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PubMed1993

Characterization of collagen-like peptides containing interruptions in the repeating Gly-X-Y sequence.

Glycine is found as every third residue along the entire length of triple helices in fibrillar collagens, but the triple-helix regions of nonfibrillar collagens and other proteins usually contain one or more interruptions in this repeating pattern. A set of four peptides was designed to model the effect of interruptions in the (Gly-X-Y)n repeating pattern on triple-helix formation, stability, and folding. Into the middle of the stable triple-helical peptide (Pro-Hyp-Gly)10, an interruption was introduced representing one of the four possible categories: a glycine deletion, a deletion of a hydroxyproline (Y position), an alanine insertion, or a glycine to alanine substitution. As shown by sedimentation equilibrium, NMR, and CD studies, the introduction of an interruption still allowed formation of trimers in solution, but with marked decrease in stability. The degree of destabilization and the thermodynamic basis for the loss of stability depended on the type of interruption. The glycine substitution and alanine insertion were the least disruptive, followed by the hydroxyproline deletion, with the glycine deletion being the most destabilizing. Our results suggest that the breaks in these peptides affect both the triple-helical conformation and the monomer conformation. These studies provide a basis for considering the structural and functional consequences of different kinds of interruptions in collagen.

Amino Acid Sequence↗
PubMed2000

Isolation, characterization, and inheritance of microsatellite loci in alpine larch and western larch.

Microsatellite loci or simple sequence repeat loci (SSRs) were isolated in alpine larch (Larix lyallii Parl.) and western larch (Larix occidentalis Nutt.). In total, 14 SSR loci were characterized; two [(TCT)4, A7] came from published Larix DNA sequence data, one (CA)17 was obtained from a partial non-enriched alpine larch total genomic DNA library, and the remaining 11 loci were obtained from larch genomic DNAs enriched for (CA)n repeats. The SSR regions in these clones could be divided into three categories: perfect repeat sequences without interruption, imperfect repeat sequences with interruption(s), and compound repeat sequences with adjacent tandem simple dinucleotides. Eight of the 14 loci analyzed were found to be polymorphic and useful markers after silver-staining polyacrylamide gel electrophoresis. In addition, several SSR primers developed for alpine larch were able to successfully amplify polymorphic loci in its related species, western larch, and among other closely related taxa within the Larix genus. The inheritance of microsatellite loci was verified by analysis of haploid megagametophyte and diploid embryo tissues of progeny obtained from controlled crosses between western larch and alpine larch. All microsatellite loci analyzed had alleles that segregated according to expected Mendelian frequencies. Two species-specific markers (UAKLly10a and UAKLla1) allow easy and rapid identification of specific genetic entry of alpine larch and western larch at any stage in the sporophyte phase of the life cycle. Therefore, these markers are efficient in identifying the parental species and to validate controlled crosses between these two closely related species. These results are important in tree improvement programs of alpine larch and western larch aimed at producing genetically improved hybrid stock for reforestation in Western Canada and U.S.A.

Cloning, Molecular↗
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↗
PubMed2000

Microsatellite loci of the cattle tick Boophilus microplus (Acari: Ixodidae).

This brief communication reports the identification of microsatellite loci in the economically important tick species Boophilus microplus. The data are potentially useful in distinguishing different strains of B. microplus. Eight polymorphic loci were isolated in larvae, male and female adults analysed individually from 12 field isolates and laboratory strains from Australia (n = 8), Brazil, Mexico, Papua New Guinea and Zimbabwe. Nucleotide sequencing of alleles at these microsatellite loci revealed that non-repeat bases interrupted dinucleotide and tetranucleotide repeats in some loci. Loci with non-repeat bases interrupting them were shorter compared with loci that were not interrupted. Thus the presence of non-repeat bases in a repeated sequence seems to constrain the evolution of additional repeats by slip-strand misparing at these loci.

Animals↗
PubMed1994

The exon-intron organization of the human X-linked gene (FLN1) encoding actin-binding protein 280.

