The purpose of this project is to characterize mammalian and avian TGF-Betas in terms of the chemistry, biology, and molecular biology of each of the component members and to understand their mechanisms of expression. The emphasis has been on cloning TGF-Beta cDNA homologs in rodent and avian species and using these cDNAs to generate riboprobes for use in in situ hybridization studies, and on characterizing previously cloned avian TGF-Beta cDNAs and their corresponding mRNAs. Rat TGF-BL CDNA and murine TGF-Bs 2 and 3 cDNAs have been cloned by polymerase chain reaction (PCR) amplification of reverse-transcribed mRNAs extracted from adult rat liver and 15-day-old mouse embryos, respectively. The C-terminal mature 112 amino acids of rat TGF-Beta1 and murine TGF-Betas 2 and 3 both show 98% identity with human TGF-Betas 1, 2 and 3, respectively. A 1.9 Kb mRNA species has also been identified in infarcted rat heart which shows distinct hybridization to TGF-Beta cDNA. The 1.9 Kb MRNA has been used to clone a 630 bp cDNA fragment using PCR amplification; the sequence of this fragment has been found to be identical to the mature coding region plus a portion of the latency associated peptide (LAP) region of the 2.4 Kb rat TGF-BI mRNA. In addition, the chicken TGF-B cDNA family has been extended to include TGF-Beta2 cDNA in addition to TGF-Bs 1, 3 and 4 cDNAs. Chicken TGF-B2 cDNA has been cloned from the same chicken embryo chondrocyte cDNA library that was used to clone chicken TGF-Betas 1, 3 and 4 cDNAs. The C-terminal 112 amino acids of chicken TGF-Beta2 show 98% identity with human TGF-Beta2. RNA Northern blot analysis using TGF-B cDNAs and immunohistochemical staining using TGF-B antibodies suggest that chicken TGF-Betas 2, 3 and 4 mRNAs and TGF-Bs 1, 2, 3 and 4 proteins are co-expressed in chicken cartilage and heart. In situ hybridization is being used to identify cell-specific localization of the chicken TGF-Beta mRNAs.
