cDNA cloning, expression pattern, genomic structure and chromosomal location of RAB6KIFL, a human kinesin-like gene

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal 35 kDa, plant 55 kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.     

Coupled analysis of gene expression and chromosomal location

Microarray technology can be used to assess simultaneously global changes in expression of mRNA or genomic DNA copy number among thousands of genes in different biological states. In many cases, it is desirable to determine if altered patterns of gene expression correlate with chromosomal abnormalities or assess expression of genes that are contiguous in the genome. We describe a method, differential gene locus mapping (DIGMAP), which aligns the known chromosomal location of a gene to its expression value deduced by microarray analysis. The method partitions microarray data into subsets by chromosomal location for each gene interrogated by an array. Microarray data in an individual subset can then be clustered by physical location of genes at a subchromosomal level based upon ordered alignment in genome sequence. A graphical display is generated by representing each genomic locus with a colored cell that quantitatively reflects its differential expression value. The clustered patterns can be viewed and compared based on their expression signatures as defined by differential values between control and experimental samples. In this study, DIGMAP was tested using previously published studies of breast cancer analyzed by comparative genomic hybridization (CGH) and prostate cancer gene expression profiles assessed by cDNA microarray experiments. Analysis of the breast cancer CGH data demonstrated the ability of DIGMAP to deduce gene amplifications and deletions. Application of the DIGMAP method to the prostate data revealed several carcinoma-related loci, including one at 16q13 with marked differential expression encompassing 19 known genes including 9 encoding metallothionein proteins. We conclude that DIGMAP is a powerful computational tool enabling the coupled analysis of microarray data with genome location.     

Characterization and chromosomal mapping of a mouse ortholog of the late-infantile ceroid-lipofuscinosis gene CLN2

Late-infantile ceroid-lipofuscinosis (CLN2) is an autosomal recessively inherited, neurodegenerative disease in humans. The CLN2 locus has been mapped to Chromosome (Chr) 11p15, and its sequence and genomic organization have recently been reported. In the present study, the cDNA sequence, exon/intron organization, and chromosomal localization of a mouse ortholog of the CLN2 gene are described. The mouse cDNA contains an open reading frame that predicts a protein product of 562 amino acids. The mouse and human coding regions are 86% and 88% identical at the nucleic acid and amino acid levels, respectively. One less codon appears in the mouse cDNA when compared with the human ortholog. The mouse gene (Cln2) spans more than 6 kb and consists of 13 exons separated by introns ranging in size from 111 to 1259 bp. Length polymorphism in an (AC)_n microsatellite in intron 3 of the mouse Cln2 gene was used to perform segregation analysis with The Jackson Laboratory DNA Panel Mapping Resource. On the basis of this analysis, the Cln2 gene was localized to a region of mouse Chr 7 that corresponds to human Chr 11p15. Characterization of the mouse Cln2 gene will facilitate generation of a mouse model for late-infantile ceroid-lipofuscinosis by gene targeting and identification of functionally important regions of the Cln2 protein. Received: 25 May 1999 / Accepted: 22 July 1999     

Characterization of the human talin (TLN) gene: genomic structure, chromosomal localization, and expression pattern

Talin is a high-molecular-weight cytoskeletal protein, localized at cell-extracellular matrix associations known as focal contacts. In these regions, talin is thought to link integrin receptors to the actin cytoskeleton. Talin plays a key role in the assembly of actin filaments and in spreading and migration of various cell types. Talin proteins are found in a wide variety of organisms, from slime molds to humans. The human Talin (HGMW-approved symbol TLN) gene was previously mapped to chromosome 9p, but little was known of its sequence and genomic structure. To characterize human TLN further, we have isolated a single bacterial artificial chromosome clone, harboring the entire gene. The gene extends over more than 23 kb and consists of 57 exons. We have localized TLN to human chromosome band 9p13 by both fluorescence in situ hybridization and radiation hybrid mapping. Northern blot analysis detected TLN expression in various human tissues, including leukocytes, lung, placenta, liver, kidney, spleen, thymus, colon, skeletal muscle, and heart. Based on its chromosomal location, expression pattern, and protein function, we considered TLN as a candidate gene for cartilage-hair hypoplasia (CHH), an autosomal recessive metaphyseal chondrodysplasia, previously mapped to 9p13. We sequenced the entire TLN coding sequence in several CHH patients, but no functional mutations were detected.     

