CGG Triplet Research Articles

Cloning, Characterization and Properties of Plasmids Containing CGG Triplet Repeats from the FMR-1 Gene

The FMR-1 gene for the human fragile-X syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5′ non-translated region. The severity of the disease is correlated with the length of the CGG tract. The cloning of 28 stable plasmids containing (CGG) n inserts (where n=6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies in Escherichia coli. Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG) 81, (CGG) 11AGG(CGG) 60CAG(CGG) 8, from a cDNA from a fragile-X patient or from expansions or deletions of these sequences in E. coli. The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector. Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-X syndrome.

Linkage analysis of the fragile X gene FMR-1 and schizophrenia: no evidence for linkage but report of a family with schizophrenia and an unstable triplet repeat.

We have examined 23 families multiply affected with schizophrenia for linkage to the FMR-1 gene on the X chromosome. Alleles at the FMR-1 CGG triplet repeat were analysed by the polymerase chain reaction, and methylation status at the FMR-1 locus in individuals with evidence of expanded or unstable repeats was analysed by Southern hybridization. Two-point LOD score analyses with a range of X-linked single gene models and a non-parametric affected sib-pair method revealed no evidence for linkage. In one family, however, a fragile X premutation was found, and one individual with schizophrenia and developmental delay was a mosaic for the full and premutation. We conclude that although mutations within the FMR-1 gene do not have a major aetiological role in schizophrenia in our collection of pedigrees, it is possible that FMR-1 mutations can modify the clinical phenotype of schizophrenia.

Anomalous Rapid Electrophoretic Mobility of DNA Containing Triplet Repeats Associated with Human Disease Genes

Eight human genetic diseases have been associated with the expansion of CTG or CGG triplet repeats. The molecular etiology behind expansion is unknown but may involve participation of an unusual DNA structure in replication, repair, or recombination. We show that DNA fragments containing CTG triplet repeats derived from the human myotonic dystrophy gene migrate up to 20% faster than expected in nondenaturing polyacrylamide gels, suggesting the presence of an unusual DNA helix structure within the CTG triplet repeats. The anomalous migration is dependent upon the number of triplet repeats, the length of the flanking DNA, and the percentage and temperature of the polyacrylamide. The effect could be reduced by the addition of actinomycin D. Applying a reptation model for electrophoresis, the results are consistent with a 20% increase in persistence length of the DNA. PCR products containing CTG or CGG repeats from the spinocerebellar ataxia type I gene (SCA1) or the fragile X FMR1 gene, respectively, also showed higher electrophoretic mobility. These are the first sequences of defined length for which a dramatic increase in mobility can be attributed to sequence-dependent structural elements in DNA.

Rat PPARδ Contains a CGG Triplet Repeat and Is Prominently Expressed in the Thalamic Nuclei

We have isolated a new rat sequence containing motifs of a nuclear hormone receptor from a brain cDNA library. The deduced amino acid sequence encoded by the cDNA clone showed a strong homology to the human NUCI and the mouse peroxisome proliferator activated receptor δ (PPARδ). We therefore refer to this new clone as rat PPARδ (rPPARδ). The new feature of rPPARδ is a 14 CGG triplet repeat on the 5′ untranslated region, not previously reported in either NUCI or mPPARδ. We found that rPPARδ was expressed as a 3.5-kb transcript which showed a wide distribution in adult rat tissues. Abundant expression was detected in brain, heart, skeletal muscle, kidney and lung. Weaker expression was noted in the liver, spleen and testis. To determine the specific brain localization of rPPARδ we performed in situ hybridization analysis. Prominent expression was observed in the thalamus, particularly in the posterior part of the ventral medial nucleus, a site responsive to pain and cold stress. These results raise the possibility that PPARδ might play a role in modulating response to thermal and pain sensations.

Pausing of DNA Synthesis in Vitro at Specific Loci in CTG and CGG Triplet Repeats from Human Hereditary Disease Genes

Several human hereditary neuromuscular disease genes are associated with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase beta paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70 degrees C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausing increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.