We have determined the exon-intron organization of the human X-linked gene (FLN1) encoding actin-binding protein 280 (filamin), a ubiquitous protein that plays an important role in the mechanochemical activities of cells through its association with actin filaments and membrane components. The gene is composed of 47 exons spanning approximately 26 kb. The first and part of the second exon are untranslated. The actin-binding domain at the N-terminus is encoded by exons 2 to 5. The 96-amino-acid repeats corresponding to the elongated rod backbone of the protein are encoded by the remaining 42 exons: size, location, and boundaries of the exons cannot be easily correlated with the repeated structure, while sequences interrupting the repeats (the two hinge segments preceding repeats 16 and 24 and the 8-amino-acid (aa) segment interrupting the 15th repeat) were encoded by separate exons, suggesting that they may be recent additions to the X-linked protein. The 8-aa segment is encoded by exon 29, which is alternatively spliced.

Amino Acid Sequence↗
PubMed2001

Sequence variation in humans and other primates at six short tandem repeat loci used in forensic identity testing.

A large number of alleles from the six different short tandem repeat (STR) loci FGA, D3S1358, vWA, CSF1PO, TPOX and TH01, used in human identity testing were sequenced to provide support for the robustness of fluorescent STR DNA typing by allele size. Sequence information for some of these loci (FGA, vWA, TH01) is an extension of published work, whereas no extensive sequence information is available with respect to the D3S1358, CSF1PO, and TPOX loci. Sequencing of alleles at each locus has provided quantitative data with respect to the true nucleotide length of common alleles, and of alleles that vary in length from the common alleles. All alleles that were identified as "off-ladder" alleles through fluorescent typing at these STR loci have proven to be true length variant alleles. Sequencing at the D3S1358 and CSF1PO loci allowed for the establishment of a common nomenclature for these loci. A correlation between percent stutter and the length of the core tandem repeat is demonstrated at the FGA locus. Alleles in which the core tandem repeat is interrupted by a repeat unit of different sequence have a reduced percent stutter. DNA samples from three non-human primates (chimpanzee, orangutan, and gorilla) were compared to the human sequences, and shown to differ markedly across loci with respect to their homology. The effects of primer binding site mutations on the amplification efficiency at a particular locus, and methods used to interpret amplification imbalance of heterozygous alleles at a locus is also addressed.

Animals↗
PubMed2024

Sequencing the orthologs of human autosomal forensic short tandem repeats provides individual- and species-level identification in African great apes.

BACKGROUND: Great apes are a global conservation concern, with anthropogenic pressures threatening their survival. Genetic analysis can be used to assess the effects of reduced population sizes and the effectiveness of conservation measures. In humans, autosomal short tandem repeats (aSTRs) are widely used in population genetics and for forensic individual identification and kinship testing. Traditionally, genotyping is length-based via capillary electrophoresis (CE), but there is an increasing move to direct analysis by massively parallel sequencing (MPS). An example is the ForenSeq DNA Signature Prep Kit, which amplifies multiple loci including 27 aSTRs, prior to sequencing via Illumina technology. Here we assess the applicability of this human-based kit in African great apes. We ask whether cross-species genotyping of the orthologs of these loci can provide both individual and (sub)species identification. RESULTS: The ForenSeq kit was used to amplify and sequence aSTRs in 52 individuals (14 chimpanzees; 4 bonobos; 16 western lowland, 6 eastern lowland, and 12 mountain gorillas). The orthologs of 24/27 human aSTRs amplified across species, and a core set of thirteen loci could be genotyped in all individuals. Genotypes were individually and (sub)species identifying. Both allelic diversity and the power to discriminate (sub)species were greater when considering STR sequences rather than allele lengths. Comparing human and African great-ape STR sequences with an orangutan outgroup showed general conservation of repeat types and allele size ranges. Variation in repeat array structures and a weak relationship with the known phylogeny suggests stochastic origins of mutations giving rise to diverse imperfect repeat arrays. Interruptions within long repeat arrays in African great apes do not appear to reduce allelic diversity. CONCLUSIONS: Orthologs of most human aSTRs in the ForenSeq DNA Signature Prep Kit can be analysed in African great apes. Primer redesign would reduce observed variability in amplification across some loci. MPS of the orthologs of human loci provides better resolution for both individual and (sub)species identification in great apes than standard CE-based approaches, and has the further advantage that there is no need to limit the number and size ranges of analysed loci.

Animals↗
PubMed2006

Surfactant protein A without the interruption of Gly-X-Y repeats loses a kink of oligomeric structure and exhibits impaired phospholipid liposome aggregation ability.

