An osteopenic nonfracture syndrome with features of mild osteogenesis imperfecta associated with the substitution of a cysteine for glycine at triple helix position 43 in the pro alpha 1(I) chain of type I collagen.

Mutations affecting the pro alpha 1(I) or pro alpha 2(I) collagen genes have been identified in each of the major clinical types of osteogenesis imperfecta. This study reports the presence of a heritable connective tissue disorder in a family with an osteopenic syndrome which has features of mild osteogenesis imperfecta but was considered idiopathic osteoporosis in the proband. At age 38, while still premenopausal, she was found to have osteopenia, short stature, hypermobile joints, mild hyperelastic skin, mild scoliosis, and blue sclerae. There was no history of vertebral or appendicular fracture. Hip and vertebral bone mineral density measurements were consistent with marked fracture risk. Delayed reduction SDS-PAGE of pepsin-digested collagens from dermal fibroblast cultures demonstrated an anomalous band migrating between alpha 1(I) and alpha 1(III). This band merged with the normal alpha-chains upon prereduction, indicating an unexpected cysteine residue. Cyanogen bromide peptide mapping suggested that the mutation was in the smaller NH2-terminal peptides. cDNA was reverse transcribed from mRNA and amplified by the polymerase chain reaction. A basepair mismatch between proband and control alpha 1(I) cDNA hybrids was detected by chemical cleavage with hydroxylamine:piperidine. The cysteine substitution was thus localized to alpha 1(I) exon 9 within the cyanogen bromide 4 peptide. Nucleotide sequence analysis localized a G----T point mutation in the first position of helical codon 43, replacing the expected glycine (GGT) residue with a cysteine (TGT). The prevalence of similar NH2-terminal mutations in subjects with this phenotype which clinically overlaps idiopathic osteoporosis remains to be determined.

Osteoporosis and familial idiopathic scoliosis: association with an abnormal alpha 2(I) collagen.

A positive family history is considered a risk factor for osteoporosis (OP) although the genetic or biochemical basis for this relationship remains undefined. Various mutations affecting normal synthesis of type I collagen have been reported in osteogenesis imperfecta (OI), a heritable disorder of connective tissue. Family A, in which the proband and a daughter are afflicted with OP and idiopathic scoliosis was examined for defects in collagen metabolism. Dermal fibroblast cultures were established to investigate de novo collagen synthesis. SDS-PAGE revealed an abnormally migrating alpha 2(I) chain and procollagen in two generations. Examination of the kinetics of type I collagen pC & N-propeptide processing demonstrated a rate 2x control in the proband. The phenotype family A is not OI. It shares features with families B & C, having familial clustering of OP. However, collagen synthesis was not abnormal in family B & C. These data suggest that in family A the alpha 2(I) structural defect may be related to defective skeletal matrix formation.

Pulmonary hypoplasia and osteogenesis imperfecta type II with defective synthesis of alpha I(1) procollagen.

Perinatal lethal osteogenesis imperfecta (OI type II), a heritable disorder of connective tissue occurs approximately once in 60,000 live births. Phenotypic characteristics include defective cranial ossification and severe skeletal deformity due to intrauterine rib and long bone fractures. Lethal OI may be associated with intracranial hemorrhage or severe respiratory insufficiency. Pulmonary hypoplasia has been previously observed in OI type II, but has not been defined clinically. The infant described herein was born with OI type II and pulmonary hypoplasia. Pathological examination of airway branching patterns indicated that lung development had progressed to only the 10 week stage with immature acinar development. Investigation type I collagen synthesis by cultured dermal fibroblasts revealed the presence of electrophoretically abnormal alpha 1(I) polypeptides. These findings suggest that biochemically regulated processes, as well as mechanical factors, may impeded pulmonary development in similar cases of OI type II.

Proteins associated with rabbit reticulocyte mRNA caps during translation as investigated by photocrosslinking.

This laboratory previously detected by UV crosslinking a number of proteins associated with cytoplasmic mRNA in mammalian cells, and the data suggested that they are involved in translation. To find out which proteins are associated with caps we made use of reticulocyte mRNA specifically labeled in the cap with 32P together with a cell-free translation system and UV crosslinking. Approximately 8 bands corresponding to proteins crosslinked to the cap itself have been detected by polyacrylamide gel electrophoresis after UV crosslinking and digestion with RNases or tobacco pyrophosphatase. All but one were specific for methylated caps. One was similar in size and partial peptide map to a cap-binding protein, CBP I, previously identified in other laboratories, and most of the others corresponded to proteins previously known to be associated with mRNA but not known to be associated with caps. The results suggest that most mRNA-associated proteins are associated with caps or poly(A). Also, the number of cap-associated proteins may be greater than previously suspected.

Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity.

The type I killer strains of Saccharomyces cerevisiae secrete a dimeric 19-kDa protein that kills sensitive cells by disrupting cytoplasmic membrane function. This toxin is encoded by the double-stranded RNA plasmid M1-dsRNA, which also determines specific immunity to toxin. A preprotoxin, the 35-kDA in vitro translation product of denatured M1-dsRNA, is presumed to be the primary in vivo gene product. To facilitate studies on preprotoxin structure and maturation, we have inserted a partial cDNA copy of M1-dsRNA into the yeast vector p1A1, bringing it under control of the phosphate-repressible PHO5 promoter. This in-frame gene fusion encodes all of the preprotoxin sequence except for its N-terminal secretion leader, which is replaced by the leader sequence of PHO5. Transformation of sensitive yeast strains lacking M1-dsRNA with such fusion plasmids converts them to phosphate-repressible, immune killers, demonstrating that both toxin and immunity determinants are contained within the preprotoxin molecule. L-1-Tosylamido-2-phenylethyl chloromethyl ketone retards glycosylation of preprotoxin to toxin, facilitating size comparisons and indicating that processing of the normal precursor involves three glycosylation events but does not involve cotranslational leader peptidase action. In contrast, the PHO5 leader is apparently removed from the fusion preprotoxin.

Yeast killer dsRNA plasmids are transcribed in vivo to produce full and partial-length plus-stranded RNAs.

In vivo transcripts of the L (4.5 kb) and M (1.9 kb) dsRNA plasmids were examined in type I killers of Saccharomyces cerevisiae. Transcripts for both plasmids include full-length (l,m) and partial-length (la,ma) single-stranded species. Both L-dsRNA transcripts (l,la) have in vitro mRNA activity for L-P1, previously shown to be identical to ScV-P1, the 88,000 dalton major capsid protein of the virus-like particles containing L- and M1-dsRNAs. 1, but not 1a, is bound to poly(U)-sepharose and may be polyadenylated. Other L-dsRNA gene products and their transcripts may exist. For M1-dsRNA, both species (m, ma) have in vitro mRNA activity for M1-P1, the 32,000 dalton pre-protoxin encoded by M1-dsRNA. Both m and ma are bound to poly(U)-Sepharose and ma is probably a 5' terminal fragment of m. A functional model for M1-dsRNA killer plasmid structure is presented.