Crystal structure of a PPR1-DNA complex: DNA recognition by proteins containing a Zn2Cys6 binuclear cluster.

PPR1 is a yeast transcription factor that contains a six-cysteine, two-zinc (Zn) domain, homologous to a similar structure in GAL4. Like GAL4, it binds to DNA sites with conserved CGG triplets symmetrically placed near each end. Whereas the GAL4 site has 11 intervening base pairs, the PPR1 site has 6. The crystal structure of a 95-residue fragment of PPR1 in specific complex with DNA shows that the protein binds to a symmetrical 14-bp recognition site as a nonsymmetrical homodimer. An amino-terminal Zn domain interacts with a conserved CGG triplet near each end of the site through major groove contacts, and the carboxy-terminal residues mediate dimerization through a coiled-coil element and an extended strand. A linker region, connecting the Zn domain and the coiled-coil, folds into a beta-hairpin. This hairpin packs differently on the two subunits and leads to a striking asymmetry, which is largely restricted to the dimerization and linker regions of the protein. Comparison with the GAL4-DNA structure shows that their specificities for sites of different length are determined by the preferred folds of their respective linker segments and by residues at the amino-terminal ends of their coiled-coils. None of these residues contact DNA in PPR1, and they contact only the sugar phosphate backbone in GAL4. We propose that this novel mode of DNA site selection is employed by other proteins that contain a Zn2Cys6 binuclear cluster.

The yeast activator HAP1--a GAL4 family member--binds DNA in a directly repeated orientation.

The yeast transcriptional activator HAP1 contains a DNA-binding domain homologous to GAL4, PPR1, and related factors. By selecting random HAP1-binding sites, we found that HAP1, like GAL4, binds to two CGG triplets. Unlike GAL4, the CGGs in the HAP1 consensus are in a direct and not inverted orientation. Sites with inverted CGGs were not recovered, and mutations converting the direct repeat of CGGs to an inverted repeat greatly reduce HAP1-binding affinity. Also, the 6-bp spacer between the CGGs contains a consensus TA that is positioned asymmetrically. Dimethylsulfate protection patterns on six of these sites show protections and enhancements that also lie in a directly repeated orientation, suggesting that the two HAP1 DNA recognition domains of a HAP1 homodimer are oriented in a directly repeated configuration on the DNA. Moreover, substitution of the HAP1 dimerization domain with that of PPR1, which forms coiled-coils and dimerizes symmetrically, did not diminish the ability of the protein to bind selectively to a direct repeat. This result suggests that one DNA-binding domain of the HAP1 homodimer must be able to swivel 180 degrees relative to the dimerization domain to make specific contacts with the second CGG triplet. Our results present a novel example of domain swiveling in one of the two identical subunits of a homodimer to accommodate specific DNA contacts to both CGG triplets of a direct repeat.

Robust amplification and ethidium-visible detection of the fragile X syndrome CGG repeat using Pfu polymerase.

We report on a method for ethidium bromide detection of FMR1 normal and premutation-size CGG triplet repeats. A set of PCR conditions has been optimized for the polymerase of the hyperthermophilic bacterium, Pyrococcus furiosus. Using this protocol, normal-size alleles ranging from 5 to 50 repeats, as well as most of premutation-size alleles, varying from > 50 to approximately 200 repeats, could be detected by ethidium bromide staining of agarose gels. Since the protocol requires neither autoradiography nor polyacrylamide gel electrophoresis, it promises to provide a rapid means for the exclusion of fragile X syndrome in males with mental retardation.

