Isolation of a cDNA for a novel human RING finger protein gene, RNF18, by the virtual transcribed sequence (VTS) approach(1).
We have recently developed a novel database system, designated as the virtual transcribed sequence (VTS) which efficiently extracts many genes from public human genome databases, and tested the feasibility of this novel computational approach (N. Miyajima, C. Burge, T. Saito, Biochem. Biophys. Res. Commun. 272 (2000) 801; http://host45.maze.co.jp/vts/). In this study, using the VTS approach, we isolated a cDNA for a novel human gene with RING finger motif (C(3)HC(4)), which is not deposited in public EST databases. The isolated cDNA clone is 2163 bp in length, and contains an open reading frame of 452 amino acids. We designated the novel gene as RNF18. A database search showed that the RNF18 gene had the moderate similarity to SS-A/Ro52 protein, which is a ribonucleoprotein reactive with autoantibodies in patients with Sjögren's syndrome and systemic lupus erythematosus. Tissue distribution analyses by Northern blot and RT-PCR methods demonstrated that the RNF18 messenger RNA was preferentially expressed in testis. The exon-intron boundaries of RNF18 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The isolated cDNA consists of eight exons that span about 11 kb of the genome DNA. The precise chromosomal location of the RNF18 gene was determined by PCR-based radiation hybrid mapping, and the gene was located to centromere region of chromosome 11 between markers NIB1900 and D11S1350. Taken together, the VTS approach should provide a novel cDNA cloning strategy for isolating unidentified genes, which are not found even in EST databases but are detectable computationally.
Yoshikawa
T
T
Biological Technology Laboratory, Helix Research Institute, Kisarazu, Chiba, Japan.
Seki
N
N
Azuma
T
T
Masuho
Y
Y
Muramatsu
M
M
Miyajima
N
N
Saito
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000819:agonists* / Q000737:chemistry; D002880:Chromosomes, Human, Pair 11; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005821:Genetic Techniques; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013737:Testis / Q000378:metabolism*
D002352:Carrier Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; C416947:TRIM49 protein, human