Molecular biology of the alpha-L-fucosidase gene and fucosidosis.

Human alpha-L-fucosidase, a lysosomal enzyme, hydrolyzes alpha-L-fucose from glycolipids and glycoproteins. Its activity is deficient in human fucosidosis an autosomal recessive disease. In order to understand the molecular basis of this lysosomal storage disorder we have cloned several cDNAs coding for human alpha-L-fucosidase from a human hepatoma and a human liver cDNA library constructed in lambda gt11. Compiling the cDNA sequences of these clones we have identified 1,829 base pairs (bp) encoding human alpha-L-fucosidase. This includes an open reading frame of 1,172 bp, a consensus polyadenylation signal AAT AAA and a poly(A)+ tail. The sequence is incomplete at the 5'-end, and clones encoding the amino terminus of the native protein, the propeptide and leader signal have not yet been isolated. The open reading frame encodes for 390 amino acids with a calculated Mr of 45,557. This represents 78-95% of the mature processed alpha-L-fucosidase. The availability of these cDNA clones has enabled us to map the structural gene for alpha-L-fucosidase to chromosome 1p34.1-1p36.1 by Southern blot analysis of DNA from human-rodent somatic cell hybrids and by in situ hybridization. Furthermore, a Pvu II restriction fragment length polymorphism (RFLP) has been identified at the human alpha-L-fucosidase gene locus. Analysis of mRNA by Northern blotting gives a major species of 2.25 kb. In 4 patients with fucosidosis no mRNA signal was detected and Western blots gave no immunoreactive enzyme. Southern blotting after Eco RI digestion in two fucosidosis families revealed a banding abnormality (extra 6-kb band).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of human RNA transcripts among heterogeneous nuclear RNA from man-mouse somatic cell hybrids.

In man-mouse hybrid line from our cell library, the only cytological detectable portion of the human genome is the X chromosome, and the only genetic markers regularly expressed are coded by genes known to be ?X-linked. A component of the heterogeneous nuclear RNA of these cells was found to be complementary to repetitive human DNA sequences by means of RNA-DNA hybridization on nitrocellulose filters. The same procedure also permitted the identification of hybrid cell DNA sequences that are complementary to human heterogeneous nuclear RNA. This experimental approach, coupled with hybridization studies in situ, is expected to yield critical data on the distribution and the specificity of the repetitive DNA sequences present in the human genome and to provide a new tool for cytological mapping of human chromosomes.