Skip Navigation

This Article
Right arrow Print PDF (7595K)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (24)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Grange, D. K.
Right arrow Articles by Marini, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grange, D. K.
Right arrow Articles by Marini, J. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 1990, Vol. 18, No. 14 4227-4236
© 1990

MOLECULAR BIOLOGY

Detection of point mutations in type I collagen by RNase digestion of RNA/RNA hybrids

Dorothy K. Grange, Gary S. Gottesman, Mary Beth Lewis and Joan C. Marini*

Unit on Connective Tissue Disorders, Human Genetics Branch, National Institute of Child Health and Human Development Bethesda, MD 20892, USA

*To whom correspondence should be addressed at Human Genetics Branch, NICHD, Building 10, Room 9S242, 9000 Rockville Pike, Bethesda, MD 20892, USA

Received February 7, 1990. Revised June 21, 1990. Accepted June 21, 1990.

We have developed a strategy for the detection, localization and sequence determination of point mutations in the mRNA coding for the {alpha} 1(1) and {alpha}2(l) chains of type I collagen. Point mutations are detected by RNase A cleavage of mismatches in RNA/RNA hybrids. The mRNAs coding for the fibrillar collagens present special problems for hybrid analysis because of their large size and their GC-rich and repetitive sequences. We have generated a series of overlapping antisense riboprobes covering the entire pro{alpha}1(l) and pro{alpha}2(l) mRNAs. Uniformly labelled normal antisense riboprobes are hybridized with the total fibroblast RNA of patients with possible mutations in type I collagen. Mismatches in the resulting RNA/RNA hybrids are cleaved with RNase A and the labelled riboprobe cleavage products are examined electrophoretically. The sensitivity and specificity of the system were demonstrated by the detection and localization of a known point mutation in the codon for {alpha}1 (I) glycine 988 (1). DNA for sequencing the mutations localized by hybrid analysis may be obtained by either (1) generation of a fibroblast cDNA library and isolation of both alleles by plaque screening, or (2) a more rapid method using first strand cDNA synthesis from poly (A + )-mRNA, followed by PCR amplification of the mutation-containing region of the DNA/RNA hybrid. This strategy for detection and isolation has wide application not only for mutations causing connective tissue disorders, but also for mutations in other large and repetitive genes.

We have used this strategy for the detection and sequencing of a point mutation in {alpha}2(1) mRNA associated with a case of lethal osteogenesis imperfecta. The G->A point mutation in the codon for {alpha}2(l) glycine residue 805 results in the substitution of an aspartic acid at this position and is consistent with the proband's collagen protein data.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Med. Genet.Home page
W A Cabral, S Milgrom, A D Letocha, E Moriarty, and J C Marini
Biochemical screening of type I collagen in osteogenesis imperfecta: detection of glycine substitutions in the amino end of the alpha chains requires supplementation by molecular analysis.
J. Med. Genet., August 1, 2006; 43(8): 685 - 690.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. A. Cabral, M. V. Mertts, E. Makareeva, A. Colige, M. Tekin, A. Pandya, S. Leikin, and J. C. Marini
Type I Collagen Triplet Duplication Mutation in Lethal Osteogenesis Imperfecta Shifts Register of alpha Chains throughout the Helix and Disrupts Incorporation of Mutant Helices into Fibrils and Extracellular Matrix
J. Biol. Chem., March 14, 2003; 278(12): 10006 - 10012.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Forlino, F. D. Porter, E. J. Lee, H. Westphal, and J. C. Marini
Use of the Cre/lox Recombination System to Develop a Non-lethal Knock-in Murine Model for Osteogenesis Imperfecta with an alpha 1(I) G349C Substitution. VARIABILITY IN PHENOTYPE IN BrtlIV MICE
J. Biol. Chem., December 31, 1999; 274(53): 37923 - 37931.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Q. Wang, A. Forlino, and J. C. Marini
Alternative Splicing in COL1A1 mRNA Leads to a Partial Null Allele and Two In-frame Forms with Structural Defects in Non-lethal Osteogenesis Imperfecta
J. Biol. Chem., November 8, 1996; 271(45): 28617 - 28623.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Bouma, W. A. Cabral, W. G. Cole, and J. C. Marini
COL5A1 Exon 14 Splice Acceptor Mutation Causes a Functional Null Allele, Haploinsufficiency of alpha 1(V) and Abnormal Heterotypic Interstitial Fibrils in Ehlers-Danlos Syndrome II
J. Biol. Chem., April 13, 2001; 276(16): 13356 - 13364.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.