Abstract
The structure and expression of rat sterol carrier protein 2 (SCP2) were studied by cloning and sequencing SCP2 cDNAs and by hybridizing the SCP2 cDNA to RNA from a variety of rat tissues. Initial screening of a rat liver cDNA library with an oligonucleotide probe derived from the rat SCP2 protein sequence revealed an 825-base pair cDNA clone coding for the complete SCP2 protein sequence. Our results suggest that rat liver SCP2 is 143 amino acid residues long, including a 20-amino acid residue prepeptide (pre-SCP2). Using the rat SCP2 cDNA as a probe for Northern blot hybridization analyses under high stringency conditions with rat liver poly(A) RNA, four mRNA species differing in their sizes and levels of expression are detected. The lengths of the corresponding mRNAs are 0.8, 1.4, 2.1, and 2.7 kilobases (kb), respectively. In all other tissues analyzed mRNAs hybridizing to the SCP2 cDNA probe were detected in levels much lower than in the liver. In these tissues the vast majority of the expressed SCP2 mRNAs consists of the 0.8-kb mRNA. Results from Southern blotting hybridization analyses with rat genomic DNA suggest that the four different mRNA species are products of a single gene. Extensive screening of rat liver cDNA libraries using rat liver SCP2 cDNA probes led to the isolation of 10 additional clones, all of them containing cDNA insertions longer than 0.8 kb. Restriction mapping and nucleotide sequencing analyses of these cDNAs indicate that eight extend upstream of the SCP2 cDNA, and two extend 5' as well as 3'. Translation of the sequence extending most upstream suggests that this cDNA is a nearly full-length cDNA coding for a protein of 547 amino acids (SCP chi). SCP chi contains a 404-amino acid amino-terminal extension as compared with pre-SCP2 whereas its carboxyl-terminal part is identical with pre-SCP2. Sequencing of the 3' extension cDNAs suggests that polyadenylation had occurred 651 nucleotides downstream as compared with the remaining cDNAs. A probe from this region hybridizes to the 1.4- and 2.7-kb mRNAs. From our data we conclude that the 1.4- and 2.7-kb mRNA species are generated by alternative polyadenylation of the 0.8- and 2.1-kb mRNAs, respectively. In contrast, the 2.1- and 2.7-kb mRNAs contain an approximately 1300-nucleotide extension upstream of the 0.8- and 1.4-kb mRNAs, suggesting differential splicing of the primary SCP2 gene transcript leading to expression of pre-SCP2 and SCP chi from a single gene.
Highlights
The structure and expression of rat sterol carrier differential splicing of the primary SCPz gene tranprotein 2 (SCPz) were studiebdy cloningand sequenc- script leading to expressoiof npre-SCPz and SCP, from ing SCP2cDNAs and by hybridizing theSCP2cDNA to a single gene
Since the carboxylterminal leucine residueis missing from thesequence obtained by conventional protein sequencing [21], it may be possible that this amino acid residue is removed from rat liver SCP, spleen,and 10 pg of DNA was digested with various restriction post-translationally.Ontheotherhand,dataobtained by enzymes (Boehringer Mannheim andNew England Biolabs), electro- mass spectrometry support theconclusion that thecarboxylphoresed in 1%agarose gels, transferred ontonylon membranes [32], and hybridized with "P-labeled probes
IsolationandCharacterization of the Rat SCP, cDNAInitial screeningof approximately 5 X IO5clones of a rat liver cDNA library witha radiolabeled oligonucleotidecorresponding to the amino acid sequence extending from positions 84 t o 91 to the ratSCP, protein sequence [21,22] resulted in the FIG. 1
Summary
From the ZZnstitut fur Arterioskleroseforschung an derUniuersitat Munster, Munster 0-4400, Federal Republic of Germany and the iilnstitut f u r Klinisch Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfalisch Wilhelms-Uniuersitat, Munster 0-4400, Federal Republic of Germany. DNA and RNA Hybridization Blotting Analyses-Total RNA was isolated from various rat tissues[29], electrophoresed in 1% agaroseformaldehyde gels [30], and transferred ontonylon membranes (Biotended product of 184 nucleotides is obtained (see Fig. 4C) From these datwae conclude that ratliver SCP, is synthesized with a 20-amino acid prepeptide that is removedfrom the protein co- or post-translationally. ChromosomalDNA was prepared [31] fromrat quence of rat SCP, is Lys-Ala-Lys-Leu. Since the carboxylterminal leucine residueis missing from thesequence obtained by conventional protein sequencing [21], it may be possible that this amino acid residue is removed from rat liver SCP, spleen,and 10 pg of DNA was digested with various restriction post-translationally.Ontheotherhand,dataobtained by enzymes (Boehringer Mannheim andNew England Biolabs), electro- mass spectrometry support theconclusion that thecarboxylphoresed in 1%agarose gels, transferred ontonylon membranes [32], and hybridized with "P-labeled probes.
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