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Gene cloning using cDNA libraries in a differential competition hybridization strategy: application to cloning XP-A
A Rinaldy1, M L Dodson, T L Darling
1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville 37232.
Abstract:
A competition hybridization strategy using size-cut cDNA libraries as both probe and competitor was designed for the cloning of genes whose mRNAs are either regulated transcriptionally or vary in abundance as a function of cell line or cell cycle. We used this strategy to construct cDNA libraries from a particular size fraction of mRNA from three members of a xeroderma pigmentosum (XP), complementation group A family. Size-cut cDNA libraries derived from the father's, mother's, and child's fibroblast cell line were used in a competition scheme to screen two lambda gt11 human cDNA libraries. Of the 15 positive lambda gt11 clones which have been characterized, at least 14 clones represent the same gene which is present in greater abundance in both the mother and father XP obligate heterozygotes relative to the homozygous affected child.
Insights
This study developed a competition hybridization method to clone genes with varying mRNA levels. The technique identified a gene with higher abundance in xeroderma pigmentosum (XP) heterozygote parents compared to their affected child.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Gene expression regulation is crucial for cellular function.
- Understanding differential gene abundance aids in disease research.
- Xeroderma pigmentosum (XP) is a genetic disorder affecting DNA repair.
Purpose of the Study:
- To develop a novel competition hybridization strategy for cloning genes with differential mRNA expression.
- To identify genes with altered abundance in a xeroderma pigmentosum (XP) complementation group A family.
Main Methods:
- Utilized size-cut complementary DNA (cDNA) libraries as probes and competitors in a hybridization scheme.
- Screened lambda gt11 human cDNA libraries using samples from XP family members (father, mother, child).
- Characterized positive clones to identify specific genes and their expression levels.
Main Results:
- Successfully cloned 15 positive lambda gt11 clones.
- Identified at least 14 clones representing a single gene.
- This gene showed significantly higher abundance in XP obligate heterozygote parents compared to the homozygous affected child.
Conclusions:
- The competition hybridization strategy is effective for isolating genes with differential expression.
- The identified gene's altered abundance may be relevant to xeroderma pigmentosum (XP) pathogenesis.
- Further research is warranted to elucidate the role of this gene in XP.