Ronald R. Minor

Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-α2(I) and Severe Osteogenesis Imperfecta

RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the probands RNA had a unique cleavage pattern in the region of COL1A2 encoding the C‐propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991‐3994 (“CTAG”) were replaced with “TGTCATTGG.” The first seven bases of the inserted sequence were identical to nucleotides 4002‐4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse‐transcription polymerase chain reaction (RT‐PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Increased density of pC‐α2(I) suggested comigration with the similarly sized pro‐α2(I) derived from the mutant allele. Furthermore, α‐chains were overhydroxylated and the ratio of α1(I):α2(I) was 3.2:1, consistent with the presence of α1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro‐α2(I) C‐propeptide and confirmed a diagnosis of OI.

Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.

Acetylcholine receptor regulation in L5 muscle cells is independent of increases in collagen secretion induced by ascorbic acid

Ascorbic acid is the active component of fetal brain extract that induces increased acetylcholine receptor (AChR) expression in L5 rat clonal muscle cell cultures. The induction of AChR expression, as determined by 125I-alpha-bungarotoxin binding, occurs with a delay of 20-25 h. We report that the delayed increase in AChR can be triggered by a 5-h exposure to ascorbic acid. These studies suggest that intermediary processes may be involved. Ascorbic acid treatment also causes a threefold increase in collagen secretion in L5 cultures by 3 h. The rapid increase in collagen secretion and the delayed induction of surface AChR suggested that there may be a link between these two responses. However, although bacterial collagenase eliminates secreted collagen, it had no effect on the increase in surface AChR. Thus, the ascorbic acid effect on elevating AChR expression is independent of its effect on collagen secretion.