Abstract
This study describes a homozygous, G----A transition at the moderately conserved +5 position within the splice donor site of intron 14 in the human alpha 1(I) collagen gene. The mutation reduced the efficiency of normal splice-site selection since the exon upstream of the mutation was spliced alternatively. Moreover, the extent of alternative splicing was sensitive to the temperature at which the mutant cells were grown, suggesting that the mutation directly affected spliceosome assembly. To achieve exon skipping, this effect must be propagated so as to disrupt the selection of a second splice site in the adjacent intron.
Highlights
University Center, of Michigan, This study describes a homozygous, G+A transition at the moderately conserved +5 position within the splice donor site of intron 14 in the human al(I) collagen gene
Methods for preparation of total RNA, cDNA synthesis, the polymerase chain reaction (PCR), Ml3 cloning and DNA sequence analysis, and allele-specific oligonucleotide hybridization are detailed in Ref
A novel peptide was identified in the cyanogen bromide (CNBr) peptide map of proal chains recovered from the medium and cell layer of 01 cells (Fig. IA)
Summary
Detailed methods for much of the work presented here can be found in previous publications. Methods for preparation of total RNA, cDNA synthesis, the polymerase chain reaction (PCR), Ml3 cloning and DNA sequence analysis, and allele-specific oligonucleotide hybridization are detailed in Ref. 15. The relative mobility of DNA standards was plotted against (log bp)-I, and a linear regression analysis [19] was used to obtain an equation for the plot. The 01 and control PCR inserts were excised from the pUCl8 vector as blunt-end XbuI fragments and ligated to the expression vector pSVL (Pharmacia LKB Biotechnology Inc.). The vector was digested previously with XbaI/EcoRI and the EcoRI site blunt-ended by a fillin reaction These efforts created 01 and control minigene constructs that included the al(I) sequence extending from within exon 13 to within exon 15. Ultrasound examination of the fetus was consistent with a diagnosis of short-limbed dwarfism, and the pregnancy was terminated electively. The physical and xray findings were compatible with a diagnosis of 01 II
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