Cloning and expression analysis of a novel gene, RP42, mapping to an autism susceptibility locus on 6q16

We isolated a novel mouse gene, RP42, in a systematic search for genes expressed in proliferating neuroblasts whose human orthologs map to susceptibility loci for autism. This gene is intronless and encodes a putative 259-amino-acid protein that exhibits 30-36% overall sequence identity to a fission yeast and a nematode protein (GenPept Accession Nos. CAA17006 and CAB54261). Nevertheless, no homology to any known gene was found. RP42 has developmentally regulated expression, particularly in proliferating neuroblasts from which neocortical neurons originate. Its human ortholog is located in a cluster of embryonic neuronally expressed genes on the 6q16 chromosome, making it a positional candidate susceptibility gene for autism.     

Cloning and characterization of additional members of the G protein-coupled receptor family

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.     

Sequence analysis of a KpnI family member near the 3' end of human beta-globin gene.

We determined the complete nucleotide sequence (6125 bp) of a full-length member of human KpnI family, designated T beta G41, which is located about 3 kb downstream from the beta-globin gene. Comparison of the sequence with the KpnI family sequence compiled by Singer revealed that a new 131 bp sequence is present in the T beta G41. Hybridization analyses showed that a few thousand of human KpnI family members are carrying this additional sequence. Computer search of DNA databases for T beta G41-homologous sequence showed that some T beta G41-homologous sequences were closely associated with pseudogenes. The T beta G41 sequence also showed significant sequence homology with ChBlym-1, a transferrin-like transforming gene of chicken. Furthermore, an amino acid sequence deduced from the T beta G41 nucleotide sequence revealed a relatively-high homology to those of human transferrin and lactotransferrin.     

Estrogen-dependent and cell-specific regulation of gene expression in RUCA-I endometrial adenocarcinoma cells

Estrogens are believed to play a crucial role in growth regulation and differentiation of the normal endometrial tissue as well as in the carcinogenesis of the endometrium. Therefore, the influence of estrogens and antiestrogens on gene expression in the estrogen receptor-positive rat endometrial adenocarcinoma cell line RUCA-I was investigated. Differentially expressed genes were detected by differential display PCR of RNA of untreated, estradiol-treated and antiestrogen-treated RUCA-I cells. By means of the PCR technique, 14 differentially expressed fragments could be detected. Three of these 14 differentially expressed fragments were confirmed by Northern blotting. The steady state mRNA levels of the three gene fragments named AH41, AH42 and AH44 were downregulated by the antiestrogen ICI 164384. Further characterization revealed that the fragment AH41 is not expressed in stromal cells but in the human and rodent epithelial cell lines, BG-1 and RUCA-II. A comparison of the cDNA sequence of fragment AH41 with the EMBL database showed no high homology to known genes. Therefore, fragment AH41 has to be regarded as a fragment of a novel, estradiol-sensitive gene.     

Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes

We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH) was used to map GPR20 to chromosome 8q, region 24.3–24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22–q31.1, and GPR23 to chromosome X, region q13–q21.1.© 1997 Elsevier Science B.V. All rights reserved.     

A Novel Human Gene Encoding a G-Protein-Coupled Receptor (GPR15) Is Located on Chromosome 3

We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have namedGPR15.A comparison of the amino acid sequence of the receptor encoded byGPR15with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1.GPR15was mapped to human chromosome 3q11.2–q13.1.     

The C1orf9 gene encodes a putative transmembrane member of a novel protein family

Here we report the characterization of a human mRNA encoding a novel protein denoted C1orf9 (chromosome 1 open reading frame 9). The cDNA sequence, derived from a testis cDNA library, contains 5700 bp which encodes an open reading frame of 1254 amino acids. The deduced protein contains a putative N-terminal signal peptide and one putative transmembrane region, indicating membrane localization. No significant homology was found with known characterized proteins. However, a 150 amino acid region has significant homology to deduced protein sequences from other organisms, including Caenorhabditis elegans (43% identity), Saccharomyces cerevisiae (47% identity), Schizosaccharomyces pombe (48% identity), and two proteins from Arabidopsis thaliana (42% and 40% identity), suggesting a novel family of conserved domains. The C1orf9 gene was assigned to chromosome 1q24. The gene spans approximately 78.7 kb and is organized into at least 24 exons. Expression analysis revealed a single C1orf9 mRNA species of approximately 6.0 kb with a predominant expression in pancreas and testis, and only low levels of expression in other tissues examined.     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

Testis-specific expression of an intronless gene encoding a human poly(A) polymerase.

A mouse intronless gene, encoding a testis-specific poly(A) polymerase (mPAPT), was previously identified. mPAPT may play a role as a putative enzyme that is responsible for polyadenylation regulation during mouse spermatogenesis. In order to understand how PAPT genes are conserved in mammals, we isolated a human cDNA homolog encoding a human PAPT (hPAPT), which was specifically expressed in the testis. The structure of hPAPT was very similar to that of mPAPT. The about 100 residues at the C-terminal region of a nuclear poly(A) polymerase, PAP II, were missing in both PAPT proteins. An analysis of the genomic DNA showed that the hPAPT gene is an intronless gene that is similar to the mPAPT gene. Interestingly, the sequence homology between hPAPT and mPAPT was much lower than the homology between hPAP II and mPAP II. The phylogenetic analysis suggests that PAPTs arose through retrotransposition after the amphibian-amniote split during evolution.     

Cloning,molecular characterization,and spatial and developmental expression analysis of GPR41 and GPR43 genes in New Zealand rabbits

Short-chain fatty acids (SCFAs) play a regulatory role in various physiological processes in mammals and act as endogenous ligands for the G protein-coupled receptors (GPR) 41 and 43. The role of GPR41 and GPR43 in mediating SCFA signaling in the rabbit remains unclear. The present study was to investigate the sequence of the GPR41 and GPR43 messenger RNA (mRNA) and their expression pattern in different tissues and developmental stages in New Zealand rabbit. Comparison of genomic sequences in GenBank using the Basic Local Alignment Search Tool program suggested that the New Zealand rabbit GPR41 mRNA has high similarities with the human (84%), bovine (84%) and Capra hircus (84%) genes. Similarly, GPR43 mRNA has high similarity with the rat (84%) and mouse (84%) genes. Real-time PCR results indicated that GPR41 and GPR43 mRNA were expressed throughout rabbit’s whole development and were expressed in several tissues. G protein-coupled receptor 41 and GPR43 mRNA were most highly expressed in pancreas (P<0.05) and s.c. adipose tissue (P<0.05), respectively. The expression levels of GPR41 mRNA was down-regulated in duodenum, cecum (P<0.05) and pancreas and up-regulated in jejunum, ileum, adipose tissue and spleen during growth. G protein-coupled receptor 43GPR43 mRNA was highly expressed in the duodenum, jejunum, ileum, colon, cecum and lung at 15^th day (P<0.05), whereas the expression levels in the pancreas and spleen increased later after birth, with the highest expression at 60^th day (P<0.05).     

Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics. A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.