GENOMICS 46, 234–239 (1997) ARTICLE NO. GE975038 Cloning, Sequencing, Gene Organization, and Localization of the Human Ribosomal Protein RPL23A Gene Wufang Fan,* ,1 Mari Christensen,* Evan Eichler,* Xiaoxiao Zhang,† and Greg Lennon * * The Human Genome Center, L-452, Lawrence Livermore National Laboratory, Livermore, California 94551; and †Laboratory of Medical Genetics, The Panum Institute, 2200 Copenhagen, Denmark Received May 5, 1997; accepted September 18, 1997 and Perry, 1985). Because many pseudogene copies ex- The intron-containing gene for human ribosomal ist, it is relatively difficult to localize a functional rp protein RPL23A has been cloned, sequenced, and local- gene in a complex genome. In mammals including hu- ized. The gene is approximately 4.0 kb in length and mans, only a handful of intron-containing rp genes contains five exons and four introns. All splice sites have been cloned and mapped until recently (Wiede- exactly match the AG/GT consensus rule. The tran- mann et al., 1987; Antoine and Fried, 1992; Foe et al., script is about 0.6 kb and is detected in all tissues ex- 1992; Davies and Fried, 1993, 1995; Gallagher et al., amined. In adult tissues, the RPL23A transcript is dra- 1994; Annilo et al., 1995; Nolte et al., 1996; Mazuruk matically more abundant in pancreas, skeletal muscle, et al., 1996). and heart, while much less abundant in kidney, brain, The rp genes not only play a vital role in protein placenta, lung, and liver. A full-length cDNA clone of synthesis, but their mutations may also be implicated 576 nt was identified, and the nucleotide sequence was in some diseases. It has been speculated for a long time found to match the exon sequence precisely. The open that haploinsufficiency of rp genes may play an im- reading frame encodes a polypeptide of 156 amino portant role by underlying the Turner phenotype in acids, which is absolutely conserved with the rat humans, which is predominantly a consequence of X- RPL23A protein. In the 5 flanking region of the gene, chromosome monosomy (Fisher et al., 1990). In Dro- a canonical TATA sequence and a defined CAAT box sophila melanogaster, it has been demonstrated that were found for the first time in a mammalian ribo- the rp gene L9 is located in Minute locus M(2)32D, and somal protein gene. The intron-containing RPL23A transferring a functional L9 gene into mutant flies can gene was mapped to cytogenetic band 17q11 by fluo- rescue the Minute phenotype completely (Schmidt et rescence in situ hybridization.  1997 Academic Press al., 1996). Furthermore, analysis of the 5 end of the human RPL7A gene has revealed that it is able to acti- vate the trk proto-oncogene receptor kinase domain INTRODUCTION (Kozma et al., 1988). Therefore, some rp genes may be related to oncogenesis through regulatory pathways. The ribosome is a cellular organelle responsible for In this report, we present the data about cloning, synthesis of proteins in all cells. Its structural organi- sequencing, gene organization, and chromosomal local- zation consists of about 80 ribosomal proteins (rp) and ization of the intron-containing human RPL23A gene. four RNA species (18S RNA in the 40 S r-subunit and The potential promoter elements in the 5 flanking re- 28S, 5S, and 5.8S RNA in the 60S r-subunit), which gion are also shown and discussed. reflects a complex coordinating mechanism that has not yet been elucidated fully. Isolating all rp cDNAs and chromosomally mapping their intron-containing MATERIALS AND METHODS genes in higher eukaryotes are, therefore, the funda- mental steps to elucidate the mechanism further. BAC and cosmid clones. BAC clone 94912 was isolated from a Ribosomal genes, in general, seem to be present in total human BAC library (Research Genetics, Huntsville, AL) repre- families composed of multiple intronless pseudogenes senting a 2.61 coverage of the haploid human genome. Cosmid clone 103C6 was identified from a chromosome 17-specific genomic library and only one intron-containing functional gene (Ma- (17NC01, Los Alamos National Laboratory, Los Alamos, NM). A ra- zuruk et al., 1996). The pseudogene often lacks a func- dioactively labeled human RPL23A full-length cDNA probe was used tional promoter and is usually located around multiple for screening the above genomic libraries. repetitive elements (Dudov and Perry, 1984; Wagner Northern analysis. Northern analysis was performed by standard methods. The RNA blot was purchased from Clontech (Palo Alto, CA). The probe was labeled with [ 32 P]dCTP by Taq polymerase extension 1 To whom correspondence should be addressed. Telephone: (510) 422-5712. Fax: (510) 422-2282. (Medori et al., 1992). 234 0888-7543/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.