Chick cartilage chondroitin sulfate proteoglycan core protein. II. Nucleotide sequence of cDNA clone and localization of the S103L epitope.

A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.     

Poly(A) synthesis by RNA polymerase II from rat liver

Multiple forms of DNA-dependent RNA polymerase were resolved by DEAE-Sephadex chromatography. In addition to RNA polymerases, an active poly(A) polymerase was also fractionated. RNA polymerases were examined for their capacity to synthesize poly(A). None of the freshly prepared enzymes could efficiently make poly(A) in presence or absence of exogenous primers. However, “aging” of polymerase II by simple incubation at 37°C resulted in the loss of RNA polymerizing activity with a corresponding increase in poly(A) synthesizing activity. Transformation of RNA polymerase to poly(A) polymerase resulted in reduced capacity to transcribe native DNA and altered chromatographic behavior. The results suggest that subunits of polymerase II obligatory to DNA-dependent RNA synthesis were degraded by “aging” and that a stable subunit of the RNA polymerase could preferentially make poly(A).