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.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-6585-8 |
| Date | 20 November 2001 |
| Creators | Muona, A. (Anu) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2001 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
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