Tracing the pathway between mutation and phenotype in osteogenesis imperfecta: isolation of mineralization-specific genes.
The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblasts seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs (approximately 300 bp) corresponding to the 3' ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones.
Culbert
A A
AA
Wellcome Trust Centre for Cell-Matrix Research School of Biological Sciences, University of Manchester, United Kingdom.
Wallis
G A
GA
Kadler
K E
KE
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002113:Calcification, Physiologic / Q000235:genetics*; D002642:Chick Embryo; D003001:Cloning, Molecular; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D015891:Cystatins / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D005353:Fibronectins / Q000096:biosynthesis / Q000235:genetics; D015723:Gene Library; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D008969:Molecular Sequence Data; D010013:Osteogenesis Imperfecta / Q000235:genetics*; D053495:Osteopontin; D010641:Phenotype; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000235:genetics*; D012867:Skin / Q000166:cytology; D012879:Skin Physiological Phenomena; D012886:Skull / Q000166:cytology / Q000502:physiology*
D015891:Cystatins; D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D005353:Fibronectins; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D010750:Phosphoproteins; D012333:RNA, Messenger; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D003094:Collagen; D000469:Alkaline Phosphatase