DNA fingerprinting using non-radioactive oligonucleotide probes specific for simple repeats.
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Biotin↗
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Results for “simple sequence repeat”
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Functional roles for the TATA promoter and enhancers in basal and Tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat.
We have analyzed the contributory role of the human immunodeficiency virus type 1 (HIV-1) promoter and enhancers in basal and Tat-induced transcription. We found that a minimal promoter competent for basal expression is contained within sequences spanning nucleotides -43 to +80. Basal expression from this HIV-1 promoter was boosted more by the additional presence of the NF-kappa B elements than by the Sp1 elements. The minimal long terminal repeat promoter (-43 to +80), while having an intact TAR sequence, was not Tat inducible. However, the simple addition of short synthetic enhancer motifs (AP1, Oct, Sp1, and NF-kappa B) conferred Tat responsiveness. This ability to respond to Tat was in part dependent on the presence of the HIV-1 promoter. Changing the HIV-1 TATA to other eucaryotic TATA or non-TATA initiators minimally affected basal expression but altered Tat inducibility. Our findings suggest a specific context of functional promoter and enhancer elements that is optimal for Tat trans activation of the HIV-1 long terminal repeat. Our results do not allow conclusions about whether Tat acts at the level of initiation or at the level of elongation to be drawn.
Base Sequence↗
An oligo-screening strategy to fill gaps found during shotgun sequencing projects.
During the course of projects to sequence human and nematode cosmids we encountered great difficulties generating contiguous sequence in regions with repetitive DNA (Alu repeats in humans and tandem or inverted repeats in the nematode). We have developed a simple and efficient strategy to fill gaps. By screening M13, plasmid or phagemid libraries with oligonucleotides flanking the gap, clones are identified that contiguate the cosmid sequence. Our method has been integrated into the GAP4 sequence assembly program. The strategy reduces both time and costs in large scale sequencing projects.
Animals↗
Insulin and epidermal growth factor receptors contain the cysteine repeat motif found in the tumor necrosis factor receptor.
The insulin receptor (INSR) and epidermal growth factor receptor (EGFR) are representatives of two structurally related subfamilies of tyrosine kinase receptors. Using the Wisconsin GCG sequence analysis programs, we have demonstrated that the cysteine-rich regions of INSR and EGFR conform to the structural motif found in the tumor necrosis factor receptor (TNFR) family. The study also revealed that these regions were not composed of simple repeats of eight cysteine residues as previously proposed and that the second Cys-rich region of EGFR contained one fewer TNFR repeat than the first. The sequence alignments identified two cysteine residues in INSR that could be responsible for the additional disulfide bonds known to be involved in dimer formation. The published data on the alignments for the fibronectin type III repeat region of the INSR together with previous cysteine mutagenesis studies indicated that there were two disulfide bonds linking the alpha and beta chains of the INSR, but only one alpha-beta linkage in the insulin-like growth factor 1 receptor (IG1R). Database searches and sequence alignments showed that the TNFR motif is also found in the cysteine-rich repeats of laminins and the noncatalytic domains of furin-like proteases. If the starting position of the repeat is altered the characteristic laminin repeat of eight cysteine residues can be shown to consist of a TNFR-like motif fused to the last half of an EGF-like repeat. The overlapping regions of these two motifs are known to have identical disulfide bonding patterns and similar protein folds.
Amino Acid Sequence↗
Poly(A) tailing of ancient DNA: a method for reproducible microsatellite genotyping.
Microsatellites could be of great potential use in the analysis of ancient remains, but so far such analyses have failed to be reproducible mainly because of the high degree of ancient DNA (aDNA) degradation. During PCR, annealing of the primers to the complementary sequences of microsatellites occurs together with cross-annealing of partially degraded repeated sequences. This could create chimeric alleles that do not correspond to the authentic ones. Here we report a simple method for processing aDNA fragments prior to PCR that greatly reduces the production of chimeric alleles. This approach eliminates aDNA molecules broken within the repeats as targets for Taq polymerase by adding poly(A) tails at the 3(') ends of the DNA fragments, which disrupts the homology in the region and thus prevents annealing out of register. We have analyzed one dinucleotide- (D6S337) and two trinucleotide-containing loci (IT15 and SCA1) using poly(A)-tailed and the same untreated aDNA as template. aDNAs were isolated from 28 human remains, 600 and 7000 years of age. In repeated experiments with untreated aDNAs we obtained three to five times more alleles compared to poly(A)-tailed aDNAs. According to our results, modification of aDNA by poly(A) tailing is an efficient pretreatment for accurate genotyping.
