Identification of sonic hedgehog-responsive genes using cDNA microarray.

Sonic hedgehog (Shh) is a secreted signaling protein that plays important roles in a variety of developmental processes and also in pathogenesis of some human cancers and congenital diseases. Molecules that function downstream of Shh, however, still remain elusive. Here we searched for Shh-responsive genes by using an in-house cDNA microarray. Two genes were newly identified to be Shh responsive in neuroepithelial cell line MNS-70: the metal-binding protein Ceruloplasmin (Cp) and the serine protease inhibitor inter-alpha-trypsine inhibitor heavy chain H3 (ITIH3). In MNS-70 cells, expression of ITIH3 was regulated by Gli zinc-finger transcription factors downstream of Shh, whereas Cp appeared to be regulated by Gli-independent pathways. Cp mRNA was detected in the developing mouse brain, where its expression domain was closely adjacent to that of Shh. These results demonstrate that microarray technology provides a useful tool for studying expression of developmentally regulated genes.

Human and mouse homologues of the Drosophila melanogaster tweety (tty) gene: a novel gene family encoding predicted transmembrane proteins.

We have cloned cDNA for TTYH1, a human homologue of the Drosophila melanogaster tweety (tty) gene. The 450-residue predicted protein shows 27% amino acid sequence identity (51% similarity) to the Drosophila protein, which contains an additional C-terminal repetitive region. A second Drosophila homologue exhibits 42% identity (65% similarity) to the tty protein. Mouse (Ttyh1), macaque, and Caenorhabditis elegans homologues were also identified, and the complete coding sequence for the mouse gene was determined. The mouse protein is 91% identical to the human protein. Hydrophobicity analysis of the tty-related proteins indicates that they represent a new family of membrane proteins with five potential membrane-spanning regions. The yeast FTR1 and FTH1 iron transporter proteins and the mammalian neurotensin receptors 1 and 2 have a similar hydrophobicity profile, although there is no detectable sequence homology to the tty-related proteins. This suggests that the tweety-related proteins could be involved in transport of iron or other divalent cations or alternatively that they may be membrane-bound receptors. TTYH1 was mapped to chromosome 19q13.4 by FISH and by radiation hybrid mapping using the Stanford G3 panel.

cDNA cloning, expression profile, and genomic structure of human and mouse RNF10/Rnf 10 genes, encoding a novel RING finger protein.

RING finger (C3HC4-type zinc finger) is a variant zinc finger motif present in a new family of proteins including transcription regulators. A new member of the RING finger protein family was identified through a mouse expressed sequence tag (EST) database search, and its full-length cDNA was isolated from a mouse brain full length-enriched cDNA library. The gene was designated as Rnf10, for RING finger protein 10. The cDNA clone consists of 3110 nucleotides and encodes an open reading frame (ORF) of an 804-amino acid protein. A database search revealed that human KIAA0262 protein (accession number, D87451) has strong homology to mouse Rnf10. To confirm that mouse Rnf10 is the homolog or an isolog of human KIAA0262, a human RNF10 cDNA was cloned in our hands from a fetal brain cDNA pool. The newly isolated cDNA contained an ORF for 811 amino acids which had almost identical structure to mouse Rnf10 protein, indicating that the human ORF codes for RNF10 protein. This finding was also supported by comparative chromosome mapping in which both genes were localized in a conserved linkage homology region between mouse and human. Comparison of the RNF10 and KIAA0262 proteins revealed that both were transcribed from the same gene and that the longer RNF10 ORF would be the authentic form. The complete genomic organization of RNF10 was determined to consist of 17 exons spanning at least 40kb in the genome.

Isolation and characterization of a human cDNA encoding a protein homologous to the 7.2-kDa protein (subunit X) of bovine ubiquinol-cytochrome C reductase.

Through large-scale sequencing of clones randomly selected from a library of human cDNAs, we have isolated a novel human gene termed hUQCR10. Its open reading frame encodes 63 amino acids that share 88.5% identity with the sequence of bovine ubiquinol-cytochrome C reductase 7.2-kDa protein (subunit X). A single 0.6-kb transcript was expressed in all human tissues examined, but was particularly abundant in heart and skeletal muscle, tissues that consume a large amount of oxygen. The gene product therefore may play a significant role in the cellular respiratory system. In support of this hypothesis, our immunohistochemical analysis revealed that the hUQCR10 protein is located in mitochondria. A homology search using computer programs determined the chromosomal localization of the gene at 22q12.

Isolation and chromosomal mapping of a novel human gene showing homology to Na+/PO4 cotransporter.

We isolated a cDNA clone which shows a significant similarity with the renal Na+/phosphate cotransporter (NPT) from a human intestine mucosa cDNA library. The cDNA is 2626 bases long, with one open reading frame encoding a protein of 497 amino acids. The deduced amino acids sequence shows an overall homology of 48% with the human renal NPT1 protein. This gene is expressed in intestine, colon, liver, and pancreas. Thus, this gene may code for intestinal type NPT or closely related proteins. The chromosomal location of the gene was determined on the chromosome 6p21.3-p22 region by polymerase chain reaction-based analysis with both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel.

Isolation, tissue expression, and chromosomal assignment of a novel human gene which encodes a protein with RING finger motif.

We identified a novel gene encoding a RING finger (C3HC4-type zinc finger) protein from a human neuroblastoma full-length enriched cDNA library. This cDNA clone consists of 1919 nucleotides with an open reading frame of a 485-amino acid protein. From reverse transcription (RT)-polymerase chain reaction (PCR) analysis, the messenger RNA was ubiquitously expressed in various human adult tissues. The chromosomal location of the gene was determined on the chromosome 6p21.3 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein.

The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of Lhcb1*3 gene expression and in the function of the circadian oscillator in Arabidopsis thaliana. By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (beta) subunit of the protein kinase CK2 and have designated it as CKB3. CKB3 is the only reported example of a third beta-subunit of CK2 found in any organism. CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an in vitro interaction assay. Other subunits of CK2 also show an interaction with CCA1 in vitro. CK2 beta-subunits stimulate binding of CCA1 to the CCA1 binding site on the Lhcb1*3 gene promoter, and recombinant CK2 is able to phosphorylate CCA1 in vitro. Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA-protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1 in vivo.

Effectiveness of formula ophiopogoins in the treatment of xerostomia and pharyngoxerosis.

A multicenter study was carried out in 24 institutions in Kanagawa Prefecture, Japan. The subjects were 113 male and 145 female patients, ranging in age from 28 to 86 years (mean 63.7) who complained of dryness of the mouth and throat. The following illnesses were diagnosed: xerostomia and pharyngoxerosis in 161, radiation xerostomia and pharyngoxerosis in 56, chronic pharyngitis with dry mouth and throat in 22, and Sjögren's syndrome in 7. Eleven patients had other symptoms. The subjects were given a 3-g pack of granules of formula ophiopogoins three times a day before a meal. Their subjective symptoms and objective signs were examined prior to, 2, and 4 weeks after the beginning of therapy.