Fragile X Syndrome

Fragile X syndrome (Martin-Bell syndrome)¹is the most common cause (one in 1500 males) of mental retardation from a single gene defect. The discovery of the fragile X mental retardation—1 (<i>FMR1</i>) gene and its mutation (CGG triplet repeat enlargement) in 1991^1-4has prompted intensive molecular studies of both the gene function and the mechanism by which the triplet repeat further expands from generation to generation.^5-8In this issue, the article by Warren and Nelson⁹summarizes the rapid increase in our molecular knowledge of the disease. In another article, Taylor et al¹⁰have retrospectively investigated female carriers of fragile X mutations for correlation of mental retardation syndrome with DNA-based methods of measuring the triplet repeat mutation. There is a great deal to be learned from both articles. See also pp 507 and 536. The physician is at a disadvantage in making a clinical diagnosis of fragile

How do “Zn2 cys6” proteins distinguish between similar upstream activation sites? Comparison of the DNA-binding specificity of the GAL4 protein in vitro and in vivo.

The GAL4 protein of Saccharomyces cerevisiae is the prototype of a family of transcription factors that contain a "Zn2Cys6" coordination complex in the DNA-binding domain. GAL4 activates the transcription of genes involved in galactose and melibiose metabolism by binding to sites that contain one or more copies of a sequence 5'-CGGN5TN5CCG-3'. Other Zn2Cys6 proteins in S. cerevisiae also recognize sequences containing two CGG triplets, but with different spacings between them. In this report we investigate the mechanism by which GAL4 distinguishes its bona fide binding site from similar sequences as well as from bulk genomic DNA. In vitro, GAL4 recognizes with moderate to high affinity a variety of sites of the general formula (A/C)GGN10-12CCG. This level of specificity is apparently insufficient for the activator to carry out its biological role. However, many of the sites to which GAL4 binds in vitro do not support GAL4-activated transcription in vivo. In most cases there is not a quantitative correlation between the relative affinity of a site for GAL4 in vitro and the level of GAL4-dependent transcription supported by it in vivo. These data imply that there is some mechanism in vivo by which the intrinsic binding specificity of GAL4 is modified.

Fine structure of the human FMR1 gene.

The fragile X syndrome is due to a CGG triplet expansion in the first exon of FMR1, resulting in hypermethylation and extinction of gene expression. To further our understanding of the gene's involvement in the syndrome, we report the physical structure of this locus. A high resolution restriction map of the FRAX(A) locus has been prepared encompassing approximately 50 kb. Using exon-exon PCR and restriction analysis, the FMR1 gene has been determined to consist of 17 exons spanning 38 kb of Xq27.3. Each intron-exon boundary has been sequenced. In general, the splice donors and acceptors located in the 5' portion of the gene demonstrate greater adherence to consensus than those in the 3' end, providing a possible explanation for the finding of alternative splicing in FMR1. The elucidation of the exon composition of the FMR1 gene and its flanking region will enhance detection of coding sequence mutations possible in fragile X phenocopy individuals.

Sequence preferences of covalent DNA binding by anti-(+)- and anti-(-)-benzo[a]pyrenediol epoxides

The sequence preferences of formation of piperidine-labile adducts of guanine by individual (+)- and (-)-isomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [anti-(+)- and anti-(-)-BPDE] were examined by techniques analogous to chemical DNA sequencing. Data were obtained on over 1200 bases with anti-(-)-BPDE and 1000 bases with anti-(+)-BPDE. Guanines on average yielded more labile adducts than other bases, and the reactivities of guanines with both anti-(+)- and anti-(-)-BPDE isomers were found to be distinctly nonrandom with respect to DNA sequence. The most and least reactive guanines, defined in terms of the upper and lower 10 percentiles of reactivity, differed on average by a factor of 17. This range of guanine reactivities was correlated with distinct sequence preferences, which differed in part for the two isomers. The strongest determinant for preferred reaction of anti-(-)-BPDE to form a labile adduct at a guanine was the presence of a 3'-flanking guanine, but a thymine 5'-flanking a guanine also generally enhanced reactivity. The triplets containing central guanines most preferred by anti-(-)-BPDE were AGG, CGG, and TG(G greater than T greater than C,A). anti-(+)-BPDE also formed labile adducts preferentially at AGG and CGG triplets, but not at TGN triplets. Significant effects of next-nearest-neighbor bases on guanine reactivities were also noted.