Structure, expression and chromosomal location of the Oct-4 gene.

The map position of Oct-4 on mouse chromosome 17 is between Q and T regions in the Major Histocompatibility Complex (MHC), and it is physically located within 35 kb of a class I gene. Several Oct-4-related genes are present in the murine genome; one of them maps to chromosome 9. The genomic structure and sequence of Oct-4 determined in t-haplotypes reveals five exons, and shows no significant changes in the t12 mutant haplotype making it unlikely that Oct-4 and the t12 early embryonic lethal are the same gene. By in situ hybridization, detectable onset of zygotic Oct-4 expression does not occur until compaction begins at 8-cells, suggesting that there might be other regulatory factors responsible for initiating Oct-4 expression.     

Evolution, expression, and chromosomal location of a novel receptor tyrosine kinase gene, eph.

Partial sequence analysis of the genomic eph locus revealed that the splicing points of kinase domain-encoding exons were completely distinct from those of the other protein tyrosine kinase members reported, suggesting that this is the earliest evolutionary split within this family. In Northern (RNA) blot analysis, the eph gene was expressed in liver, lung, kidney, and testis of rat, and screening of 25 human cancers of various cell types showed preferential expression in cells of epithelial origin. Overexpression of eph mRNA was found in a hepatoma and a lung cancer without gene amplification. Comparison of cDNA sequences derived from a normal liver and a hepatoma that overproduces eph mRNA demonstrated that two of them were completely identical throughout the transmembrane to the carboxy-terminal portions. Southern blot analysis of DNAs from human-mouse hybrid clones with an eph probe showed that this gene was present on human chromosome 7.     

Identification, genomic organization, chromosomal mapping and mutation analysis of the human INV gene, the ortholog of a murine gene implicated in left-right axis development and biliary atresia

Determination of left-right axis is a precocious embryonic event, and all phenotypic anomalies resulting from disruption of the normal lateralization process are collectively referred to as the lateralization defect. A transgenic mouse with lateralization defect and hepatic, kidney, and pancreatic anomalies has resulted from disruption of the inv gene by insertion of a transgene. The human ortholog is thus a good candidate for lateralization defect in humans, in particular in cases with associated hepatic anomalies. Here, we have identified, mapped, and characterized the INV human gene and screened a series of heterotaxic patients (with or without biliary anomalies) for mutation in this gene. In a German family of Turkish origin, we have found that all available affected and unaffected individuals are heterozygous for a mutation in the splicing donor site of intron 12 in the INV gene resulting in two different aberrant splicing isoforms. This can be explained either by a randomization of lateralization defects or, as suggested earlier, di- or trigenic inheritance, although we have been unable to detect, in this family, a mutation in genes known to be involved in the human lateralization defect ( LEFTY1, LEFTY2, ACVR2B, NODAL, ZIC3, and CFC1). In contrast to the mouse, the affected individuals have no biliary anomalies, and the absence of mutation in a series of seven cases with lateralization defect and biliary anomalies demonstrates that INV is not frequently involved in such a phenotype in humans.     

The human colipase gene: isolation, chromosomal location, and tissue-specific expression.

The digestion of dietary triglycerides occurs in the duodenum through the action of triglyceride lipase, a pancreatic exocrine protein. The activity of pancreatic lipase is inhibited by the bile salts normally found in the gut lumen. Another pancreatic exocrine protein, colipase, restores the lipolytic activity of triglyceride lipase. The synthesis and secretion of both triglyceride lipase and colipase is increased by dietary fats and secretin. An increase in mRNA accompanies the increased activity, suggesting that the genes for triglyceride lipase and colipase contain nucleotide elements responsive to dietary fats or secretin or both. To study the regulation of colipase expression, we have first isolated the gene for human colipase from a cosmid library with a cDNA probe. The gene was localized to chromosome 6 and is organized into three exons contained in a single 3.3-kb BamHI fragment. The 5'-flanking region of the gene contains a TATA box, a GC box, and a 28-bp region with homology to the rat pancreatic-specific enhancer. This region directs the tissue-specific expression of the chloramphenicol acetyltransferase gene in a transfected rat pancreatic acinar cell line, AR42-J. The same construct is inactive in HEPG2, C2C12, and COS-1 cells. These results demonstrate that the isolated gene for human colipase contains tissue-specific promoter activity in the 5'-flanking DNA. The 28-bp region specifically binds to a factor in nuclear extracts.     

