REGULATION OF FIBRIL-ASSOCIATED COLLAGENS IN CORNEA

Project Details

Description

Corneal transparency depends on the small uniform diameter and the
regular spacing of collagen fibrils. The "fibrillar" collagens are
involved in determining fibril diameter; the "fibril-associated"
collagens, a new class, may be responsible for interfibrillar
relationships, such as spacings. We have isolated cDNAs for two putative
"fibril associated" collagens expressed in cornea. One is for type XIV
collagen, a known member of a fibril-associated family; the other
potentially encodes a cornea-specific molecule with multiple, small
collagenous domains. The genes for these collagens will be characterized
and their exon/intron structures defined. Cis-acting elements (DNA
sequences) that are important for transcriptional regulation of the genes
will be identified. The region 5' to the transcriptional start site
(containing putative promoters/enhancers/silencers) will be examined, as
well as the first intron, which may .also be involved in transcriptional
regulation. These regions will be ligated to a reporter gene and the
constructs transfected into skin and corneal fibroblasts. To precisely
identify the regulatory sequences within these regions and to determine
whether the sequences function as promoters, enhancers, or silencers,
systematic nested deletions of portions of the regulatory regions of
these constructs will be made and evaluated by transfection. The
presence of potential cis-elements unique to cornea will be examined by
in vivo footprinting, a method which allows identification and sequencing
of regions of genes to which DNA-binding proteins (trans-acting factors)
are bound. The technique employs the polymerase chain reaction for
amplification, and thus can be performed on the amounts of tissues
available from embryos. Once obtained, the DNA sequences of these
cis-acting regions will be utilized to screen a corneal cDNA expression
library to isolate cDNAs expressing DNA binding proteins (trans-acting
factors). If trans-acting factors unique to cornea are found they will
be tested for functionality in co-transfection experiments. For this,
non-corneal cells, such as skin fibroblasts will be simultaneously
co-transfected with a construct encoding the transacting factor driven by
a constitutive promoter, and a construct of the promoter/enhancer regions
of the cornea-specific collagen gene promoter linked to a reporter gene.
If transcription of the reporter gene is observed, it will indicate that
a trans=acting factor unique to cornea is able to effect directly the
regulation of a cornea-specific gene in a non-corneal cell.
StatusFinished
Effective start/end date5/1/9111/30/12

Funding

  • National Institutes of Health: $269,373.00
  • National Institutes of Health: $203,953.00
  • National Institutes of Health: $378,483.00
  • National Institutes of Health: $170,383.00
  • National Institutes of Health: $447,590.00
  • National Institutes of Health: $443,425.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $339,608.00
  • National Institutes of Health: $438,584.00
  • National Institutes of Health: $469,538.00
  • National Institutes of Health: $339,608.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $233,235.00
  • National Institutes of Health: $339,608.00
  • National Institutes of Health: $77,750.00
  • National Institutes of Health: $477,898.00
  • National Institutes of Health
  • National Institutes of Health: $238,896.00
  • National Institutes of Health: $180,974.00

ASJC

  • Medicine(all)

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