Abstract
A mouse liver genomic library was probed with a 450-base pair AccI----3' gene-specific fragment of a mouse bone marrow-derived mast cell proteoglycan cDNA to isolate 15-18-kilobase (kb) genomic clones containing the gene that encodes the peptide core of mouse secretory granule proteoglycans. Based on the nucleotide sequences of its 2.0-3.5-kb subcloned fragments, this mouse gene consists of three exons. The first exon contains 41 base pairs of untranslated nucleotides that are present in the 5' region of the transcript and also encodes the hydrophobic 25-amino acid signal peptide. The second exon encodes a 48-amino acid sequence that would be predicted to be the N terminus of the peptide core after the signal peptide has been removed in the endoplasmic reticulum. The third exon encodes a 79-amino acid sequence that includes the 15 amino acids immediately preceding an alternating serine-glycine 21-amino acid sequence for glycosaminoglycan attachment, and the subsequent C-terminal 43 amino acids; this exon also contains the 424 untranslated nucleotides present in the 3' region of the transcript. Primer extension and S1 nuclease protection analyses were performed to determine the transcription initiation site of the mouse gene. Rat-1 fibroblasts were cotransfected with the selectable marker pSV2 neo and a lambda clone (lambda MG-PG1) to obtain two rat-1 fibroblast cell lines that had the mouse secretory granule proteoglycan gene integrated into their genomes. RNA blot analysis of both cell lines revealed the presence of the 1.0-kb secretory granule proteoglycan peptide core mRNA transcript, indicating that lambda MG-PG1 contained the entire mouse secretory granule proteoglycan peptide core gene including some of the regulatory elements in its promoter region. The gene that encodes the peptide core of mouse secretory granule proteoglycans is the first proteoglycan gene to have its complete exon/intron organization determined and to be transfected and expressed in another cell type.
Highlights
Cloning and Characterizationof the Mouse Gene That Encodes the Peptide Core of Secretory Granule Proteoglycans and Expression of This Gene in Transfected Rat-1 Fibroblasts*
The second exon encodes a 48-amino acid sequence that would bepredicted to be the N terminus of the peptide core after the signal peptide has been sulfated proteoglycans are stored inthe secretory granules of mast cells [6,7],ionically bound to basically charged carboxypeptidase A [8]and serine proteases ( 5, 9) that are enzymatically active at neutral pH, the proteaseresistance of the peptide core may be important for maintaining the configuration of the proteoglycan-protease complex and preventing removed in the endoplasmicreticulum
When bone marrow-derived mast cells (BMMC) are activated by encodes a 79-amino acid sequence that includes the 15 calcium ionophore or by perturbation of their IgE Fc recepamino acids immediately preceding analternating tors, the proteoglycans and proteases are exocytosed from the serine-glycine 2 1-amino acid sequence for glycosami- secretory granules as a macromolecular complex [5, 8]
Summary
RNA blot analysis [10] revealed that various rodent mast cells that synthesize secretory granule proteoglycans bearing different types of glycosaminoglycans express the gene that encodes the peptide core of a rat L2 yolk sac tumor chondroitin sulfate A proteoglycan [11, 12]. A full length cDNA was isolated from a rat basophilic leukemia cell cDNA library and sequenced [13] to demonstrate that the same gene was used by this transformed mucosal-like mast cell [14] to encode the peptide cores of secretory granule chondroitin sulfate diB/ heparin hybrid proteoglycans [15, 16]. The abbreviations used are: BMMC, bone marrow-derived mast cell; bp, base pair(s); kb, kilobase(s); Pipes, 1,4-piperazinediethanesulfonic acid Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
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