Pulmonary surfactant protein A (SP-A) belongs to the collectin subgroup of the C-type lectin superfamily. SP-A oligomerizes as an octadecamer, which forms a flower bouquet-like structure. A collagen-like domain of human SP-A consists of 23 Gly-X-Y repeats with an interruption near the midpoint of this domain. This interruption causes a kink, but its role remains unknown. To define the importance of the kink region of SP-A, two mutated proteins were constructed to disrupt the interruption of Gly-X-Y repeats: SP-ADEL, which lacks the Pro47-Cys48-Pro49-Pro50 sequence at the interruption, and SP-AINS, in which two glycines were introduced to insert Gly-X-Y repeats (Gly-Pro47-Cys48-Gly-Pro49-Pro50). Electron microscopy using rotary shadowing revealed that both mutants form octadecamers that lack a bend in the collagenous domain. Electrophoretic analysis under nondenaturing conditions and gel filtration chromatography demonstrated that SP-AINS consisted of a large assembly of oligomers whereas SP-ADEL formed mainly octadecamers. Both SP-ADEL and SP-AINS mutants as well as wild-type SP-A bound to liposomes containing dipalmitoylphosphatidylcholine and galactosylceramide at equivalent levels, but the abilities of the mutants to induce phospholipid liposome aggregation were significantly less developed than that of the wild type. The mutants SP-ADEL and SP-AINS augmented liposome uptake by alveolar type II cells and inhibited secretion of phospholipids from type II cells at a level comparable to that of wild-type SP-A. These results indicate that the interruption of Gly-X-Y repeats in the SP-A molecule is critical for the formation of a flower bouquet-like octadecamer and contributes to SP-A's capacity to aggregate phospholipid liposomes.

Animals↗
PubMed2026

Dissecting the relationship between haplotypes around ATXN2 CAG repeats and the number of CAA interruptions by long-read sequencing.

BACKGROUND: CAG repeat expansions in ATXN2 are implicated as risk factors for several neurological diseases, including spinocerebellar ataxia type 2 (SCA2) when >=33 CAG repeats are present, and amyotrophic lateral sclerosis (ALS) when 27-33 CAG repeats are present. However, how haplotypes around the repeats and CAA interruptions within the repeats are associated with disease phenotypes remains poorly understood. Previous studies on haplotypes around ATXN2 were limited to SNPs very close to the repeats (<5kb) or were based on statistical inference only. METHODS: Here, we used long-read sequencing on the Oxford Nanopore Technologies (ONT) platform to simultaneously infer haplotypes around ATXN2, the number of CAG repeats, and the number of CAA interruptions, along with NYGC ALS Consortium NGS dataset. We further sequenced 41 individuals (EUR = 39) with neurological diseases with intermediate repeats by ONT. RESULTS: We found that haplotypes around ATXN2 and the number of interruptions show ethnicity-specific and ALS-specific distribution. Three CAA interruptions are present at low prevalence (~1%) in control populations in multiple ancestry groups, but high prevalence (~55%) in ALS individuals with intermediate repeats. Furthermore, we examined 159 individuals with ALS (~90% European ancestry) with intermediate ATXN2 repeats and found a unique haplotype in ALS individuals with three CAA interruptions, which can be tagged by an SNV, rs148019457. We also validated that the rs148019457-G allele is only present in haplotypes with three CAA interruptions. CONCLUSIONS: In summary, our study shows that 3 CAA interruptions are rarely seen in healthy controls but are common in those with expanded ATXN2 CAG repeats who have neurological disorders, and that rs148019457 tags a specific haplotype with 3 CAA interruptions within expanded ATXN2 CAG repeats in individuals of European ancestry. These results have implications for the development of precision genomic medicine for neurological disorders, and the tag SNP may help identify those with interruptions from existing population genotyping data.

ATXN2↗
PubMed1991

Nucleotide and deduced amino acid sequence of human threonyl-tRNA synthetase reveals extensive homology to the Escherichia coli and yeast enzymes.

The nucleotide sequence of a cDNA coding human threonyl-tRNA synthetase has been determined. The predicted protein sequence is highly homologous to that of the yeast cytoplasmic, yeast mitochondria and Escherichia coli threonyl-tRNA synthetases. In particular, the three structural motifs recently shown to be common to class II aminoacyl-tRNA synthetases are present in the threonyl-tRNA synthetases from all sources. Primer extension and S1 nuclease analyses indicate that transcription initiates approximately 220-230 nucleotides upstream of the putative initiator methionine codon. This region contains a 10-nucleotide interrupted inverted repeat flanked by a 13-nucleotide interrupted direct repeat.

