Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member.

Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have been 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3' untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.     

Molecular cloning, characterization, and genetic mapping of an endogenous murine mammary tumor virus proviral unit I of C3H/He mice.

We have cloned and characterized a novel endogenous murine mammary tumor virus proviral unit of the C3H/He strain of mice. The cloned proviral unit is 16 kilobase pairs (kbp) in size and is composed of a 5.6-kbp 5' EcoRI segment of an endogenous provirus with 10.4-kbp flanking cellular sequences. A comparison of the restriction map of the cloned proviral DNA with that of an endogenous provirus of the GR strain of mice has revealed minor differences in restriction sites on the two proviruses. The restriction enzyme SstI, which does not cleave the 5' EcoRI fragment of GR DNA, cleaves the C3H/He proviral sequences once; MspI has an additional site in the C3H/He proviral sequences. By using a subcloned fragment containing unique cellular sequences as a hybridization probe, we (i) mapped the C3H/He proviral unit to chromosome 14 by using mouse-hamster somatic cell hybrids, and (ii) demonstrated that this proviral unit is also present in the genome of DBA/2 mice. From these results we conclude that the C3H/He strain of mice acquired this proviral unit from DBA stock by genetic transmission. Our data also indicate that the murine mammary tumor virus sequences present in the gag-specific proviral unit of C3H/He mice extend at least 2.45 kbp downstream of the EcoRI site in the genomic DNA. Since the structural organization and chromosomal location of this proviral unit are distinct from those of previously reported proviral units represented by similar-sized (16.7-kbp) EcoRI fragments, we tentatively propose to designate this proviral unit Mtv-7a.     

Structure of a cluster of mouse histone genes.

The four mouse histone genes (2 H3 genes, an H2b gene and an H2a gene) present in a cloned 12.9 kilobase fragment of DNA have been completely sequenced including both 5' and 3' flanking regions. These genes are expressed in cultured mouse cells and the 3' and 5' ends of the mRNA have been determined by S1 nuclease mapping. These genes code for a minor fraction of the histone mRNAs expressed in cultured mouse cells. They comprise at most 5-8% of the total histone mRNA of each type. The two H3 genes code for H3.2 and H3.1 histone proteins, while the H2b gene codes for an H2b.1 protein with a single amino acid change (val-leu) at position 18. Only the 3' portion of the H2a gene is contained in the clone and there is an amino acid change (alanine-proline) at position 126. Comparison of the 5' and 3' flanking sequences reveals a conserved sequence at the 3' end of the mRNA which forms a hairpin loop structure. The codon usage in the genes is non-random and there has been no discrimination against CG doublets in the coding region of the genes.     

The human apolipoprotein AII gene: structural organization and sites of expression.

The complete nucleotide sequence of the human apolipoprotein All gene together with 911 bases of 5' flanking sequence and 687 bases of 3' flanking sequence have been determined. The mRNA coding region is interrupted by three introns of 169, 293 and 395bp. The Intro-exon structure of the apo All gene is similar to that of the apo AI, apo CIII and apo E genes: three introns separate 4 coding sequences specifying the 5' untranslated region, pre-peptide, a short N-terminal domain and a C-terminal domain composed of a variable number of lipid-binding amphipathic helices. Intron II carries a 33bp dG-dT repetitive element adjacent to the 3' splice junction which has the potential to adopt the Z-DNA conformation. The 5' and 3' terminuses of the mRNA have been identified by primer extension and S1 nuclease mapping. A number of short direct repeats are found in the 5' flanking region and an inverted repeat occurs between the CAAT and TATA boxes. Downstream of the the gene is an Alu family repeat containing a polymorphic MspI site, the deletion of which is associated with increased circulating levels of apoAII. ApoAII gene expression was demonstrated in adult human liver and HepG2 cells but not in human small intestine. Of ten Rhesus monkey tissues examined apo All mRNA was detected only in liver.     

Structure and arrangement of the beta-tubulin genes of Leishmania tropica.

We studied the organization and arrangement of the genes encoding beta-tubulin in the protozoan parasite Leishmania tropica and examined the structure and orientation of the beta-tubulin mRNA relative to the gene. There were found to be eight to nine beta-tubulin genes arranged in an array of direct tandem repeat units with a length of 3.8 kilobase pairs, and they were extremely homologous, if not identical, in sequence. These repeat units did not contain the alpha-tubulin genes. The transcribed sequences within the beta-tubulin genes were localized, and the orientation of the major alpha-tubulin mRNA was mapped on the gene by S1 nuclease analysis.     

Tn2610, a transposon involved in the spread of the carbenicillin-hydrolyzing beta-lactamase gene

We have found a new transposon, Tn2610, on pCS200 in clinical isolates of Escherichia coli, which encodes the carbenicillin-hydrolyzing beta-lactamase gene in combination with the resistance determinants to streptomycin and sulfonamide. Tn2610 has a molecular size of 24 kilobase pairs and is flanked by long inverted repeat sequences of 3 kilobase pairs in length. Genetical and physical analyses indicate that Tn2610 is a single transposable unit encoding the multiple resistance determinants and that is different from any previously described transposon. The characteristic DNA structure observed in various complex resistance transposons involved in the transposition of the carbenicillin-hydrolyzing beta-lactamase gene is discussed.     

Recruitment of the Puf3 protein to its mRNA target for regulation of mRNA decay in yeast

The Puf family of RNA-binding proteins regulates mRNA translation and decay via interactions with 3' untranslated regions (3' UTRs) of target mRNAs. In yeast, Puf3p binds the 3' UTR of COX17 mRNA and promotes rapid deadenylation and decay. We have investigated the sequences required for Puf3p recruitment to this 3' UTR and have identified two separate binding sites. These sites are specific for Puf3p, as they cannot bind another Puf protein, Puf5p. Both sites use a conserved UGUANAUA sequence, whereas one site contains additional sequences that enhance binding affinity. In vivo, presence of either site partially stimulates COX17 mRNA decay, but full decay regulation requires the presence of both sites. No other sequences outside the 3' UTR are required to mediate this decay regulation. The Puf repeat domain of Puf3p is sufficient not only for in vitro binding to the 3' UTR, but also in vivo stimulation of COX17 mRNA decay. These experiments indicate that the essential residues involved in mRNA decay regulation are wholly contained within this RNA-binding domain.     

Structural isoforms of a membrane transport protein from Leishmania enriettii.

A membrane transport protein of the glucose transporter superfamily from Leishmania enriettii is encoded by a family of tandemly repeated genes. The first gene in this tandem repeat codes for a structural isoform that contains a unique amino-terminal hydrophilic domain, probably located in the cytoplasm; the remainder of the protein is identical to the polypeptide encoded by the internal genes in the tandem repeat. The unique isoform is represented by a distinct stable RNA.