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Results for “Triplet interruption”

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PubMed1995

Polymorphic CAC/T repetitive sequences in the pig genome 1.

Three genomic clones were isolated from a size-selected pig DNA library by hybridization with a DNA-fingerprint probe. Analysis at the sequence level revealed that all three clones contain interrupted stretches of triplet repeats mainly composed of CAC and CAT triplets. Evaluation of the corresponding loci for polymorphism by Southern blot hybridization showed considerable length variation. For two loci the polymorphism was also demonstrated by polymerase chain reaction (PCR) amplification. The PiGMaP reference pedigree was typed for all three loci.

Animals↗

PubMed2002

Premutation and intermediate-size FMR1 alleles in 10572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles.

We previously reported a 1:259 prevalence of female carriers of FMR1 premutation-size alleles (greater than 54 triplet repeats) in the general population. We now have screened 10 572 independent males from the same population for similar alleles using high-throughput Southern blotting. We identified 13 male carriers of an allele with more than 54 repeats. This corresponds to a prevalence of 1:813 males (95% confidence interval 1:527 to 1:1781). Haplotype analysis of four markers flanking the triplet array revealed that the prevalence of the major fragile X mutation-associated haplotype was increased among FMR1 alleles of 40-54 repeats. Although sequencing of highly unstable premutation alleles from fragile X families revealed only pure CGG tracts, this was not the case for alleles of similar size that were identified in males from the general population. Forty-eight out of forty-nine alleles of 40 or more triplets had one or two AGG interruptions. This observation, combined with the observation of the enrichment of major fragile X syndrome haplotypes in all alleles of this size, is evidence that the loss of an AGG interruption in the triplet repeat array is not necessary for expansion of normal alleles of 29-30 triplets to intermediate size. The loss of AGG interruptions thus appears to be a late event that leads to greatly increased instability and may be related to the haplotype background of specific FMR1 alleles.

Fragile X Messenger Ribonucleoprotein 1↗

PubMed1995

Atypical Gly-X-Y sequences surround interruptions in the repeating tripeptide pattern of basement membrane collagen.

The triple-helical domains of type IV collagen chains have more than 20 sites at which the repeating (Gly-X-Y)n pattern is interrupted. Analysis of alpha 1 (IV) and alpha 2 (IV) chains indicates the residues in the three Gly-X-Y triplets preceding or following interruptions differ statistically from the rest of the chain. Unusually high frequencies of charged residues are seen at a number of X and Y sites, with the charge density being particularly high C-terminal to the interruption site. Analyses were carried out on individual categories of interruptions, classified as insertions or deletions in the Y position. All of the residues in the X and Y positions of the triplets flanking insertion sites are atypical, with a high concentration of charged residues. Triplets flanking sites where there has been a deletion in the Y position show unusually high frequencies of charged residues at some sites, hydrophobic residues at other sites, and an invariant imino acid N-terminal to the interruption. The presence of atypical sequences surrounding interruptions could be important at a molecular level, related to triple-helix stability, or at a supramolecular level, related to the association of molecules to form networks in basement membranes.

Animals↗

PubMed1993

Characterization of an intronless collagen gene family in the marine sponge Microciona prolifera.

Two independent clones from the genomic DNA of a marine sponge Microciona prolifera were isolated by hybridization to the Caenorhabditis elegans Col-1 gene and one clone was obtained from genomic DNA by PCR. They contain open reading frames (MpCol1, MpCol2, MpCol3, MpCol4) capable of coding for a family of collagens different from those previously found in sponges. Southern blotting of genomic DNA suggested the presence of several other homologous genes. cDNA clones covering most of the triple-helical coding domain and the 3' untranslated region of MpCol1 were isolated by specific primers and reverse PCR. Two cDNA clones end in the middle of an AATAAA sequence 170 bp downstream from the translation stop codon of MpCol1. The putative NH2-terminal noncollagenous peptide is composed of only seven amino acid residues. The 1074-bp triple-helical coding region is not interrupted by intervening sequences. It codes for a polypeptide of 120 Gly-Xaa-Yaa triplets with only one short interruption near the COOH terminus. A putative N-glycosylation sequence (Asn-Gly-Ser), three Arg-Gly-Asp triplets known as cell recognition peptides, frequent Lys residues in the Yaa position (which are templates for hydroxylation), several Lys-Gly-Asn/Xaa-Arg peptides known as the lysyl oxidase recognition site, and long stretches without imino acids could be found within the triple-helical domain. The short COOH-terminal noncollagenous domain closely resembles that of nematode cuticular collagens and vertebrate nonfibrillar collagens. Our results strongly support the idea that the diversity of collagen genes and gene families found in higher organisms already existed in sponge.