Animals↗
Simple repetitive sequences in the genome: structure and functional significance.
The current explosion of DNA sequence information has generated increasing evidence for the claim that noncoding repetitive DNA sequences present within and around different genes could play an important role in genetic control processes, although the precise role and mechanism by which these sequences function are poorly understood. Several of the simple repetitive sequences which occur in a large number of loci throughout the human and other eukaryotic genomes satisfy the sequence criteria for forming non-B DNA structures in vitro. We have summarized some of the features of three different types of simple repeats that highlight the importance of repetitive DNA in the control of gene expression and chromatin organization. (i) (TG/CA)n repeats are widespread and conserved in many loci. These sequences are associated with nucleosomes of varying linker length and may play a role in chromatin organization. These Z-potential sequences can help absorb superhelical stress during transcription and aid in recombination. (ii) Human telomeric repeat (TTAGGG)n adopts a novel quadruplex structure and exhibits unusual chromatin organization. This unusual structural motif could explain chromosome pairing and stability. (iii) Intragenic amplification of (CTG)n/(CAG)n trinucleotide repeat, which is now known to be associated with several genetic disorders, could down-regulate gene expression in vivo. The overall implications of these findings vis-à-vis repetitive sequences in the genome are summarized.
Animals↗
Cointegrase, a naturally occurring, truncated form of IS21 transposase, catalyzes replicon fusion rather than simple insertion of IS21.
The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon. IS21 spontaneously forms tandem repeats designated (IS21)2. Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism. Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase. In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA. The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions. Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene. Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2. By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements. Transposase had intermediate, uniform activity in both pathways. The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase. Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood.
Base Sequence↗
The role of introns in repeat protein gene formation.
Genes composed of tandem repetitive sequence motifs are abundant in nature and are enriched in eukaryotes. To investigate repeat protein gene formation mechanisms, we have conducted a large-scale analysis of their introns and exons. We find that a wide variety of repeat motifs exhibit a striking conservation of intron position and phase, and are composed of exons that encode one or two complete repeats. These results suggest a simple model of repeat protein gene formation from local duplications. This model is corroborated by amino acid sequence similarity patterns among neighboring repeats from various repeat protein genes. The distribution of one- and two-repeat exons indicates that intron-facilitated repeat motif duplication, in which the start and end points of duplication are located in consecutive intronic regions, significantly exceeds intron-independent duplication. These results suggest that introns have contributed to the greater abundance of repeat protein genes in eukaryotic versus prokaryotic organisms, a conclusion that is supported by taxonomic analysis.
Animals↗
Nature of a paramyxovirus replication promoter influences a nearby transcription signal.
The genomic and antigenomic 3' ends of the Sendai virus replication promoters are bi-partite in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or gene end (ge) site, respectively, and a simple hexameric repeat. Studies of how mRNA synthesis initiates from the first gene start site (gs1) have been hampered by the fact that gs1 is located between two essential elements of the replication promoter. Transcription initiation, then, is separated from the replication initiation site by only 56 nt on the genome, so that transcription and replication may sterically interfere with each other. In order to study the initiation of Sendai virus mRNAs without this possible interference, Sendai virus mini-genomes were prepared having tandem promoters in which replication takes place from the external one, whereas mRNA synthesis occurs from the internal one. Transcription now initiates at position 146 rather than position 56 relative to the genome 3' end. Under these conditions, it was found that the frequency with which mRNA synthesis initiates depends, in an inverse fashion, on the strength of the external replication promoter. It was also found that the sequences essential for replication are not required for basic mRNA synthesis as long as there is an external replication promoter at which viral RNA polymerase can enter the nucleocapsid template. The manner in which transcription and replication initiations influence each other is discussed.
