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PubMed2003

A highly polymorphic STR locus in Cannabis sativa.

We report on the first short tandem repeat (STR) locus to be isolated from the plant Cannabis sativa. The STR locus, isolated by a hybrid-capture enrichment procedure, was found to contain a simple sequence repeat motif of 6 bp. This 6 bp repeat motif showed no variation in repeat length but with minor variations in repeat unit sequences. The data show the locus to be highly polymorphic with the number of repeat units ranging from 3 to 40 in 108 screened samples. The observed heterozygosity was approximately 87.04%. The forward and reverse primers (CS1F and CS1R) produced no PCR products in cross-reaction study from 20 species of plants, including highly related species such as Humulus japonicus and Nicotiana tabacum. This hexanucleotide repeat DNA locus could be used to identify cannabis samples and predict their genetic relationship as the test is specific to C. sativa and is highly reproducible.

Alleles↗

PubMed2025

The Complete Chloroplast Genome and the Phylogenetic Analysis of Panicum bisulcatum (Thumb.) (Poaceae).

The chloroplast (cp) genome of Panicum bisulcatum (Thumb.), a significant agricultural weed, was sequenced and characterized to elucidate its genomic architecture, evolutionary dynamics, and phylogenetic relationships. The complete cp genome was assembled as a circular DNA molecule of 138,489 bp, exhibiting a typical quadripartite structure comprising a large single-copy (LSC, 82,260 bp), a small single-copy (SSC, 12,569 bp), and a pair of inverted repeats (IR, 21,830 bp each) regions. It encodes 135 genes, including 89 protein-coding genes, 49 tRNAs, and 8 rRNAs. Functional annotation revealed that most genes are involved in photosynthesis and genetic system. A total of 51 simple sequence repeats (SSRs) and 62 long repeats (LRs) were identified, providing potential molecular markers. Comparative analysis of IR boundaries highlighted both conserved features and species-specific expansion/contraction events among Panicum species. Phylogenomic analysis robustly placed P. bisulcatum within the genus Panicum, showing a closest relationship with P. incomtum and confirming the monophyly of the genus. Furthermore, single nucleotide polymorphism (SNP) analysis with its closest relative, P. incomtum, revealed 4659 SNPs, with a dominance of synonymous substitutions, indicating the action of purifying selection. This study provides the first comprehensive cp genomic resource for P. bisulcatum, which will facilitate future studies in species identification, phylogenetic reconstruction, population genetics, and the development of sustainable management strategies for this weed.

Phylogeny↗

PubMed2002

Microsatellite isolation and characterization in sunflower (Helianthus annuus L.).

Development of microsatellite markers for sunflower (Helianthus annuus L.) was performed to estimate their frequency, nature (structure), levels of polymorphism, usefulness for genotype identification, and calculation of genetic relationships between inbred lines representing the species diversity. Isolation was performed from a small-insert genomic library followed by hybridization screening using oligonucleotide probes containing different nucleotide arrays. In this work, 503 unique microsatellite clones were sequenced and 271 PCR primer sequences bordering the microsatellite repeat were designed. For polymorphism assessment, 16 H. annuus germplasm accessions were checked and 170 of the primers tested were shown to be polymorphic for the selected lines. The polymorphic microsatellites produced an average of 3.5 alleles/locus and an average polymorphism information content (PIC) of 0.55. The most frequently found motifs within polymorphic simple-sequence repeats (SSRs) were: (GA)n, (GT)n, (AT)n, followed by trinucleotides (ATT)n, (TGG)n, and (ATC)n, and the tetranucleotide (CATA)n. Most of the 170 SSRs obtained showed important differences in the 16 reference inbred lines used for their characterization. In this work, 20 of the most informative SSRs destined to sunflower genotyping and legal fingerprinting purposes are fully described.

DNA Primers↗

PubMed2004

Phylogeny and strain typing of Escherichia coli, inferred from variation at mononucleotide repeat loci.

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.

Bacterial Typing Techniques↗

PubMed1992

Organization, structure, and function of 95 kb of DNA spanning the murine T-cell receptor C alpha/C delta region.