Amino Acid Sequence↗

PubMed1994

Mutations in the alpha 2(IV) basement membrane collagen gene of Caenorhabditis elegans produce phenotypes of differing severities.

Type IV collagen forms a network that provides the major structural support of basement membranes. We have determined the nucleotide alterations and phenotypes of 17 mutant alleles of the Caenorhabditis elegans alpha 2(IV) collagen gene let-2. All 17 mutations are within the triple helical (Gly-X-Y) repeat domain of the molecule. Fifteen of the mutations are replacements of Gly-X-Y repeat glycines with aspartate, glutamate or arginine, and they cause a wide range of phenotypes. The mildest alleles are nearly wild-type at 15 and 20 degrees C but embryonic lethal at 25 degrees C, while the most severe allele is embryonic lethal at all three temperatures. Mutations resulting in severe phenotypes are generally located in areas of lower calculated thermal stability of the type IV collagen molecule. An alanine to threonine substitution at position X of a Gly-X-Y triplet immediately following an interruption results in a severe phenotype. This mutation is unusual because substitutions at positions X or Y have not generally been found to cause strong phenotypes in C. elegans or human collagens. An intron splice acceptor mutation causes a strict embryonic lethal phenotype, but does not completely abolish gene function. Pairs of independent mutations affect each of three glycines, indicating a non-random distribution of mutations in the molecule. It is suggested that this clustering results because many glycine substitutions may cause dominant lethal or sterile phenotypes.

Alleles↗

PubMed1994

Cryptic and polar variation of the fragile X repeat could result in predisposing normal alleles.

Fragile X syndrome results from the expansion of a CGG repeat that is normally interrupted by occasional AGG triplets. Linkage disequilibrium suggests that certain normal haplotypes may contribute unequally to the pool of fragile X chromosomes. Sequence analysis of normal alleles demonstrates haplotype-specific variation of the cryptic nature of the repeat. Variation occurs principally at the 3' end of the repeat, suggesting stability differences between the leading and lagging strands of DNA replication. Normal alleles with greater than 24 perfect 3' CGG repeats appear more frequently on haplotypes overrepresented among fragile X chromosomes. Such alleles are found in 2% of normal chromosomes and could comprise the ancestral allelic pool from which fragile X chromosomes are derived. These data also may imply that equilibrium between certain predisposed alleles and fragile X expansions has not yet been attained, indicating ongoing evolutionary change at this locus.

Alleles↗

PubMed2000

Interruption of the fragile X syndrome expanded sequence d(CGG)(n) by interspersed d(AGG) trinucleotides diminishes the formation and stability of d(CGG)(n) tetrahelical structures.

Fragile X syndrome is caused by expansion of a d(CGG) trinucleotide repeat sequence in the 5' untranslated region of the first exon of the FMR1 gene. Repeat expansion is thought to be instigated by formation of d(CGG)(n)secondary structures. Stable FMR1 d(CGG)(n)runs in normal individuals consist of 6-52 d(CGG) repeats that are interrupted every 9-11 triplets by a single d(AGG) trinucleotide. By contrast, individuals having fragile X syndrome premutation or full mutation present >54-200 or >200-2000 monotonous d(CGG) repeats, respectively. Here we show that the presence of interspersed d(AGG) triplets diminished in vitro formation of bimolecular tetrahelical structures of d(CGG)(18)oligomers. Tetraplex structures formed by d(CGG)(n)oligomers containing d(AGG) interspersions had lower thermal stability. In addition, tetraplex structures of d(CGG)(18)oligomers interspersed by d(AGG) triplets were unwound by human Werner syndrome DNA helicase at rates and to an extent that exceeded the unwinding of tetraplex form consisting of monotonous d(CGG)(18). Diminished formation and stability of tetraplex structures of d(AGG)-containing FMR1 d(CGG)(2-50)tracts might restrict their expansion in normal individuals.

Base Sequence↗

PubMed1984

Structure of human-basement-membrane (type IV) collagen. Complete amino-acid sequence of a 914-residue-long pepsin fragment from the alpha 1(IV) chain.

