Multiple abnormal beta-hexosaminidase alpha chain mRNAs in a compound-heterozygous Ashkenazi Jewish patient with Tay-Sachs disease

Abnormal beta-hexosaminidase alpha chain cDNA clones were isolated from fibroblasts of an Ashkenazi Jewish patient with Tay-Sachs disease. Four abnormal cDNA clones were sequenced in their entirety. We showed previously that three of these mRNAs retained intron 12 with a mutation from G to C at the 5' donor site and that the patient was heterozygous with respect to this splicing defect (Ohno, K., and Suzuki, K., (1988) Biochem. Biophys. Res. Commun. 153, 463-469). One clone retained, in addition to intron 12, intron 13, which was truncated and polyadenylated due to a polyadenylation signal within intron 13. The fourth clone did not contain intron 12 and was missing exon 12. Some of these abnormal mRNAs were also missing one or more of upstream exons. The regions of exon 12-intron 12 and of upstream exons were evaluated in a total of 30 clones, including those completely sequenced, by restriction mapping and Southern analysis with appropriate probes. Of the 25 cDNA clones that included the exon 12-intron 12 region, 11 contained the exon 12-intron 12 sequence with the junctional transversion, and 11 were missing both exon 12 and intron 12. Among the 12 clones that included the region of exon 3-exon 9, 7 were missing one or more of upstream exons. Three clones gave results expected of normal cDNA in the region of exons 12 and 13. One of the three, furthermore, was 3.6-kilobases long and contained the completely normal beta-hexosaminidase alpha chain mRNA sequence on the 3' side and an abnormal 1.7-kilobase segment at the 5' end. These findings suggest that the splicing defect results in either retention of intron 12 or skipping of exon 12 in approximately equal proportions and that remote upstream exons are also frequently excised out. The three clones that were normal in the exon 12-intron 12 region could have derived from the other yet-to-be-characterized mutant allele. However, we were unable to obtain firm evidence that the abnormal upstream sequence is directly related to Tay-Sachs disease.     

Simian immunodeficiency virus displays complex patterns of RNA splicing.

The human and simian immunodeficiency viruses encode at least six gene products that apparently serve regulatory functions. To evaluate the regulation of simian immunodeficiency virus gene expression at the level of RNA splicing, we used the polymerase chain reaction to amplify and clone cDNAs corresponding to a large array of mRNAs from infected cells. We identified mRNAs that used splice acceptor sites upstream of the initiator codons for tat, rev, vpr, nef, vif, and vpx, suggesting that these proteins may be expressed from different mRNAs. We also provide hybridization data suggesting that the same splice acceptor site may be used for both rev and env mRNAs. Furthermore, we isolated both tat and rev cDNAs that utilized three alternative splice acceptor sites at the start of coding exon 2, indicating that different versions of these proteins may be encoded. Finally, approximately 10 to 20% of simian immunodeficiency virus mRNAs spliced an intron from their untranslated 5' ends, and sequences contained within this intron constituted a portion of the tat-responsive TAR element. Thus, alternative pre-mRNA splicing adds a level of complexity to simian immunodeficiency virus expression, which may affect several levels of gene regulation.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene.

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens disease at the molecular level. zeta-Crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several zeta-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3' end of the coding region. This deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic zeta-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the zeta-crystallin gene disclosed a dinucleotide deletion of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered zeta-crystallin protein. This is the first time a genomic mutation in an enzyme/crystallin gene has been directly linked to a congenital cataract.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

Nonallelic members of the cytochrome c multigene family of the rat may arise through different messenger RNAs

We determined the nucleotide sequences of three nonallelic cytochrome c genes (from recombinant clones Ch4A-RC5, 6 and 8) isolated from the rat cytochrome c gene family. In contrast with a fourth gene (from Ch4A-RC4), which has an intron and correctly encodes rat cytochrome c, these three appear to be pseudogenes and resemble mRNA molecules in two respects: they are all missing the intron of clone 4, and sequence homology with clone 4 in their 3' noncoding regions abruptly ends at two different A-rich tracts reminiscent of poly(A) tails. We also detect three cytochrome c mRNAs of sizes 1400, 1100 and 700 nucleotides in several tissues of the adult rat. The size differences among the mRNAs can be accounted for by length heterogeneity in their 3' noncoding regions. Two of the 3' ends map to the two points where the mRNA-like genes diverge from clone 4 at poly(A) tracts. Furthermore, short direct repeats flank the genes of clones 5, 6 and 8 at the positions where their sequences diverge. The observations suggest that these members of the cytochrome c multigene family may arise through insertion into the genome of DNA copies of cytochrome c mRNAs.     

Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression.

The genes encoding mouse and human acetylcholinesterases have been cloned from genomic and cosmid libraries. Restriction analysis and a comparison of sequence with the cDNAs have defined the exon-intron boundaries. In mammals, three invariant exons encode the signal peptide and the amino-terminal 535 amino acids common to all forms of the enzyme whereas alternative exon usage of the next exon accounts for the structural divergence in the carboxyl termini of the catalytic subunits. mRNA protection studies show that the cDNA encoding the hydrophilic catalytic subunits represents the dominant mRNA species in mammalian brain and muscle whereas divergent mRNA species are evident in cells of hematopoietic origin (bone marrow cells and a erythroleukemia cell line). Analyses of mRNA species in these cells and the genomic sequence have enabled us to define two alternative exons in addition to the one found in the cDNAs; they encode unique carboxyl-terminal sequences. One mRNA consists of a direct extension through the intervening sequence between the common exon and the 3' exon deduced from the cDNA. This sequence encodes a subunit lacking the cysteine critical to oligomer formation. Another mRNA results from a splice that encodes a stretch of hydrophobic amino acids immediately upstream of a stop codon. This exon, when spliced to the upstream invariant exons, should encode glycophospholipid-linked species of the enzyme. Homologous sequence, identity of exon-intron junctions, and identity of position of the stop codon are seen for this region in mouse and human. Polymerase chain reactions carried out across the expected intron region and mRNA protection studies show that this splice occurs in mouse bone marrow and erythroleukemia cells yielding the appropriate cDNA.     

Two genes for the high mobility group protein HMG-Y are present in the genome of Canavalia gladiata D.C.

Two cDNAs encoding high-mobility-group (HMG) proteins that correspond to animal HMG-Y proteins were isolated from maturing seeds of Canavalia gladiata D.C. The deduced amino acid sequences of these cDNAs showed similarity to other plant HMG-I/Y proteins reported to date. The mRNAs for the HMG-Y proteins were detected in leaves, stems, roots, pods and seeds of C. gladiata. The level of the mRNA was high in the maturing seeds of 30 days after flowering and 2-day germinated seeds. Two genomic clones were isolated from DNA of C. gladiata and both were shown to represent single-copy genes consisting of two exons and one intron. This is the first report of the genomic sequences for HMG-I/Y protein in plants.