Molecular cloning and nucleotide sequence of a complete human uroporphyrinogen decarboxylase cDNA.

P H Roméo,N Raich,A Dubart,D Beaupain,M Pryor,J Kushner,M Cohen-Solal,M Goossens

doi:10.1016/s0021-9258(18)67589-1

Abstract

We have cloned and sequenced a full-length cDNA coding for human uroporphyrinogen decarboxylase. The deduced 367-amino acid sequence is consistent with the molecular weight, the partial amino acid sequence of cyanogen bromide peptides, and the total amino acid composition of the purified enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome, and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. We have also demonstrated that the level of uroporphyrinogen decarboxylase mRNA is markedly increased in tissues or cell lines of erythroid origin and that this is due to a tissue-specific transcriptional activation of the uroporphyrinogen decarboxylase gene.

Highlights

We have cloned and sequenced a full-length cDNA carboxymethyl side chains of uroporphyrinogen to yield cocoding for human uroporphyrinogen decarboxylase
This paper reports the cloning and analysis of a cDNA sequence complementary to uroporphyrinogen decarboxylase mRNA obtained from human spleen
A recent nucleotide sequence of this cDNA, and we show that the predicted amino acid sequence of the enzyme agrees with the partial amino acid sequencethat we have determined by direct protein sequencing

Summary

MATERIALS ANDMETHODS

General Procedures-A previous communication [8] described the methods used for in vitro translation in a messenger-dependent rabbit reticulocyte cell-free system, uroporphyrinogen decarboxylase immunoprecipitation from in vitro translation products, and SDS-polyacrylamide gel electrophoresis. The cDNAs were excised from the recombinant plasmid, isolated by agarose gel electrophoresis, electroeluted, and nick-translated as described [14]. Messenger RNA obtained from the spleen was fractionated by preparative gel electrophoresis (la), and fractions containing mRNA enriched in uroporphyrinogen decarboxylase sequences were pooled and ethanol-precipitated. Double-stranded cDNAs longer than 800 bp were purified by polyacrylamide gel electrophoresis followed by electroelution They were inserted in the PstI site of pBR322 using the homopolymeric tailing and hybridization method [21]. To measure the relative rate of transcription, the following cloned DNAs were immobilized on Genescreen Plus (New England Nuclear): i) pUD3 cDNA which represents the DNA complementary to uroporphyrinogen decarboxylase mRNA (this cDNA recognizes a unique geneby Southern hybridization analysis), ii) pBR322, and iii) plasmid-containing sequences coding for 28 S ribosomal RNA. Amino acid analysis of the entire protein was performed after hydrolysis of the sample in 6 N HCI for 24 h

RESULTS

DISCUSSION

Trp CYS "

Hf L