We have analyzed the organization, structure, and function of the murine T-cell receptor C alpha/C delta region. This region spans 94.6 kb of DNA and contains the C alpha and C delta genes, as well as the V delta 5, J delta 2, and 50 different J alpha gene segments. Within this sequence we have identified 15 new J alpha gene segments, 40 new 5' RNA splice signals, and 40 new DNA rearrangement signals for the J alpha gene segments. The murine C alpha/C delta sequence contains an exceptionally high level of coding sequence with over 5.7% of the total sequence found in the exons. This is much more than that found in the beta-globin locus and the HPRT locus. Using the sequence data obtained from the C alpha/C delta region, we have designed simple assays to test for J alpha gene segment transcription and to determine the level of polymorphism for simple repeat sequences among different inbred strains of mice using the polymerase chain reaction. Furthermore, comparisons of this 95 kb of sequence with the available sequence from homologous regions of other species have led to the identification of a highly conserved sequence that is present throughout vertebrates and in the mouse binds lymphocyte-specific nuclear proteins. Comparisons of a 10-kb region, which includes the C alpha gene in human and mouse, average 66% sequence similarity. These studies support the contention that large-scale DNA sequencing projects of homologous regions of mouse and human will provide powerful new tools for studying the biology and evolution of loci such as the T-cell receptor and for identifying and posing new questions about the functions of conserved sequences.

Amino Acid Sequence↗

PubMed2004

Genomic instability in oral squamous cell carcinoma: relationship to betel-quid chewing.

Genomic instability in repeated DNA sequences is exhibited by a variety of cancer types, including oral squamous cell carcinoma. Exposure to carcinogenic compounds may further increase the instability. We have used Inter-Simple Sequence Repeat (Inter-SSR) PCR methodology to detect genetic alterations in 37 oral cancer patients who had chewed betel-quid. Thirty-eight percent of DNA from tumors had genomic alterations in the sequences flanked by (CA)(8) and (GT)(8) repeats. Patients with tumor DNAs harboring genomic alterations had a two-fold higher consumption of betel-quid than patients without alterations in tumor DNA. Matched normal and tumor DNAs were also screened for microsatellite instability where four patients (10.8%) showed alterations in at least one microsatellite marker but there was no relationship between this phenotype and betel-quid chewing. These data indicate that exposure to carcinogens present in the betel-quid may contribute to genomic instability detected by inter-SSR PCR in a subset of oral tumors.

Adult↗

PubMed2000

Modulation of EGFR gene transcription by secondary structures, a polymorphic repetitive sequence and mutations--a link between genetics and epigenetics.

The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation and motility of normal as well as tumor cells. The transduction of extracellular signals to the cytoplasm via the receptor not only depends on ligand binding, but is also determined by the receptor density on the cell surface. Therefore, in terms of cancer diagnosis and therapeutic approaches targeting EGFR it is decisive to know how the expression level of EGFR is controlled. We found that transcription activity declines with increasing numbers of CA dinucleotides of a highly polymorphic CA repeat in the first intron epidermal growth factor receptor gene. In vivo data from cultured cell lines support these findings, although other regulation mechanisms can compensate this effect. In addition, we showed that RNA elongation terminates at a site closely downstream of the simple sequence repeat (SSR) and that there are two separate major transcription start sites. Model calculations for the helical DNA conformation revealed a high bendability in the EGFR polymorphic region, especially if the CA stretch is extended. These data suggest that the CA-SSR can act like a joint bringing the promoter in proximity to a putative repressor protein bound downstream of the CA-SSR. The data suggest that this polymorphism is a marker for cancer linking genetic and epigenetic risk. Furthermore in breast cancer, heterozygous tumours with short CA-SSR showed an elevated EGFR-expression in contrast to tumours with longer CA-SSR. Tumours with loss of heterozygosity in intron 1 of egfr revealed an increased EGFR expression if the longer allele was lost. Moreover, deceased egfr gene dosages were significantly correlated to poor prognosis in breast cancer.

Animals↗

PubMed1990

Transcription of a satellite DNA on two Y chromosome loops of Drosophila melanogaster.

Primary spermatocyte nuclei of Drosophila melanogaster exhibit three giant lampbrush-like loops formed by the kl-5, kl-3 and ks-1 Y chromosome fertility factors. Detailed mapping of satellite DNA sequences along the Y chromosome has recently shown that AA-GAC satellite repeats are a significant component of the kl-5 and ks-1 loop-forming regions. To determine whether these simple repeated sequences are transcribed on the loop structures we performed a series of DNA-RNA in situ hybridization experiments to fixed loop preparations using as a probe cloned AAGAC repeats. These experiments showed that the probe hybridizes with homologous transcripts specifically associated with the kl-5 and ks-1 loops. These transcripts are detected at all stages of development of these two loops, do not appear to migrate to the cytoplasm and are degraded when loops disintegrate during the first meiotic prophase. Moreover, an examination of the testes revealed that the transcription of the AAGAC sequences is restricted to the loops of primary spermatocytes; the other cell types of D. melanogaster spermatogenesis do not exhibit nuclear or cytoplasmic labeling. These experiments were confirmed by RNA blotting analysis which showed that transcription of the AAGAC sequences occurs in wild-type testes but not in X/O testes. The patterns of hybridization to the RNA blots indicated that the transcripts are highly heterogeneous in size, from large (migration at limiting mobility) to less than 1 kb. We discuss the possible function of the AAGAC satellite transcripts, in the light of the available information on the Y chromosome loops of D. melanogaster.