The complete amino acid sequence of the 914-residue-long pepsin fragment alpha 1 (IV)95 from the alpha 1 chain of human placental basement membrane (type IV) collagen is presented. This sequence contains 12 interruptions of the collagenous triplet sequence Gly-Xaa-Yaa which varied in length from 1 to 11 residues. The distribution of amino acids between the Xaa and Yaa position was similar to that found in interstitial collagens but the extent of proline and lysine hydroxylation differed. Computer comparisons of the alpha 1 (IV)95 sequence with those of the interstitial collagen chains did not reveal any homology, whereas a comparison with the partial sequences of mouse tumor and bovine lens capsule alpha 1 (IV) showed an approximately 85% identity. The unique sequence characteristics of type IV collagen are discussed in relation to its macromolecular structure and to the interstitial collagens.

Amino Acid Sequence↗

PubMed1997

DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines.

We have used DNase I footprinting to assess the formation of triple helices at 15mer oligopurine target sites which are interrupted by several (up to four) adjacent central pyrimidine residues. Third strand oligonucleotides were designed to generate complexes containing central (X.TA)nor (X.CG)n triplets (X = each base in turn) surrounded by C+.GC and T.AT triplets. It has previously been shown that G.TA and T.CG are the most stable triplets for recognition of single TA and CG interruptions. We show that these triplets are the most useful for recognizing consecutive pyrimidine interruptions and find that addition of each pyrimidine residue leads to a 30-fold decrease in third strand affinity. The addition of 10 microM naphthylquinoline triplex-binding ligand stabilizes each complex so that all the oligonucleotides produce footprints at similar concentrations (0.3 microM). Targets containing two pyrimidines are only bound by oligonucleotides generating (G.TA)2 and (T.CG)2 with a further 30-fold decrease in affinity. (G.TA)2 is slightly more stable than (T.CG)2. In the presence of the triplex-binding ligand the order of stability is (G.TA)2 > (C.TA)2 > (T.TA)2 > (A.TA)2 and (T.CG)2 > (C.CG)2 > (G.CG)2 = (A.CG)2. No oligonucleotide footprints are generated at target sites containing three consecutive pyrimidines, though addition of 10 microM triplex-binding ligand produces stable complexes with oligonucleotides generating (G.TA)3, (T.CG)3 and (C.CG)3, with a further 30-fold reduction in affinity. No footprints are generated at targets containing four Ts, though the ligand induces a weak interaction with the oligonucleotide generating (T.CG)4.

Base Sequence↗

PubMed2007

CAG and CTG repeat polymorphism in exons of human genes shows distinct features at the expandable loci.

Although the trinucleotide repeats are present in the exons of numerous human genes, the allele distribution is not well known, and the factors responsible for their intergenic and intragenic variability are not well understood. We have analyzed the length and sequence variation within the most commonly occurring CAG and CTG repeats in a large number of human genes selected to contain the longest reported repeat tracts. Our study revealed that in genes other than those implicated in the Triplet Repeat Expansion Diseases (TREDs), the very long and highly polymorphic repeats are rather infrequent. The length of pure repeat tract in the most frequent allele was found to correlate well with the rate of the repeat length polymorphism, and CAA triplets were shown to be the most frequent CAG repeat interruptions. As both the CAG and CAA triplets code for glutamine, our results may suggest that the selective pressure disfavors the long uninterrupted CAG repeats in genes and transcripts but not the long normal polyglutamine tracts in proteins. This may indicate that hairpin structures formed in ssDNA and RNA by long pure CAG repeats would be selected against as they may impede normal cellular processes.

3' Untranslated Regions↗

PubMed1997

The Friedreich ataxia GAA triplet repeat: premutation and normal alleles.