Characterization of the calmodulin gene family in wheat: structure, chromosomal location, and evolutionary aspects

Calmodulin is a ubiquitous transducer of calcium signals in eukaryotes. In diploid plant species, several isoforms of calmodulin have been described. Here, we report on the isolation and characterization of calmodulin cDNAs corresponding to 10 genes from hexaploid (bread) wheat (Triticum aestivum). These genes encode three distinct calmodulin isoforms; one isoform is novel in that it lacks a conserved calcium binding site. Based on their nucleotide sequences, the 10 cDNAs were classified into four subfamilies. Using subfamily-specific DNA probes, calmodulin genes were identified and the chromosomal location of each subfamily was determined by Southern analysis of selected aneuploid lines. The data suggest that hexaploid wheat possesses at least 13 calmodulin-related genes. Subfamilies 1 and 2 were both localized to the short arms of homoeologous-group 3 chromosomes; subfamily 2 is located on all three homoeologous short arms (3AS, 3BS and 3DS), whereas subfamily 1 is located only on 3AS and 3BS but not on 3DS. Further analysis revealed thatAegilops tauschii, the presumed diploid donor of the D-genome of hexaploid wheat, lacks a subfamily-1 calmodulin gene homologue, whereas diploid species related to the progenitors of the A and B genomes do contain such genes. Subfamily 3 was localized to the short arm of homoeologous chromosomes 2A, 2B and 2D, and subfamily 4 was mapped to the proximal regions of 4AS, 4BL and 4DL. These findings suggest that the calmodulin genes within each subfamily in hexaploid wheat represent homoeoallelic loci. Furthermore, they also suggest that calmodulin genes diversified into subfamilies before speciation ofTriticum andAegilops diploid species.     

Structure,expression, chromosomal location and product of the gene encoding ADH1 inPetunia

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated fromPetunia hybrida cv. V30 by screening aPetunia genomic library with a maizeAdh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of twoAdh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay inPetunia protoplasts. We have designated this genePetunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction ofAdh1 mRNA.     

Characterization and expression of a genomic pectin methyl esterase-encoding gene in Aspergillus niger

The genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344 was cloned by probing a genomic DNA library with a cDNA coding for PME. The recombinant phage clone was isolated and a 6-kb HindIII fragment was subcloned and characterized. The gene consists of seven exons and six introns. The nucleotide sequences of the coding regions were identical to those found in the pmeA cDNA. Cotransformation of A. niger was achieved with the vector, pAN7-1, and transformants were then tested for PME production. Transformants which produced more PME than the untransformed recipient strain were subjected to Southern-blot and Northern-blot analysis. The results show that there is a reasonable correlation between gene copy number, mRNA levels and PME production. PME was produced by A. niger transformants in an active 43-kDa form, which is similar to that of the mature protein isolated from the strain, RH5344. On the basis of the results of affinity labeling of PME with sugar-specific lectins and the amino acid sequence data, it has been revealed that PME is a glycoprotein and the protein-bound glycans are oligosaccharides with a high mannose content.     

Characterization and comparative genomic analysis of intronless Adams with testicular gene expression

ADAM (a disintegrin and metalloprotease) family members with testis-specific or -predominant gene expression are divided phylogenically into two groups: ADAMs 2, 3, 5, 27, and 32 (the first group) and ADAMs 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the second group). We cloned and sequenced cDNAs for previously unidentified mouse Adams that belong to the second group. We found that all the Adam genes in the second phylogenic group are transcribed by both somatic and germ cells in mouse testis, representing a unique expression pattern different from that of the first-group Adams. Genomic analyses revealed that all the second-group Adam genes lack introns interrupting protein-coding sequences and many of them are present as multicopy genes, resulting in total of 14 functional mouse genes in this phylogenic group. Comparing the mouse and human ADAM genes, we found that a number of these mouse Adam genes do not have human orthologues and, even if they exist, some orthologues are pseudogenes in human. These results suggest the differential expansion of the second-group Adam genes in the mouse genome during evolution and a relationship between these Adams and male reproduction unique to mouse.