Animals↗

PubMedDate not supplied

Modulation of EGFR gene transcription by a polymorphic repetitive sequence--a link between genetics and epigenetics.

The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation and motility of normal as well as tumor cells. The transduction of extracellular signals to the cytoplasm via the receptor not only depends on ligand binding, but is also determined by the receptor density on the cell surface. Therefore, with regard to cancer diagnosis and therapeutic approaches targeting EGFR it is important to know how the expression level of EGFR is controlled. We found that transcription activity declines with increasing numbers of CA dinucleotides of a highly polymorphic CA repeat in the first intron of the epidermal growth factor receptor gene. In vivo data from cultured cell lines support these findings, although other regulation mechanisms can compensate this effect. In addition, we showed that RNA elongation terminates at a site closely downstream of the simple sequence repeat (SSR) and that there are two separate major transcription start sites. Model calculations for the helical DNA conformation revealed a high bendability in the EGFR polymorphic region, especially if the CA stretch is extended. These data suggest that the CA-SSR can act like a joint, bringing the promoter in proximity to a putative repressor protein bound downstream of the CA-SSR. The data indicate that this polymorphism may be a marker for cancer, linking genetic and epigenetic risk factors. Furthermore, in breast cancer, heterozygous tumors with short CA-SSR showed an elevated EGFR-expression in contrast to tumours with longer CA-SSR. Tumours with loss of heterozygosity in intron 1 of egfr revealed an increased EGFR expression if the longer allele was lost. Moreover, decreased EGFR gene levels were significantly correlated with poor prognosis in breast cancer.

Biomarkers, Tumor↗

PubMed1998

Characterization of novel minisatellite repeat loci in Atlantic salmon (Salmo salar) and their phylogenetic distribution.

We present here the sequence and characterization of various minisatellite-like tandem repeat loci isolated from the genome of Atlantic salmon (Salmo salar). Their diversity of sequence and lack of core motifs common to minisatellites of other species suggest the presence of numerous and previously unidentified simple sequence repeat families in this salmonid. Evidence for their ubiquity was provided by screening of a salmon genomic library. Southern blot analysis of the phylogenetic distribution of a subset of the minisatellites found one sequence to be pervasive among vertebrates, others present only in Salmoninae or Salmonidae species, and one amplified only in Atlantic salmon. There is evidence for the positioning of microsatellite and minisatellite arrays in close proximity at many loci. Furthermore, one tandem repeat appears to have been inserted into the transposase coding region of a copy of the Tc1 transposon-like element recently identified in salmonids.

Animals↗

PubMed2002

Telomerase recognizes its template by using an adjacent RNA motif.

Telomerase adds telomeric repeats to chromosome 3' ends, forestalling the cellular senescence, apoptosis, and genomic instability that result from telomere loss caused by incomplete DNA replication. The telomerase ribonucleoprotein is dedicated to synthesis of tandem, simple-sequence repeats by virtue of its specialization for copying only a specific template region within the integral RNA. Here, using circularly permuted variants of Tetrahymena thermophila telomerase RNA, we identify the features that allow recognition of the template region within the RNA. We engineered a template-less telomerase ribonucleoprotein that can position and reverse transcribe an exchangeable RNA oligonucleotide template accurately. Only a short "template-recognition" element sequence tag is required to direct efficient use of adjacent 5' residues as a template for telomeric repeat synthesis. Our findings reveal molecular requirements for template selection by telomerase and physically resolve templating from other RNA functions in catalysis.

Animals↗

PubMed2001

Hypermutability to ionizing radiation in mismatch repair-deficient, Pms2 knockout mice.

