Abstract

The primary goals of the work proposed here are: 1) to examine the structure of the corneal endothelial short-chain collagen (CESCC) which is synthesized by rabbit corneal endothelial cells, and 2) to establish the structural and evolutionary relationship between the gene(s) encoding this collagen and genes that code for other short-chain collagens (Types IX, X, XII); 3) to examine the assembly and secretion of the CESCC molecule in the highly organized extracellular matrices of the cornea; 4) to examine the expression of the CESCC in the cornea during embryonic development; and 5) to analyze the modulated expression of the CESCC molecule in an in vitro model of posterior collagenous layer (retrocorneal fibrous membrane). To accomplish these goals, we will use a combination of DNA cloning/sequencing and protein chemistry. cDNA coding for the CESCC molecule will be constructed from rabbit corneal endothelial cell RNA. Primary structure of this molecule will be determined by nucleotide sequence analysis of isolated clones. The gene(s) coding of the CESCC molecule will be isolated from rabbit genomic libraries. Synthetic peptides will be made from the nucleotide sequence of rabbit cDNA. Specific antibodies will be generated against these peptides. Such antibodies, together with specific DNA probes, will be used to examine the synthesis of the CESCC molecule and the expression of its gene(s) in corneal development as well as in cultured endothelial cells. The study should provide information about the structure and functional role of a novel short-chain collagen of the cornea and the mechanisms regulating its synthesis. Such information should prove invaluable for understanding the assembly of the normal extracellular matrices of the cornea and alterations due to disease.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY007334-02
Application #
3264215
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1987-09-30
Project End
1990-09-29
Budget Start
1988-09-30
Budget End
1989-09-29
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115

  Publications

Muragaki, Y; Timmons, S; Griffith, C M et al. (1995) Mouse Col18a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones. Proc Natl Acad Sci U S A 92:8763-7
Ninomiya, Y; Kagawa, M; Iyama, K et al. (1995) Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. J Cell Biol 130:1219-29
Oohashi, T; Ueki, Y; Sugimoto, M et al. (1995) Isolation and structure of the COL4A6 gene encoding the human alpha 6(IV) collagen chain and comparison with other type IV collagen genes. J Biol Chem 270:26863-7
Sugimoto, M; Oohashi, T; Ninomiya, Y (1994) The genes COL4A5 and COL4A6, coding for basement membrane collagen chains alpha 5(IV) and alpha 6(IV), are located head-to-head in close proximity on human chromosome Xq22 and COL4A6 is transcribed from two alternative promoters. Proc Natl Acad Sci U S A 91:11679-83
Zieske, J D; Mason, V S; Wasson, M E et al. (1994) Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction. Exp Cell Res 214:621-33
Muragaki, Y; Abe, N; Ninomiya, Y et al. (1994) The human alpha 1(XV) collagen chain contains a large amino-terminal non-triple helical domain with a tandem repeat structure and homology to alpha 1(XVIII) collagen. J Biol Chem 269:4042-6
Oohashi, T; Sugimoto, M; Mattei, M G et al. (1994) Identification of a new collagen IV chain, alpha 6(IV), by cDNA isolation and assignment of the gene to chromosome Xq22, which is the same locus for COL4A5. J Biol Chem 269:7520-6
Rosenblum, N D; Briscoe, D M; Karnovsky, M J et al. (1993) Alpha 1-VIII collagen is expressed in the rat glomerulus and in resident glomerular cells. Am J Physiol 264:F1003-10
Liu, C Y; Olsen, B R; Kao, W W (1993) Developmental patterns of two alpha 1(IX) collagen mRNA isoforms in mouse. Dev Dyn 198:150-7
Sugimoto, M; Oohashi, T; Yoshioka, H et al. (1993) cDNA isolation and partial gene structure of the human alpha 4(IV) collagen chain. FEBS Lett 330:122-8

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