has placed the gene on the long arm of chromosome 2, at band 2q12. This is near the reported map position of the loci for IL-1 alpha and IL-1 beta (2q13----2q21). The murine gene has been mapped by analysis of restriction fragment length polymorphisms in interspecific backcrosses to the centromeric end of chromosome 1, in a region that is syntenic to a portion of human chromosome 2. The murine Il-1r1 gene has thus been separated from the IL-1 genes, which lie on murine chromosome 2.     

Genetic organization of the polymorphic equine alpha globin locus and sequence of the BII alpha 1 gene.

The equine alpha globin gene complex comprises two functional alpha genes and an alpha-like pseudogene arranged in the order 5'-alpha 2-(5kb)-alpha 1-(3kb)-psi alpha-3'. A single (embryonic) zeta-like sequence lies within a 12 kb region 5' to the alpha 2 gene. We have determined the sequence of the alpha 1 gene of the BII haplotype, one of two most common haplotypes (the other being BI) which encode alpha globins with either Tyr (BI) or Phe (BII) at codon 24 in both linked alpha genes. In BI and BII the non-allelic alpha 2 and alpha 1 genes respectively code for Gln or Lys at codon 60, thus accounting for the 4 alpha globin types seen in BI/BII heterozygotes. Genomic restriction enzyme maps of the BII alpha complex (24Phe/60Lys,Gln) and the allelic BI (24Tyr/60Lys,Gln) are identical to each other, and to those of a rarer normal haplotype, A, which encodes only alpha 24Tyr/60Gln globin, and a low expression mutant of BII which encodes only 24Phe/60Lys globin. These two latter haplotypes must therefore have a linked pair of alpha genes, as in BI and BII, but with identical coding properties, and it is suggested that this has arisen by gene conversion.     

Human synemin gene generates splice variants encoding two distinct intermediate filament proteins.

Intermediate filament (IF) proteins are constituents of the cytoskeleton, conferring resistance to mechanical stress, and are encoded by a dispersed multigene family. In man we have identified two isoforms (180 and 150 kDa) of the IF protein synemin. Synemin alpha and beta have a very short N-terminal domain of 10 amino acids and a long C-terminal domain consisting of 1243 amino acids for the alpha isoform and 931 amino acids for the beta isoform. An intronic sequence of the synemin beta isoform is used as a coding sequence for synemin alpha. Both mRNA isoforms (6.5 and 7.5 kb) result from alternative splicing of the same gene, which has been assigned to human chromosome 15q26.3. Analyses by Northern and Western blot revealed that isoform beta is the predominant isoform in striated muscles, whereas both isoforms (alpha and beta) are present in almost equal quantities in smooth muscles. Co-transfection and immunolabeling experiments indicate that both synemin isoforms are incorporated with desmin to form heteropolymeric IFs. Furthermore synemin and desmin are found aggregated together in certain pathological situations.     

Sequencing of full-length cDNA encoding the alpha and beta subunits of human casein kinase II from human platelets and megakaryocytic cells. Expression of the casein kinase IIalpha intronless gene in a megakaryocytic cell line

Casein kinase II (CKII) is a ubiquitous protein kinase composed of two subunits, alpha and beta, that can use both ATP and GTP as phosphoryl donors. Two genes located on two separate chromosomes were identified for CKIIalpha: one on chromosome 20 band 13 with an approximate size of 20 kb and a second on chromosome 11 band 15.5-p15.4 that is the same size as the cDNA of locus 20 kb (1.2 kb) and does not contain any introns. The two genes differ in four amino acids. Recently, it has been demonstrated that a membrane-associated platelet-derived CKII phosphorylates coagulation factor Va. The mRNA encoding the platelet CKII was isolated from fresh human platelets, and the corresponding cDNAs encoding the alpha and beta subunits of human platelet CKII were produced and sequenced. The cDNA for platelet CKIIalpha was found to be 99.7% homologous to the CKIIalpha intronless gene, having the same characteristic amino acid residues at positions 128, 256, 287, and 351. However, the cDNA of platelet CKIIalpha has a different amino acid at position 236 (Arg --> His), which is not found in the intronless gene. The cDNA of the CKIIbeta subunit was completely identical with the sequence of the CKIIbeta subunit isolated from other tissues. Since platelets arise from megakaryocytes, mRNA was isolated from the megakaryocytic cell line MEG-01 and the cDNA for CKIIalpha was cloned and sequenced. The cDNA was found to be identical to the intronless gene found in platelets. We have also investigated the expression of the intronless gene in several other cell lines. Expression of the intronless gene was only found in cell line MEG-01. Our data demonstrate expression of the CKIIalpha intronless gene in megakaryocytes and platelets.     

Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss)

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.     

Characterization of a novel synGAP isoform, synGAP-beta

We cloned a cDNA encoding a novel synGAP, synGAP-d (GenBank(TM) accession number ), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3' portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into alpha1, alpha2, beta1, beta2, beta3, beta4, and gamma, and synGAP-d was classified as the beta1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the alpha1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the beta isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing alpha1 isoform. The beta isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated alpha subunit of Ca(2+)/calmodulin-dependent protein kinase II through its unique C-terminal tail.     

Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B).

Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (beta 1) encodes a 170 amino acid protein (M(r) approximately 19.3 kDa), whereas that for the testis isoform (beta 2) contains 179 residues (M(r) approximately 20.7 kDa); these two sequences show approximately 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the beta 2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (alpha 3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.     

Chromosome assignment of genes encoding the alpha and beta subunits of glycoprotein hormones in man and mouse

The chromosomal locations of the genes for the common alpha subunit of the glycoprotein hormones and the beta subunit of chorionic gonadotropin in humans and mice have been determined by restriction enzyme analysis of DNA isolated from somatic cell hybrids. The CG alpha gene (CGA), detected as a 15-kb BamHI fragment in human DNA by hybridization to CG alpha cDNA, segregated with the chromosome 6 enzyme markers ME1 (malic enzyme, soluble) and SOD2 (superoxide dismutase, mitchondrial) and an intact chromosome 6 in human-rodent hybrids. Cell hybrids containing portions of chromosome 6 allowed the localization of CGA to the q12 leads to q21 region. The greater than 30- and 6.5-kb BamHI CGB fragments hybridizing to human CG beta cDNA segregated concordantly with the human chromosome 19 marker enzymes PEPD (peptidase D) and GPI (glucose phosphate isomerase) and a normal chromosome 19 in karyotyped hybrids. A KpnI-HindIII digest of cell hybrid DNAs indicated that the multiple copies of the CG beta gene are all located on human chromosome 19. In the mouse, the alpha subunit gene, detected by a mouse thyrotropin (TSH) alpha subunit probe, and the CG beta-like sequences (CG beta-LH beta), detected by the human CG beta cDNA probe, are on chromosomes 4 and 7, respectively.     

Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules.

To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site.