DNA mismatch repair (MMR) has been shown to play a role in the cytotoxicity of ionizing radiation (IR), as cell lines established from MMR-deficient mice exhibit higher clonogenic survival after IR than do cell lines from wild-type littermates. To test whether this tolerance phenotype would render MMR-deficient animals hypermutable to IR, we compared IR mutagenesis of Pms2-deficient versus wild-type transgenic mice carrying a lambda shuttle vector for mutation detection. In Pms2 nullizygous animals, the mutation frequency in the supFG1 reporter gene was increased from 210 x 10(-5) in untreated animals to 734 x 10(-5) after 6 Gy of IR (an increase of 524 mutants per 10(5)), whereas the frequency in wild-type mice increased from 1.9 x 10(-5) to 10.2 x 10(-5) (an increase of only 8.3 mutants per 10(5)). Similarly, when the lambda cII gene was used as a reporter, the mutation frequency in nullizygous mice was increased from 16.3 x 10(-5) to 42.3 x 10(-5) after IR (an increase of 26.0 x 10(-5)), whereas the frequency in wild-type mice increased from 2.4 x 10(-5) to 9.4 x 10(-5) (an increase of only 7.0 x 10(-5)). The pattern of IR-induced mutations in the MMR-deficient animals was notable for single bp deletions and insertions in mononucleotide repeat sequences, along with a slight increase in transversions. Overall, these results suggest that MMR-deficiency confers hypermutability to IR, and that much of this hypermutability can be attributed to induced instability of simple sequence repeats. Hence, MMR influences not only the survival but also the mutability of cells in response to IR.

Adenosine Triphosphatases↗

PubMed2000

Evolutionary inventions and continuity of CORE-SINEs in mammals.

We characterized short interspersed elements (SINEs), of the CORE-suprafamily in egg-laying (monotremes), pouched (marsupials) and placental mammals. Five families of these repeats distinguished by the presence of distinct LINE-related 3'-segments shared tRNA-like promoter and the central core region. The putative active elements were reconstructed from the alignment of genomic repeats representing molecular fossils of sequences that amplified in the past and since then underwent multiple mutations. Their mode of proliferation by retroposition was indicated by the presence of: (1) internal RNA PolIII promoter; (2) simple sequence repeated tail; (3) direct repeats; and (4) subfamilies recording the evolution of elements. The copy number of CORE-SINEs in placental genomes was estimated at about 300,000; they were highly divergent and apparently ceased to amplify before radiation of these lineages. On the other hand, among almost half a million fossil elements present in marsupials and monotremes, the youngest subfamilies could still be retropositionally active. CORE-SINEs terminate in sequence repeats of a few nucleotides similar to their 3'-segment LINE-homologues, CR1, L2 and Bov-B. These three LINE elements fall into clades distinct from that of L1 elements which, similar to their co-amplifying SINEs, end in a poly(A) tail. We propose a model in which new CORE-families, with distinct 3'-segments, are created at the RNA level due to template switching between LINE and CORE-RNA during reverse transcription. The proposed mechanism suggests that such an adaptation to the changing amplification machinery facilitated the survival and prosperity of CORE-elements over long evolutionary periods in different lineages.

Animals↗

PubMed1987

Multiple forms of male-specific simple repetitive sequences in the genus Mus.

Previous reports indicate that in laboratory strains of mice, males are distinct from females in possession of repetitive DNA, notably devoid of Eco RI and Hae III sites and rich in the simple tetranucleotides GATA/GACA. We report here that such sequences originated in an ancestor common to laboratory mice, Mus hortulanus, M. spretus, and possibly also M. cookii. Interestingly, other male-specific satellite sequences were detected in M. caroli, M. cookii, M. saxicola, and M. minutoides. This novel satellite is also likely to be composed of simple repetitious sequences, but does not contain GATA and GACA. Thus, the Y chromosome appears to contain a disproportionately large amount of simple repetitious DNA. An attractive explanation for these results is that long tandem arrays of simple repeated sequences are generated at high frequency throughout the genome and that they are retained for a longer time on the Y chromosome due to the absence of homologous pairing at meiosis.

Animals↗

PubMed1996

Sensitivity, reproducibility, and accuracy in short tandem repeat genotyping using capillary array electrophoresis.

