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A novel cDNA encoding alpha subunit of the GTP-binding protein, transducin, has been cloned from a marine fish, Sparus aurata. The cDNA contains an open reading frame of 1050 nt (encoding 350 amino acid residues). A high degree of identity was found with known mammalian transducin proteins of cones (Gt2 alpha) or rods (Gt1 alpha): human Gt2 alpha (80.2%), bovine Gt2 alpha (79.3%), mouse Tt1 alpha (78.2%), mouse Gt2 alpha (78%) and bovine Gt1 alpha (77.9%). Northern blot analysis of different tissues revealed a transcript of about 2.5 kb, which is expressed only in the fish eye and not in other tissues from adult fish, supporting its identification as transducin. Ontogeny of transducin mRNA expression during early development of Sparus aurata, determined by Northern blot analysis, showed very low levels in larvae 3 days after hatching but not earlier. Levels increased 3- and 6-fold on days 4 and 6 (respectively) compared with those on day 3 and remained essentially unchanged thereafter, until day 21 after hatching (the last day studied). Our results suggest that in fish only one alpha subunit of transducin is found, which shows similar identity with cone and rod alpha subunits of mammals.

A cDNA encoding a putative alcohol dehydrogenase class III (ADH) was cloned from a cDNA library constructed from 7-day larvae RNA of the marine teleost Sparus aurata. The full length cDNA is 1350 nucleotides (nt) long and contains an ORF of 1128 nt [encoding 376 amino acid (aa) residues]. Identity of 82% was found with human class III ADH (305 of 372 aa compared), and only 62% identity with a fish (cod) ADH (234 of 375 aa compared). Northern hybridization analysis with the cDNA revealed a transcript of about 1.4-1.5 kb, which is expressed in all tissues from adult fish studied: skeletal muscle, heart muscle, kidney, gill filaments and liver, with the highest levels found in the kidney. The expression of ADH mRNA was determined also during early development of Sparus aurata by Northern blot analysis. ADH transcripts were detected in eggs and in embryos 4, 8 and 12 h after fertilization, as well as on all days post-hatching studied. The levels of expression decreased during early embryonal development, but increased 4-fold from day 1 to day 21 after hatching. The size of the transcript was identical to that of hepatic ADH. Our results suggest that maternal ADH mRNA is present in the eggs and embryos, which decreases as divisions and development occur, while after hatching ADH mRNA is expressed by the larval tissues.

Growth plates from chicks displaying hypocalcaemic rickets, hypophosphataemic rickets and dyschondroplasia were studied. Immunohistochemical staining using specific TGF-beta 3 antibodies showed disruption of TGF-beta 3 localisation in all three disorders. In hypocalcaemic rickets, transitional and hypertrophic chondrocytes showed expression of TGF-beta 3, while accumulating, proliferative chondrocytes showed little expression. In the accumulating hypertrophic chondrocytes of hypophosphataemic rickets there was a reduction in the number of chondrocytes expressing TGF-beta 3, but transitional cells stained intensely. In the accumulating transitional chondrocytes of dyschondroplastic physes there was a reduction in the number of chondrocytes containing TGF-beta 3 and, in addition, the concentration of TGF-beta 3 appeared reduced. In all three disorders, there was localisation of some TGF-beta 3 in chondrocytes that appeared to be differentiating within the areas of repair. In addition, little TGF-beta 3 was detected in osteoclasts resorbing accumulated matrix from hypocalcaemic, hypophosphataemic and dyschondroplastic growth plates. These in vivo studies show a reduction in TGF-beta 3 localisation within growth plate chondrocytes and osteoclasts in three disorders in which chondrocytes cease to differentiate. This suggests that TGF-beta 3 may be important during chondrocyte differentiation in the growth plate.

The localization of TGF- β1, - β2 and - β3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF- β isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF- β antibodies. TGF- βs in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-β3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF- β isoforms supports the hypothesis that TGF- β has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF- β may be a marker of chondrocyte differentiation. TGF- β localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF- β in the induction of synthesis of extracellular matrix.