Amino Acid Sequence↗
PubMed1998

Interruptions in the triplet repeats of SCA1 and FRAXA reduce the propensity and complexity of slipped strand DNA (S-DNA) formation.

Models for the disease-associated expansion of trinucleotide repeats involve the participation of alternative DNA structures during replication, repair, or recombination. CAT or AGG interruptions within the (CAG)n or (CGG)n repeats of SCA1 or FRAXA, respectively, confer increased genetic stability to the repeats. In this study, we report the formation of slipped strand structures (S-DNA) using genomic sequences containing pure and interrupted SCA1 and FRAXA repeats having lengths above and below the genetic stability thresholds. S-DNA forms within the repeats during annealing of complementary strands containing equal lengths of repeats. Increased lengths of pure repeats led to an increased propensity for S-DNA formation. CAT or AGG interruptions have both quantitative and qualitative effects upon S-DNA formation: they decrease the total amount of slipped structures as well as limit the specific isomers formed. This demonstrates a unifying inhibitory effect of interruptions in both (CAG)n and (CGG)n tracts. We also present transmission stability data for SCA1 and FRAXA alleles spanning the thresholds and compare these with the ability to form slipped structures. The effect of both the length and purity of the repeat tract on the propensity of slipped structure formation correlates with their effect on genetic instability and disease, suggesting that S-DNA structures may be models for mutagenic intermediates in instability.

Ataxin-1↗
PubMed1997

Molecular and clinical correlations in spinocerebellar ataxia 2: a study of 32 families.

Spinocerebellar ataxia 2 (SCA2) is caused by the expansion of an unstable CAG repeat encoding a polyglutamine tract. One hundred and eighty four index patients with autosomal dominant cerebellar ataxia type I were screened for this mutation. We found expansion in 109 patients from 30 families of different geographical origins (15%) and in two isolated cases with no known family histories (2%). The SCA2 chromosomes contained from 34 to 57 repeats and consisted of a pure stretch of CAG, whereas all tested normal chromosomes (14-31 repeats), except one with 14 repeats, were interrupted by 1-3 repeats of CAA. As in other diseases caused by unstable mutations, a strong negative correlation was observed between the age at onset and the size of the CAG repeat (r = -0.81). The frequency of several clinical signs such as myoclonus, dystonia and myokymia increased with the number of CAG repeats whereas the frequency of others was related to disease duration. The CAG repeat was highly unstable during transmission with variations ranging from -8 to +12, and a mean increase of +2.2, but there was no significant difference according to the parental sex. This instability was confirmed by the high degree of gonadal mosaicism observed in sperm DNA of one patient.

Adolescent↗
PubMed2005

Insights into the mutational history and prevalence of SCA1 in the Indian population through anchored polymorphisms.

There is a wide variation in prevalence of spinocerebellar ataxia type 1 (SCA1) in different populations. In the present study, we observed SCA1 in approximately 22% (37/167 families) of the autosomal dominant cerebellar ataxias (ADCAs) in the Indian population. We investigated the role of various genetic factors like repeat length, interruption pattern and chromosomal background in predisposing the repeats to instability in these families. We analyzed 12 markers (9 SNPs and 3 microsatellite markers) and found 3 of them, spanning a region of approximately 65 kbp to be linked with the disease locus in the Indian population. The haplotype C-4-C defined by rs1476464 (SNP9)-D6S288-rs2075974 (SNP1), which was extremely rare in nonaffected chromosomes (approximately 3%), was observed to be significantly (P<0.0000) associated with the expanded chromosomes in approximately 44% of SCA1 families. This haplotype was found in all nonhuman primates. SNP1 (C/T), which showed a skewed allelic distribution between large (LN > 30 repeats) and small normal (SN <or= 30 repeats) alleles (P<0.0000) had similar allelic distribution (P=0.3477) in LN and expanded alleles. Our study suggested that LN and expanded chromosomes linked with the ancestral C allele of SNP1 might have originated simultaneously during evolution by the lengthening of repeats. The LN alleles might have accumulated repeat stabilizing non-CAG interruptions during this process. Similar proportions of T allele in SN with single interruptions, LN and expanded chromosomes lend credence to the origin of expanded alleles from singly-interrupted chromosomes. Our analyses using markers linked (anchoring) to SCA1 suggest that prevalence of SCA1 is correlated to both repeat length and number of interruptions in the Indian population. The spectrum of these alleles also points toward the antiquity of SCA1 mutation in the Indian population.