The Human Genome Initiative has increased significantly the rate at which disease-causing genes are being mapped and sequenced. New cost-effective methods to locate the genes and to characterize disease-causing mutations require robust, reproducible, and accurate protocols for measuring DNA fragment lengths. Capillary array electrophoresis (CAE) offers rapid, high-resolution separations, high throughput, and sensitive detection. To assess the utility of CAE for the accumulation of genetic information, we tested both sizing accuracy and reproducibility using 48-capillary prototype systems. Two multiplex PCR allelic ladder standards and several CA-repeat markers were analyzed in > 100 runs. Reproducibility in typing > 8000 genotypes reveals a standard deviation of less than 0.2 bp on these systems under optimized conditions. However, sequence-dependent migration anomalies were observed at most simple sequence loci even when analyzed under denaturing conditions, resulting in a systematic bias in estimated fragment sizes. We show here that, by normalizing results to known typing controls, one can obtain locus-averaged accuracies of < 0.06 bp and normalized results within 1 bp of actual. We detect as little as a 1:30,000 dilution of a DNA quantitation standard stained with highly sensitive intercalating dyes, indicating an 80-zeptomole sensitivity limit. However, to obtain reproducible electrokinetic injection, approximately 200 attomoles of fluorescein-labeled DNA is required. These sensitivity limits, sizing precision, and accuracy, together with the 1-hr run times for 48-96 samples, indicate that CAE is a viable method for high-throughput genetic analysis of simple sequence repeat polymorphisms.

Capillary Action↗

PubMed2006

Extensive macrosynteny between Medicago truncatula and Lens culinaris ssp. culinaris.

The first predominantly gene-based genetic linkage map of lentil (Lens culinaris ssp. culinaris) was constructed using an F5 population developed from a cross between the cultivars Digger (ILL5722) and Northfield (ILL5588) using 79 intron-targeted amplified polymorphic (ITAP) and 18 genomic simple sequence repeat (SSR) markers. Linkage analysis revealed seven linkage groups (LGs) comprised of 5-25 markers that varied in length from 80.2 to 274.6 cM. The genome map spanned a total length of 928.4 cM. Clear evidence of a simple and direct macrosyntenic relationship between lentil and Medicago truncatula was observed. Sixty-six out of the 71 gene-based markers, which were previously assigned to M. truncatula genetic and physical maps, were found in regions syntenic between the Lens c. ssp. culinaris and M. truncatula genomes. However, there was evidence of moderate chromosomal rearrangements which may account for the difference in chromosome numbers between these two legume species. Eighteen common SSR markers were used to connect the current map with the most comprehensive and recent map that exists for lentil, providing the syntenic context of four important domestication traits. The composite map presented, anchored with orthologous markers mapped in M. truncatula, provides a strong foundation for the future use of genomic and genetic information in lentil genetic analysis and breeding.

Chromosome Mapping↗

PubMed2001

[Modulation of the cat gene expression in E. coli cells by simple (AC)20 repeats].

Probable relationship between modulation of gene expression by simple repeating sequences and competition capacity of the host was studied. (AC)20 repeat can both stimulate and inhibit the expression of chloramphenicol acetyl transferase gene expression in E. coli cells depending on this gene's position. Modulation of the gene expression by simple (AC)20 repeat and competitive capacity of bacteria containing this plasmid are closely related.

Chloramphenicol O-Acetyltransferase↗

PubMed2004

In silico mapping of quantitative trait loci in maize.

Quantitative trait loci (QTL) are most often detected through designed mapping experiments. An alternative approach is in silico mapping, whereby genes are detected using existing phenotypic and genomic databases. We explored the usefulness of in silico mapping via a mixed-model approach in maize (Zea mays L.). Specifically, our objective was to determine if the procedure gave results that were repeatable across populations. Multilocation data were obtained from the 1995-2002 hybrid testing program of Limagrain Genetics in Europe. Nine heterotic patterns comprised 22,774 single crosses. These single crosses were made from 1,266 inbreds that had data for 96 simple sequence repeat (SSR) markers. By a mixed-model approach, we estimated the general combining ability effects associated with marker alleles in each heterotic pattern. The numbers of marker loci with significant effects--37 for plant height, 24 for smut [Ustilago maydis (DC.) Cda.] resistance, and 44 for grain moisture--were consistent with previous results from designed mapping experiments. Each trait had many loci with small effects and few loci with large effects. For smut resistance, a marker in bin 8.05 on chromosome 8 had a significant effect in seven (out of a maximum of 18) instances. For this major QTL, the maximum effect of an allele substitution ranged from 5.4% to 41.9%, with an average of 22.0%. We conclude that in silico mapping via a mixed-model approach can detect associations that are repeatable across different populations. We speculate that in silico mapping will be more useful for gene discovery than for selection in plant breeding programs.

Analysis of Variance↗