Type XV collagen

Complete structures of the human COL15A1 and mouse Col15a1 genes, location of type XV collagen protein in mature and developing mouse tissues, and generation of mice expressing truncated type XV collagen

Anu Muona

Collagen Research Unit, University of Oulu
Biocenter Oulu, University of Oulu
Department of Medical Biochemistry, University of Oulu

Abstract

This study was initiated to elucidate the complete genomic structures of type XV collagen in man and mouse and the functional properties of their promoters, as well as to obtain knowledge of the biological role of type XV collagen during development and maturity using immunofluorescence and transgenic techniques.

The cloning and characterization of genomic clones revealed that the human COL15A1 gene is 145-kb in size and consists of 42 exons, and the mouse Col15a1 gene is 110-kb with 40 exons. The genomic organization of the two genes was found to be highly conserved, except for two regions of divergence. The nuclease S1 protection analysis revealed multiple transcription initiation sites in both genes, which is in accordance with the overall genomic structures of their 5"-flanking sequences. Transient cell transfection experiments with varying lengths of 5"-deletion constructs identified the fragments necessary for basic promoter activity in both genes and those implicated in the positive and negative regulation of the mouse Col15a1 gene. Furthermore, the involvement of transcription factor Sp1 in the gene regulation of the human COL15A1 gene was demonstrated. A mouse specific polyclonal antibody against type XV collagen was generated and utilized in the localization of type XV collagen protein in developing and mature mouse tissues. Type XV collagen was deposited early in the development and was particularly prominent in capillaries. Spatio-temporal differences in the expression of type XV collagen in various capillary types was demonstrated. Early expression was also detected in the skeletal muscle and peripheral nerves, while expression in the heart, lung, and kidney appeared to be developmentally regulated. Transgenic mice lines expressing truncated type XV collagen driven by either short or long endogenous type XV collagen promoters were generated. The two promoters conferred different tissue-specificities and expression levels, the longer one resulting in more endogenous-like expression. Despite some expression at both mRNA and protein levels, the truncated type XV collagen did not cause any obvious phenotypic or histological changes in any of the lines driven by the shorter promoter fragment. In heterozygote matings of one of the lines driven by the longer promoter fragment, however, a portion of the transgene positive mice appeared to be lost prenatally. Furthermore, pregnancy terminations in this line indicated a high number of abortions beginning at about 11 days of development. Further studies are needed before detailed conclusions on the consequences of the generated mutation can be drawn.

The elucidation of the genomic structure of the human COL15A1 gene provides the necessary database for screening mutations in patient samples for candidate diseases caused by this collagen. The genomic clones and the mouse-specific antibody against type XV collagen are valuable tools also in future projects. The knowledge of the developmental dynamics of type XV collagen is of great value, as it helps to understand the physiological consequences that the as yet unidentified mutations in type XV collagen may cause in humans.

Table of Contents
Acknowledgements
Abbreviations
List of original articles
1. Introduction
2. Review of the literature
2.1. Fibril-forming collagens
2.2. Non-fibril forming collagens
2.3. Family of type XV and XVIII collagens
2.3.1. Common structural and biochemical properties of type XV and XVIII collagens
2.3.2. Common and distinct features in the expression and tissue distribution of type XV and XVIII collagens
2.3.3. Current knowledge about the functions of type XV and XVIII collagens
2.4. Collagen genes
2.5. Mutations in human collagen genes
2.6. Collagen mutations in animals
2.6.1. Spontaneous mutations
2.6.2. Genetically engineered mutations in mouse
2.7. Collagens in development
2.8. Basement membranes – structure and function
2.8.1. Basement membrane components
2.8.2. Developmental regulation of basement membrane components
3. Outlines of the present research
4. Materials and methods
4.1. Isolation and characterization of genomic clones of human and mouse genes (I, II)
4.2. Nucleotide sequencing and sequence analysis (I, II, IV)
4.3. Nuclease S1 analysis (I, II)
4.4. Northern blot analysis (I)
4.5. Deletion constructs for promoter analysis of human and mouse genes (I, II)
4.6. Cell culture, transfections, and luciferase assays (I, II)
4.7. Expression of polypeptide fragments in E. coli for antigen production (III)
4.8. Preparation and affinity purification of antibodies (III, IV)
4.9. SDS-PAGE and Western blot analysis of cell and tissue samples (III, IV)
4.10. Tissue preparation for routine histology and immunofluorescence studies (III, IV)
4.11. Immunofluorescence staining of tissues (III, IV)
4.12. Construction of a minigene (IV)
4.13. Generation of transgenic mice (IV)
4.14. RT-PCR analysis of transgene expression (IV)
4.15. Fetal fibroblast cultures (IV)
5. Results
5.1. Exon-intron organization of the human COL15A1 gene (I)
5.2. Exon-intron organization of the mouse Col15a1 gene (II)
5.3. Identification of the transcription initiation sites for human and mouse α1(XV) collagen genes and sequence comparison of the 5´-flanking regions (I, II)
5.4. Deletion analysis of the human COL15A1 and mouse Col15a1 promoters (I, II)
5.5. Comparison of the human COL15A1 and mouse Col15a1 genes (I, II)
5.6. Preparation and characterization of a polyclonal antibody to mouse type XV collagen (III)
5.7. Localization of type XV collagen in developing and mature mouse tissues (III)
5.8. Generation of transgenic mice with a truncated type XV collagen minigene in normal background and in Col15a1-/- background (IV)
5.9. mRNA expression of the transgene in tissues and cultured fibroblasts (IV)
5.10. Morphological analysis of transgenic mice (IV)
5.11. Western blot analysis of transgene protein in tissue homogenates (IV)
6. Discussion
6.1. Comparison of the genomic organizations of the human and mouse type XV collagen genes (I, II)
6.2. Comparison of the type XV and XVIII collagen genes (I, II)
6.3. Tissue distribution and developmentally regulated expression of type XV collagen protein in mouse (III)
6.4. Transgenic mice expressing truncated type XV collagen (IV)
7. Future perspectives
References
List of Tables
1. Collagen types, their constituent chains, chain compositions, and tissue distributions.
2. Collagen genes and their chromosomal locations*.
3. Diseases caused by mutations in collagen genes or collagen processing enzymes*.
4. Animal models with spontaneous mutations for collagen diseases*.
5. Genetically engineered mutations in collagen genes in mouse.
6. Collagens in developing mouse tissues §.
7. Type XV collagen in developing and mature BM zones and elsewhere in mouse #.
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