Alleles↗
PubMed1983

Members of the KpnI family of long interspersed repeated sequences join and interrupt alpha-satellite in the monkey genome.

Three different members of a family (KpnI-family) of interspersed repeated DNA sequences were found linked to alpha-satellite sequences in cloned segments of the African green monkey genome. In two of these segments the KpnI-family member is over 6 kbp in length and one of them is flanked by alpha-satellite on both sides indicating that it was inserted into a satellite array. Hybridization of subcloned portions of the family members to restriction endonuclease digests of monkey and human DNA and to a genomic library of African green monkey DNA indicate that 1) family members are interspersed in both the monkey and human genomes, 2) some family members may include sequences in addition to those in the three characterized here, 3) some family members may contain only parts of the sequences characterized here and 4) while the overall organization of the family is similar in the human and monkey genome the majority of the family members in each of the two genomes are distinctly identified by the variant position of certain restriction endonuclease sites. This last observation suggests that within each genome there is a tendency to maintain particular versions of the sequence. Observations 2) and 3) suggest that the KpnI family is complex and includes a variety of subfamilies.

Animals↗
PubMed1998

Anthranilate N-hydroxycinnamoyl/benzoyltransferase gene from carnation: rapid elicitation of transcription and promoter analysis.

Hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) catalyses the committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.). Three HCBT cDNAs were cloned previously from suspension-cultured carnation cells that had been induced with fungal elicitor. A rapid, transient induction of hcbt transcripts upon elicitation, reaching maximal abundances within about 0.5 h and returning to basal levels within 4 h, suggested the involvement of unusual cis elements. A DNA fragment of 3.8 kb, spanning the hcbt2 gene with the 5'-flanking region of roughly 1.8 kb, was cloned from carnation plants. The gene encodes one long open reading frame lacking introns. The DNA sequence revealed a conserved TATA box, three elicitor response elements (EREs) and a 9 bp direct repeat as well as an interrupted direct repeat of 11 bp in the TATA distal region. EMSA revealed the binding of elicitor-inducible nuclear factors to the promoter region from -377 to -326 spanning two of the EREs, and their functional relevance was confirmed by transient expression assays of hcbt2 promoter-GUS reporter gene constructs in parsley protoplasts. Furthermore, an oligo(A) segment was present immediately preceding the start of translation (+140 to +150). Transient expression analysis demonstrated that the sequence upstream to -1157 at least is required in context with the 5'-UTR, particularly including the poly(A) segment, for strong expression and full elicitor induction of the hcbt2 gene. The results suggested that several sequence motifs scattered over a wide range of the 5'-flanking region and into the exonic sequence are responsible for the full elicitor regulation of the hcbt2 gene.

Acyltransferases↗
PubMed1998

Repetitive DNA sequences in the common vole: cloning, characterization and chromosome localization of two novel complex repeats MS3 and MS4 from the genome of the East European vole Microtus rossiaemeridionalis.

We have characterized two novel, complex, heterochromatic repeat sequences, MS3 and MS4, isolated from Microtus rossiaemeridionalis genomic DNA. Sequence analysis indicates that both repeats consist of unique sequences interrupted by repeat elements of different origin and can be classified as long complex repeat units (LCRUs). A unique feature of both repeat units is the presence of short interspersed repeat elements (SINEs), which are usually characteristic of the euchromatic part of the genome. Comparative analysis revealed no significant stretches of homology in the nucleotide sequences between the two repeats, suggesting that the repeats originated independently during the course of vole genome evolution. Fluorescence in situ hybridization analysis demonstrates that MS3 and MS4 occupy distinct domains in the heterochromatic regions of the sex chromosomes in M. transcaspicus and M. arvalis but collocalize in M. rossiaemeridionalis and M. kirgisorum heterochromatic blocks. The localization pattern of the repeats on the vole chromosomes confirms the independent origin of the two repeats and suggests that expansion of the heterochromatic blocks has occurred subsequent to speciation.

Animals↗