3' Untranslated Regions↗
Identification of a telomeric DNA sequence in Trypanosoma brucei.
A simple repetitive DNA sequence in the nuclear genome of Trypanosoma brucei, consisting of tandem repeats of the hexanucleotide 5' CCCTAA 3', was identified as being telomeric by several criteria. This sequence was specifically labeled with T. brucei genomic DNA as the template for in vitro nick translation by DNA polymerase I, and was present in Bal 31 nuclease sensitive, genomic restriction fragments of the large sizes expected in this organism for at least some telomeric regions. The same repeated sequence was found in six other flagellates tested. A segment of DNA from T. brucei including this telomeric sequence was cloned in pBR322 and characterized. The cloned segment contained a sequence highly homologous to the 3' ends of several variant surface glycoprotein mRNAs, upstream of the tandemly repeated hexanucleotide sequence.
Amino Acid Sequence↗
Extended microsatellite analysis in microsatellite stable, MSH2 and MLH1 mutation-negative HNPCC patients: genetic reclassification and correlation with clinical features.
BACKGROUND: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder predisposing to predominantly colorectal cancer (CRC) and endometrial cancer frequently due to germline mutations in DNA mismatch repair (MMR) genes, mainly MLH1, MSH2 and also MSH6 in families seen to demonstrate an excess of endometrial cancer. As a consequence, tumors in HNPCC reveal alterations in the length of simple repetitive genomic sequences like poly-A, poly-T, CA or GT repeats (microsatellites) in at least 90% of the cases. AIM OF THE STUDY: The study cohort consisted of 25 HNPCC index patients (19 Amsterdam positive, 6 Bethesda positive) who revealed a microsatellite stable (MSS)--or low instable (MSI-L)--tumor phenotype with negative mutation analysis for the MMR genes MLH1 and MSH2. An extended marker panel (BAT40, D10S197, D13S153, D18S58, MYCL1) was analyzed for the tumors of these patients with regard to three aspects. First, to reconfirm the MSI-L phenotype found by the standard panel; second, to find minor MSIs which might point towards an MSH6 mutation, and third, to reconfirm the MSS status of hereditary tumors. The reconfirmation of the MSS status of tumors not caused by mutations in the MMR genes should allow one to define another entity of hereditary CRC. Their clinical features were compared with those of 150 patients with sporadic CRCs. RESULTS: In this way, 17 MSS and 8 MSI-L tumors were reclassified as 5 MSS, 18 MSI-L and even 2 MSI-H (high instability) tumors, the last being seen to demonstrate at least 4 instable markers out of 10. Among all family members, 87 malignancies were documented. The mean age of onset for CRCs was the lowest in the MSI-H-phenotyped patients with 40.5 +/- 4.9 years (vs. 47.0 +/- 14.6 and 49.8 +/- 11.9 years in MSI-L- and MSS-phenotyped patients, respectively). The percentage of CRC was the highest in families with MSS-phenotyped tumors (88%), followed by MSI-L-phenotyped (78%) and then by MSI-H-phenotyped (67%) tumors. MSS tumors were preferentially localized in the distal colon supposing a similar biologic behavior like sporadic CRC. MSH6 mutation analysis for the MSI-L and MSI-H patients revealed one truncating mutation for a patient initially with an MSS tumor, which was reclassified as MSI-L by analyzing the extended marker panel. CONCLUSION: Extended microsatellite analysis serves to evaluate the sensitivity of the reference panel for HNPCC detection and permits phenotype confirmation or upgrading. Additionally, it confirms the MSS status of hereditary CRCs not caused by the common mutations in the MMR genes and provides hints to another entity of hereditary CRC.
Adaptor Proteins, Signal Transducing↗
Microsatellite size homoplasy, SSCP, and population structure: a case study in the freshwater snail Bulinus truncatus.
