Immunization with undenatured bovine zonular fibrils results in monoclonal antibodies to fibrillin.

Microfibrils were dissected from the zonular apparatus of the bovine eye, homogenized and used as an immunogen to prepare monoclonal antibodies. Initial screening of hybridomas was performed by immunoblotting to a sonicate of zonular fibrils and by immunolocalization to frozen sections of the zonular apparatus. Subsequently, monoclonal antibodies with strong immunoreactivity to zonular fibrils were shown to recognize microfibrils in a wide range of connective tissues both by immunofluorescent staining and by electron microscopic immunolocalization. All antibodies were found to recognize a single protein of 350 kDa on Western blotting of the proteins secreted by bovine aortic smooth muscle cells. A protein of the same molecular weight and properties was recognized by an antibody previously prepared by another group against fibrillin. A member of the fibrillin family therefore represents the major immunogen of intact zonular fibrils, and the results support previous evidence for a close relationship between zonular fibrils and other connective tissue microfibrils. The zonular apparatus is a suitable system to obtain purified preparations of microfibrils in order to investigate their composition and structural organization.

Fibrillin and elastin networks in extrafusal tissue and muscle spindles of bovine extraocular muscles.

PURPOSE: Bovine extraocular rectus muscles were examined to map the distribution of elastin and fibrillin in extrafusal tissue and muscle spindles. METHODS: Immunohistochemical techniques and immunolocalization were employed to pin-point the placement of molecules relative to muscle fibers. RESULTS: Strands containing elastin and fibrillin surrounded all extrafusal fibers. They also covered the external surface of intrafusal fibers, more extensively at the equator than at the pole. Within strands elastin was placed in the center, whereas fibrillin was located in microfibrils on the periphery. CONCLUSIONS: The wide distribution in extrafusal tissue of elastin and fibrillin suggests that they are factors in determining the mechanical properties of extraocular muscles. Their placement in proximity to individual intrafusal fibers should affect the viscoelastic properties of these fibers and, thus, influence the dimensions of the afferent discharge.

The genes encoding alpha 2(IX) collagen (COL9A2) map to human chromosome 1p32.3-p33 and mouse chromosome 4.

We have determined the chromosomal locations of the human and murine genes coding for alpha 2(IX) collagen, a polypeptide subunit of the heterotrimeric type IX collagen molecule. COL9A2 was mapped to human chromosome 1p32.3-p33 using fluorescence in situ hybridization. A single-strand conformational polymorphism within the murine Col9a2 gene was used to map this locus to mouse chromosome 4. We also present new sequence data, which completes the coding information for the human alpha 2(IX) chain and revises the sequence for the chicken alpha 2(IX) chain. This permits comparison of the carboxyl-terminal (NC1) domains of the alpha 1(IX), alpha 2(IX), and alpha 3(IX) chains across several species.