Human gene for the RNA polymerase II seventh subunit (hsRPB7): structure, expression and chromosomal localization

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Abstract

The human gene for the seventh largest subunit of RNA polymerase II complex, hsRPB7 was cloned, sequenced and mapped. This complex is an integral part of the transcription-coupled DNA repair mechanism and has been shown to be involved in several human genetic diseases and implicated in many others. The hsRPB7 gene consists of 8 exons and spans approximately 5.1 kb. Southern blots of genomic and cloned DNA suggest that hsRPB7 is coded for by a single gene. Using human radiation hybrids and YACs, the gene was localized to 11q13.1, within 70 kb of marker D11S1765. The sequence of the 5' flanking region does not contain a TATA element, but does contain several Sp1 binding sites, an AP-1 site and a novel inverted polymorphic GATA tandem repeat. This novel GATA repeat can be used for linkage analysis. The hsRPB7 gene seems to be highly conserved among eukaryotic species, showing general sequence conservation to yeast and Drosophila. Northern blot analysis reveals a high degree of tissue-specific expression. For example, adult retina, brain and kidney exhibit a relatively high level of expression. A moderate level of expression is observed in heart, lung, testis, cornea, retinal pigmented epithelium/choroid and placenta with a lower level of expression in the uterus, small intestine and skeletal muscle. A very low level of expression was observed in stomach and liver. Comparison between four fetal and adult tissues also demonstrate a surprising level of developmental specificity. Expression in fetal retina is considerably lower than fetal brain but similar to adult retina.

Introduction

RNA polymerase II (Pol II) is the enzymatic complex responsible for transcription of genes that result in messenger RNA (mRNA) production. RNA polymerase II is composed of 10–14 subunits ranging in size from 220 to 10 kDa [1]. This complex interacts with the promoter regions of genes as well as with a variety of elements and transcription factors to determine essentially all of the parameters that govern transcription (e.g., tissue and developmental specificity, stress response, etc., see review [2]). Presently, human cDNAs have been isolated and chromosomal localization performed on a total of seven of the Pol II complex genes 3, 4, 5. However, to date, the complete genomic sequence has been reported for only the 220 kDa [4]and 14.5 kDa [5]subunits.

During the process of screening a macula-enriched subtraction library [6], we isolated a cDNA clone that was determined, during a BLAST [7]database search, to be hsRPB7 [8]. Although hsRPB7 message expression is only two-fold enriched between macula and peripheral retina, its chromosomal localization to 11q13.1 made this gene a possible candidate for several genetic diseases located in this area. Due to the overall importance of the Pol II complex and the possible involvement of this gene in genetic disease, we have characterized and localized the human gene for hsRPB7 as well as determined its relative level of expression in a variety of human tissues.

Section snippets

5′ Rapid amplification of cDNA ends (5′ RACE)

The 5′ RACE was performed using a previously published technique [9]. Two hsRPB7 specific primers were used: 5253 (5′TGTGCCACTTCGTCTTCGAGA3′) and 5440 (5′TACAGAACGAAGTAGAGAGCTG3′).

Screening of a human genomic library

A human genomic library constructed in PWE-15 (Clontech, Palo Alto, CA) was screened using a 32P-labelled PCR product from the hsRPB7 cDNA. The PCR product was labeled with [32P]dCTP to a specific activity of approximately 5×109 cpm/μg using a PCR-labeling technique [10]. Approximately 300 000 clones were screened

Isolation of hsRPB7 cDNA and 5′ RACE

The hsRPB7 cDNA was isolated during a solid-phase subtraction between the macula and peripheral areas of the retina as previously described [6]. Northern blot analysis of total RNA from monkey macula and peripheral retina demonstrates that hsRPB7 is enriched about 2-fold in the macula versus peripheral retina (Fig. 1). The original isolate was identified by performing a GenBank search using the National Center for Biological Information BLAST server program [7]. The search identified this clone

Discussion

Our interest in cloning the hsRPB7 gene was 3-fold: its chromosomal localization, its high and differential expression in the macula region of the retina and its fundamental importance in RNA transcription. Based on our radiation hybrid and YAC analysis, the hsRPB7 gene is located in chromosome 11 band q13.1, closely associated with the marker D11S1765. Attempts to associate this gene with other markers in the area using our YACs and PAC have been unsuccessful so far. However, we have localized

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