Cloning and characterization of the mouse gene that encodes the peptide core of secretory granule proteoglycans and expression of this gene in transfected rat-1 fibroblasts.
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.
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
S
S
Avraham
K F
KF
Austen
C F
CF
Nicodemus
M C
MC
Gartner
R L
RL
Stevens
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005796:Genes; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011509:Proteoglycans / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D011509:Proteoglycans; D012333:RNA, Messenger