The extent of microsatellite size homoplasy, as well as its effect on several population genetics statistics, was investigated in natural populations using the single-strand conformation polymorphism (SSCP) method. The analysis was conducted using 240 individuals from 13 populations of the freshwater snail Bulinus truncatus at a GT(n)CT(m) compound microsatellite locus. We showed that SSCP can be used to uncover, at least partly, size homoplasy in the core sequence of this category of loci. Eight conformers (SSCP variants) were detected among the three size variants (electromorphs). Sequencing revealed that each conformer corresponded to a different combination of repeats in the GT(n) and CT(m) arrays. Part of this additional variability was detected within populations, resulting in a substantial increase in gene diversity in four populations. Additional variability also changed the values of parameters used to analyze population differentiation among populations: pairwise tests of differentiation were significant much more often with conformers than with electromorphs. On the other hand, pairwise estimates of F(st) were either smaller or larger with conformers than with electromorphs, depending on whether or not electromorphs were shared among populations. However, estimates of F(st) (or analogs) over all populations were very similar, ranging between 0.66 and 0.75. Our results were consistent with the theoretical prediction that homoplasy should not always lead to stronger population structure. Finally, conformer sequences and electromorph size distribution suggested that single-point and/or stepwise mutations occurring simultaneously in the different repeated arrays of compound microsatellites produce sequence variation without size variation and hence generate more size homoplasy than expected under a simple stepwise mutation model.
Animals↗
Two simple repeat polymorphisms at DXS337.
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Alleles↗
Apparent encoding of sequential context in rat medial prefrontal cortex is accounted for by behavioral variability.
Simple sequences can be represented via asymmetrically linked neural assemblies, provided that the elements of the sequence are unique. When elements repeat, however (e.g., A-B-C-B-A), the same element belongs to two separate "sequential contexts," and a more complex encoding mechanism is required. To enable correct sequence performance, some neural structure must provide a disambiguating signal that differentiates the two sequential contexts (i.e., B as an element of "A-B" as opposed to "C-B"). The disambiguating signal may derive from a form of working memory, or, in some cases, a simple timing mechanism may suffice. To investigate the possible role of medial prefrontal cortex in complex sequence encoding, rats were trained on a spatial sequence containing two adjacent repeated segments (e.g., A-B-C-D-B-C-E). The double-repeat procedure minimized behavioral differences in the second leg (C) of the repeat subsequence that arise in the first leg (B) because of differences in the entry point (e.g., A-B vs D-B). Far more cells were context sensitive along the first leg than along the second (36 vs 9%), and most of the differences were accounted for by systematic variations in the rat's trajectory, which were much larger along the first leg. There is thus little evidence for sequential context-discriminative activity in the medial prefrontal cortex that cannot plausibly be accounted for by context-dependent behavior. The finding that the rodent medial prefrontal cortex is highly sensitive to sensory-behavioral variables raises doubts about previous experiments that purport to show working memory-related activity in this region.
Action Potentials↗
Detection and purification of sequence-specific DNA binding protein.
A simple assay for identifying DNA-binding proteins is described that involves loading of protein fractions onto nitrocellulose membrane using a slot-blot apparatus, incubating with 32P-labeled DNA probe in buffer in the presence of excess of nonspecific E. coli DNA at room temperature, and washing with increasing concentration of NaCl (from 50 to 500 mM) to obtain optimum signal. A simple and rapid scheme of purification of a sex and tissue-specific DNA-binding protein, which binds specifically to the GATA repeats of Bkm (banded krait minor satellite DNA), designated as Bkm-binding protein (BBP), is also described. This requires only a DNA affinity column after the initial ammonium sulfate precipitation. The insert (545 bp) of the Drosophila clone 2(8) containing 66 copies of GATA repeats was used to prepare the sequence-specific DNA-Sepharose affinity column. The slot-blot-binding assay and the simple scheme of purification described here may be used for routine screening and purification of sequence-specific DNA-binding proteins in general.
Animals↗
Length of uninterrupted repeats determines instability at the unstable mouse expanded simple tandem repeat family MMS10 derived from independent SINE B1 elements.
