Endothelin-3-converting enzyme activity of the KEL1 and KEL6 phenotypes of the Kell blood group system

The Kell blood group protein is a metalloendopeptidase that preferentially cleaves a Trp(21)-Ile(22) bond of big endothelin-3 producing bioactive endothelin-3. Kell is a polymorphic protein, and 25 different phenotypes, because of point mutations resulting in single amino acid substitutions, have been described. It was recently reported that a recombinant form of KEL1 (K, K1) phenotype, expressed in K562 and HEK293 cells, had no endothelin-3-converting activity, in contrast to the common KEL2 (k, K2) phenotype. We demonstrate that KEL1 red blood cells and also a soluble recombinant form of KEL1 protein (s-Kell KEL1) have similar enzymatic activity as the common Kell phenotype. In addition we show that KEL6 red blood cells, which are more prevalent in persons of African heritage than in Caucasians also have endothelin-3-converting enzyme activity and that the recombinant soluble form of KEL6 protein (s-Kell KEL6) has similar K(m) values as the wild-type.     

A Single Nucleotide Difference in the Gene for Myelin Proteolipid Protein Defines the Jimpy Mutation in Mouse

We have previously shown that, in the myelin-deficient jimpy mutant mouse, 74 nucleotides are absent from the mRNA for proteolipid protein (PLP) as a result of aberrant RNA processing. To define the exact site of the jimpy mutation, we have analyzed the PLP gene obtained from a jimpy mouse genomic library. We find that the nucleotide sequence that is absent from jimpy PLP mRNA is fully preserved in the jimpy PLP gene. The missing segment corresponds to a separate exon, equivalent to exon 5 of the human PLP gene. The nucleotide sequence at the 3' end of intron 4 in the jimpy PLP gene contains a single point mutation. A base change A----G in the 3' acceptor splice site has altered a position that is 100% conserved in all published splice acceptor sequences. We conclude that the primary genetic defect of the jimpy mouse is a single base change in the PLP gene disabling an invariant recognition sequence of RNA splicing.     

A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.

mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients.     

Gene conversion confined to a direct repeat of the acceptor splice site generates allelic diversity at human glycophorin (GYP) locus.

The glycophorin locus (GYP) on the long arm of chromosome 4 encodes antigens of the MNSs blood group system and displays considerable allelic variation among human populations. The genomic structure and organization of a variant glycophorin allele specifying a novel Miltenberger (Mi)-related phenotype, MiX, were examined. This variant probably arose from a gene conversion event involving a direct repeat of the acceptor splice site. Southern blot analysis indicated that MiX gene derived its 5' and 3' portions from glycophorin B or delta gene but its internal part from glycophorin A or alpha gene. Genomic sequences encompassing the rearranged regions of the MiX gene were amplified by single copy polymerase chain reaction. Direct DNA sequencing showed that during the formation of MiX gene, a short stretch of alpha exon III with a donor splice site has replaced a silent sequence in the delta gene containing a cryptic acceptor splice site. The upstream delta-alpha breakpoint is flanked by the direct repeats of the acceptor splice site, whereas the down-stream alpha-delta breakpoint is located in the adjacent intron. This segmental transfer produced a new composite exon whose expression not only transactivated a portion of silent sequence but also created intraexon and interexon hybrid junctions that characterize the antigenic specificities of MiX glycophorin. The identification of MiX as yet another delta-alpha-delta hybrid different from MiIII and MiVI in gene conversion sites suggests that shuffling of expressed and unexpressed sequences through particular genomic DNA motifs has been an important mechanism for shaping the antigenic diversity of MNSs blood group system during evolution.     

An intron mutation in the human alpha 1(I) collagen gene alters the efficiency of pre-mRNA splicing and is associated with osteogenesis imperfecta type II

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.     

Cooperation of 5' and 3' processing sites as well as intron and exon sequences in calcitonin exon recognition.

We have previously shown that the calcitonin (CT)-encoding exon 4 of the human calcitonin/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is surrounded by suboptimal processing sites. At the 5' end of exon 4 a weak 3' splice site is present because of an unusual branch acceptor nucleotide (U) and a weak poly(A) site is present at the 3' end of exon 4. For CT-specific RNA processing two different exon enhancer elements, A and B, located within exon 4 are required. In this study we have investigated the cooperation of these elements in CT exon recognition and inclusion by transient transfection into 293 cells of CALC-I minigene constructs. Improvement of the strength of the 3' splice site in front of exon 4 by the branchpoint mutation U-->A reduces the requirement for the presence of exon enhancer elements within exon 4 for CT-specific RNA processing, irrespective of the length of exon 4. Replacement of the exon 4 poly(A) site with a 5' splice site does not result in CT exon recognition, unless also one or more exon enhancer elements and/or the branchpoint mutation U-->A in front of exon 4 are present. This indicates that terminal and internal exons are recognised in a similar fashion. The number of additional enhancing elements that are required for CT exon recognition depends on the strength of the 5' splice site. Deletion of a large part of intron 4 also leads to partial exon 4 skipping. All these different elements contribute to CT exon recognition and inclusion. The CT exon is recognised as a whole entity and the sum of the strengths of the different elements determines recognition as an exon. Curiously, in one of our constructs a 5' splice site at the end of exon 4 is either ignored by the splicing machinery of the cell or recognised as a splice donor or as a splice acceptor site.