斑馬魚水晶體蛋白crystallin-βB1之選殖及表現之研究與先天性白內障生成之探討
Date Issued
2002
Date
2002
Author(s)
張百恩
DOI
902320B002146
Abstract
The aim of the project was focused on
the cloning of zebrafish crystallin-βB1 and
the study of its implication in the congenital
cataractogenesis. A fragment of zebrafish
crystallin-βB1 cDNA was cloned by RT-PCR
using degenerate primers. The 5’ and 3’
cDNA were obtained by 5’RACE and
3’RACE methods (Rapid Amplification of
cDNA Ends). Compilation of the three
fragments resulted in the full-length cDNA
sequence. The zebrafish crystallin-βB1
sequence shows high homology in amino
acid sequence when compared with those of
other vertebrates with some varied residues
in the N-terminal region. The spatialtemporal
expression of the gene in the
developing embryo was revealed by whole
mount in situ hybridization. The results
shows that this gene is initially expressed in
the developing lens at about 20 hr stage. The
transcripts of the gene are abundant in the
embryos at 48 hr stage and a single kind of
transcripts is expressed as revealed by
Northern blot analysis. On the other hand, the
genomic DNA fragment of the gene was
isolated by screening its cognate genomic
DNA library. After analyses by restriction
enzyme mapping and subcloning, the
structure of crystallin-βB1 gene was
unraveled. This gene is composed of 6 exons
and 5 introns. The upstream promoter region
was also cloned. Concerning the relation
between the cataractogenesis and the
mutation of the gene, mutated form of cDNA
was constructed by PCR. The mutant form of
the transcripts was micro-injected, however,
no evident cataract was formed in the
embryo.
Subjects
cataract
cDNA
crystallin
genome
lens
Publisher
臺北市：國立臺灣大學醫學院口腔生物科學研究所
Type
journal article
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