Mouse expanded simple tandem repeats (ESTRs) provide highly informative loci for analyzing spontaneous and induced germline mutation. We have conducted an extensive sequence database search and identified 17 new members of the highly unstable rodent-specific ESTR family called MMS10. This family has arisen by independent expansions of a common GGCAGA repeat unit from within a subset of both ancestral and modern SINE B1 elements during the course of mouse evolution. Analysis of the interspersion patterns of variant repeats along alleles of 20 of these MMS10 loci revealed two distinct classes of tandem arrays: one composed of uninterrupted GGCAGA repeats and the second with generally larger arrays interrupted by variant units. Surveys of allelic diversity at 11 representative members of these two classes of loci in various laboratory strains and BXD recombinant inbred lines revealed that the level of repeat instability was positively correlated with the length of uninterrupted repeats. Turnover processes at MMS10 loci, therefore, appear similar to the type of mechanism observed at human microsatellites. The MMS10 family thus provides a potentially useful murine model for studying dynamic mutation at simple tandem repeats.
Alleles↗
Mutation rates in the complex microsatellite MYCL1 and related simple repeats in cultured human cells.
Microsatellite instability is a phenotype observed in tumors cells that have defects in DNA mismatch repair (MMR). Most markers used for detecting microsatellite instability are mono- and dinucleotide repeats, but one tetranucleotide repeat (MYCL1) has been reported to be useful for this purpose. The MYCL1 repeat is actually a complex repeat, made up of approximately 14 GAAA tetranucleotides plus various other GA-rich repeats. In order to determine the nature of the instability of the this sequence, we have used a frameshift-reversion assay in MMR-proficient and -deficient human cells to compare the mutation rates and the types of mutation of MYCL1 to those of the related simple repeats (GAAA)17, (GA)17, and (CA)17. We found that the complex repeat was the most stable of the repeats examined in cells deficient in MMR; the tetranucleotide was less stable, while the dinucleotides were the least stable. In MMR-proficient cells, the relative rates were reversed; the MYCL1 repeat was the least stable, the tetranucleotide was more stable, and the dinucleotides were the most stable. These results suggest that MYCL1 and the pure tetranucleotide have relatively low rates of errors during replication, but that the errors in these repeats are corrected less efficiently than those in the smaller repeats. Because of their high rate of instability in MMR-proficient cells, MYCL1 and other tetranucleotide repeats appear to lack specificity for detection of tumors with defective MMR.
Base Sequence↗
ProtRepeatsDB: a database of amino acid repeats in genomes.
BACKGROUND: Genome wide and cross species comparisons of amino acid repeats is an intriguing problem in biology mainly due to the highly polymorphic nature and diverse functions of amino acid repeats. Innate protein repeats constitute vital functional and structural regions in proteins. Repeats are of great consequence in evolution of proteins, as evident from analysis of repeats in different organisms. In the post genomic era, availability of protein sequences encoded in different genomes provides a unique opportunity to perform large scale comparative studies of amino acid repeats. ProtRepeatsDB http://bioinfo.icgeb.res.in/repeats/ is a relational database of perfect and mismatch repeats, access to which is designed as a resource and collection of tools for detection and cross species comparisons of different types of amino acid repeats. DESCRIPTION: ProtRepeatsDB (v1.2) consists of perfect as well as mismatch amino acid repeats in the protein sequences of 141 organisms, the genomes of which are now available. The web interface of ProtRepeatsDB consists of different tools to perform repeat s; based on protein IDs, organism name, repeat sequences, and keywords as in FASTA headers, size, frequency, gene ontology (GO) annotation IDs and regular expressions (REGEXP) describing repeats. These tools also allow formulation of a variety of simple, complex and logical queries to facilitate mining and large-scale cross-species comparisons of amino acid repeats. In addition to this, the database also contains sequence analysis tools to determine repeats in user input sequences. CONCLUSION: ProtRepeatsDB is a multi-organism database of different types of amino acid repeats present in proteins. It integrates useful tools to perform genome wide queries for rapid screening and identification of amino acid repeats and facilitates comparative and evolutionary studies of the repeats. The database is useful for identification of species or organism specific repeat markers, interspecies variations and polymorphism.
Amino Acid Sequence↗