The most common mutation causing Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease, is the hyperexpansion of a polymorphic GAA triplet repeat localized within an Alu sequence (GAA-Alu) in the first intron of the frataxin (X25) gene. GAA-Alu belongs to the AluSx subfamily and contains several polymorphisms in strong linkage disequilibrium either with a subgroup of normal alleles, or with hyperexpanded FRDA-associated alleles. GAA repeat sizes in 300 normal chromosomes (97 from carriers and 203 from controls) were distributed in two separate groups: 83% of them contained between six and 10 triplets (small normal alleles), while the remaining 17% had more than 12 triplets, up to 36 (large normal alleles). Sequence analysis showed that no normal, stable allele contained more than 27 uninterrupted GAA triplets. All longer normal alleles were interrupted by a hexanucleotide repeat (GAGGAA). An allele containing an uninterrupted run of 34 GAA triplets was stably transmitted in four instances, but in one case underwent hyperexpansion to 650 triplets. Overall, our results suggest that the FRDA-associated expanded GAA repeats originate from normal alleles by recurrent expansions of alleles at risk.

Alleles↗

PubMed2005

Stable recognition of TA interruptions by triplex forming oligonucleotides containing a novel nucleoside.

We have prepared the 2'-aminoethoxy derivative of the S nucleoside ((2AE)S) and incorporated it into triplex-forming oligonucleotides for recognition of TA interruptions within a target oligopurine tract. Fluorescence melting, UV melting, and DNase I footprinting experiments show that (2AE)S has greater affinity than G or S for a single TA interruption. Stable triplexes are formed at pH 6.0 at an 18-mer target site containing two TA interruptions, even though this contains eight C(+).GC triplets. Although (2AE)S and S produce stable triplexes at TA interruptions, they also interact with other base pairs, in particular, CG, although the selectivity for TA improves with increased pH.( 2AE)S is the best nucleoside described so far for recognition of TA within a triple-helix target.

Base Sequence↗

PubMed1991

cDNA analysis predicts a cornea-specific collagen.

In the development of chicken corneal stroma, two or more collagens often interact, either as constituents of a single heterotypic fibril or as components of the fibril surface. The latter, fibril-associated collagens, may facilitate interactions between fibrils and the surrounding extracellular matrix or between fibrils themselves. In an effort to isolate putative nonfibrillar collagens that may have such a function, we screened a 13-day embryonic cornea cDNA library under reduced stringency conditions, using a cDNA probe for a collagenous domain of type XII collagen. We isolated a 4.2-kilobase (kb) cDNA that predicts a "collagenous" protein that has three unusual, if not unique, features. (i) The putative polypeptide encoded by this cDNA has a structural arrangement in which numerous stretches of Gly-Xaa-Yaa triplets, typical of collagens, are interrupted by non-Gly-Xaa-Yaa regions. One of the potential triple-helical domains is 246 amino acids long, but most are much smaller, consisting of 15-36 amino acids. Many are very rich in the helix-stabilizing imino acid proline. (ii) Northern blot analyses demonstrated strong cDNA hybridization to a 6.8-kb mRNA whose expression is restricted to the cornea. No hybridization was observed to mRNAs from the nine other tissues used in these analyses, even with extended exposure of the film. (iii) The cDNA contains a short (less than or equal to 425-base-pair) sequence in the 3' untranslated region of the 6.8-kb mRNA that hybridizes to a 7.8-kb mRNA that has a wide tissue distribution.

Amino Acid Sequence↗

PubMed1996

Alternative splicing of exon 12 of the COL2A1 gene interrupts the triple helix of type-II collagen in the Kniest form of spondyloepiphyseal dysplasia.

An autosomal dominant mutation in the COL2A1 gene was identified in a child with the Kniest form of spondyloepiphyseal dysplasia. A C to T transition at nucleotide 35 of exon 12 changed the codon GCG for alanine 102 of the triple helical domain of alpha 1(II) chains of type-II collagen to GTG for valine. The transition also introduced a GT dinucleotide into exon 12. Analysis of cDNA prepared from Kniest cartilage showed that in vivo the transition resulted in an alternatively spliced mRNA that lacked the 213' nucleotides from exon 12. The cartilage cDNA contained approximately equal amounts of normal cDNA and shortened mutant cDNA. The deletion of 21 nucleotides from the mutant cDNA maintained the translational reading frame but resulted in the loss of alanine 102 to lysine 108, which interrupted the repetitive glycine-X-Y triplet sequence required for formation of the triple helix. Type-II collagen molecules containing one or more mutant chains were expected, therefore, to contain interrupted triple helices with a short amino-terminal helical domain A and a large carboxy-terminal helical domain B. Kniest cartilage contained a reduced amount of pepsin-solubilized type-II collagen that consisted of overmodified alpha 1(II) chains. Peptide mapping showed that the overmodifications extended to the carboxy terminus of the alpha 1(II) chains. Pepsin digestion also yielded shortened alpha 1(II) chains corresponding to helical domain B. Kniest chondrocytes cultured in alginate beads produced type-II collagen that was not stably incorporated into the pericellular matrix. This study highlights the importance of dominant negative mutations of COL2A1 in producing Kniest dysplasia.

