The 19 F 19 F nuclear spin-spin coupling constants J FF for a set of eighteen compounds related structurally to 1,8-difluoronaphthalene were measured by 19 F NMR spectroscopy. The FF distances d FF in these compounds were determined by ab initio 3-21G* molecular orbital calculations. Consistent with the lone-pair overlap theory of the origins of through-space 19 F 19 F coupling, an exponential relationship is found between J FF and d FF (regression coefficient r 2 ) 0.991), and a linear relationship is found between J FF and the extent of the overlap interaction between the in-plane fluorine 2p lone-pair orbitals (regression coefficient r 2 ) 0.993). The magnitudes of these lone-pair interactions were estimated from molecular orbital energies obtained by ab initio 6-31G* calculations for a model consisting of a pair of HF molecules separated by various distances.

We have developed a strategy for the detection, localization and sequence determination of point mutations in the mRNA coding for the alpha 1(I) and alpha 2(I) 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(I) and pro alpha 2(I) 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(I) mRNA associated with a case of lethal osteogenesis imperfecta. The G----A point mutation in the codon for alpha 2(I) glycine residue 805 results in the substitution of an aspartic acid at this position and is consistent with the proband's collagen protein data.

We have investigated the procollagen, collagen, alpha 2(I) mRNA, and DNA of a proband with type IV OI. The proband synthesized two alpha 2(I) chains, one with normal electrophoretic migration and one more rapidly migrating. The fast alpha 2(I) chain was relatively retained within the cell and was present in collagens synthesized in the presence of alpha,alpha'-dipyridyl. The alpha 2(I) cyanogen bromide peptide CB 4-2 contained both normal and rapidly migrating components. Thermal stability of helices containing the rapidly migrating alpha 2(I) chain was reduced 6 degrees C. Parental fibroblast collagens were normal. RNA/RNA hybrids between proband total RNA and antisense riboprobe complementary to alpha 2(I) nt 236-1390 were digested with RNase A and T1. Digestion products seen exclusively in the proband suggested a structural change in the region coding for exons 16-19. The region which hybridized to the riboprobe was amplified using RNA-PCR and subcloned. Multiple restriction enzyme digestions of the two subcloned alleles suggested a structural change localized to the region coding for exons 16-17. Sequencing revealed a deletion of the 54 bp comprising exon 16 in the cDNA of one allele. The region of the proband's genomic DNA spanning exons 15-17 was amplified by PCR. The subcloned genomic fragments of each allele were distinguished by RNA/DNA hybrid analysis using a riboprobe complementary to normal genomic DNA from this region. Sequencing revealed a G+1-->A mutation at the exon 16 donor site in one allele. The mutation eliminates a StyI site.(ABSTRACT TRUNCATED AT 250 WORDS)