Alleles↗

PubMed2001

CAG repeat instability at SCA2 locus: anchoring CAA interruptions and linked single nucleotide polymorphisms.

Spinocerebellar ataxia 2 (SCA2) is an autosomal dominant neurodegenerative disorder that results from the expansion of a cryptic CAG repeat within the exon 1 of the SCA2 gene. The CAG repeat in normal individuals varies in length from 14 to 31 repeats and is frequently interrupted by one or more CAA triplets, whereas the expanded alleles contain a pure uninterrupted stretch of 34 to 59 CAG repeats. We have previously reported the presence of a limited pool of 'ancestral' or 'at risk' haplotypes for the expanded SCA2 alleles in the Indian population. We now report the identification of two novel single nucleotide polymorphisms (SNPs) in exon 1 of the SCA2 gene and their characterization in 215 normal and 64 expanded chromosomes. The two biallelic SNPs distinguished two haplotypes, GT and CC, each of which formed a predominant haplotype associated with normal and expanded SCA2 alleles. All the expanded alleles segregated with CC haplotype, which otherwise was associated with only 29.3% of the normal chromosomes. CAA interspersion analysis revealed that majority of the normal alleles with CC haplotype were either pure or lacked the most proximal 5' CAA interruption. The repeat length variation at SCA2 locus also appeared to be polar with changes occurring mostly at the 5' end of the repeat. Our results demonstrate that CAA interruptions play an important role in conferring stability to SCA2 repeat and their absence predisposes alleles towards instability and pathological expansion. Our study also provides new haplotypes associated with SCA2 that should prove useful in further understanding the mutational history and mechanism of repeat instability at the SCA2 locus.

Alleles↗

PubMed2004

Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia.

Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA*TTC)n sequences (9-105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8-107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.

DNA↗

PubMed1995

Non-sense and missense mutations in the structural genes of complement component C1q A and C chains are linked with two different types of complete selective C1q deficiencies.

To shed light on the molecular basis of two different types of complete C1q deficiency, we performed extensive Southern blot analysis and sequenced all exons of the genes coding for the A, B, and C chains of C1q on two groups of deficient patients. In one family with three cases of complete C1q deficiency we found a point mutation in the codon for glutamine (CAG) at position 186 of the A chain that led to a termination codon (TAG). No gene products of any of the three genes were found in the patients' sera, indicating that full length polypeptides of the A, B, and C chains are required to form and secrete functional C1q. A second point mutation was found in a patient with a complete functional C1q defect. The abnormal C1q molecule had been shown to have a low m.w. of approximately 150,000 and a sedimentation coefficient of below 7S. The mutation occurred in the codon for glycine in position 6 of the C chain where a single base exchange (G-->A transition) has led to an arginine residue. In the parents and son of patient G we could demonstrate the heterozygous state of the mutation by the occurrence of both bases in question, G and A. The interruption of the collagen-like triplet motif Gly-X-Y and, even more likely, the introduction of a large positively charged side chain at the N-terminus of the polypeptide may inhibit the assembly of three structural subunits consisting of two A-B dimers and one C-C dimer to form the 11S C1q macromolecule.

Adult↗

PubMed1995

Evolution of the cryptic FMR1 CGG repeat.

We have sequenced the 5' untranslated region of the orthologous FMR1 gene from 44 species of mammals. The CGG repeat is present in each species, suggesting conservation of the repeat over 150 million years of mammalian radiation. Most mammals possess small contiguous repeats (mean number of repeats = 8.0 +/- 0.8), but in primates, the repeats are larger (mean = 20.0 +/- 2.3) and more highly interrupted. Parsimony analysis predicts that enlargement of the FMR1 CGG repeat beyond 20 triplets has occurred in three different primate lineages. In man and gorilla, AGG interruptions occur with higher-order periodicity, suggesting that historical enlargement has involved incremental and vectorial addition of larger arrays demarcated by an interruption. Our data suggest that replication slippage and unequal crossing over have been operative during the evolution of this repeat.

Animals↗