CT Motifs Research Articles

Development of SSRs Based on the Whole Genome and Screening of Bolting-Resistant SSR Marker in Brassica oleracea L.

Simple sequence repeats (SSRs), also known as microsatellites, stand out as the most crucial molecular markers in both animals and plants owing to their high polymorphism, extensive information content, ease of detection through polymerase chain reaction (PCR) assays, and widespread distribution across the genome. In this study, a total of 125,443 SSR loci were identified from the whole-genome sequence of B. oleracea, and 82,948 primer pairs for SSR have been designed. Furthermore, each primer pair is designated with a unique identifier (ranging from BolSSR00001 to BolSSR82984). Our findings indicated that certain markers within them could be transferred to other cruciferous crops. In addition, a total of 336 pairs of SSR primers have been used to screen the polymorphism between the bolting-resistant and bolting-easy gene pools. After the test of verification with F2 generation individual plants, we obtained an SSR dominant marker, BolSSR040196, linked with bolting-resistant locus in cabbage, and the genetic distance between this SSR marker and the bolting-resistant locus was 10.69 cM. Moreover, BolSSR040196 is located on the C05 chromosome with a CT motif, characterized by a repeat of 9 in bolting-easy plants and 11 in bolting-resistant plants. Haplotype analysis showed that the correct prediction rate reached 82.35%. The BolSSR040196 marker can be used in marker-assisted selection (MAS) breeding, offering a straightforward and efficient approach for bolting-resistant cabbage breeding in the future.

Role of the leucine-rich repeat protein kinase 2 C-terminal tail in domain cross-talk.

Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Understanding the role of the LRRK2 tail in the crosstalk between kinase and the N-terminal domains.

LRRK2 is a multi-domain protein encompassing biology's two most critical molecular switches; a kinase and a GTPase. Mutations in LRRK2 are one of the key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to exploring the physiological role of LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is a common feature in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and can serve as a multi-functional protein: protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and are using peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB region with a combination of Gaussian MD simulations and peptide arrays. This Ct-Motif contains multiple phosphorylation sites including two auto-phosphorylation sites (Thr2083 and Thr2524), a 14-3-3 binding site (Thr2524) and a putative PKA phosphorylation site. These two sites together form a part of a dynamic “CAP” that regulates the N-lobe of the kinase domain. We also explore how the cellular localization of LRRK2RCKW and its docking onto microtubules is altered by mutations in the Ct helix, if at all. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Genome-wide mining of potentially-hypervariable microsatellites and validation of markers in Momordica charantia L.

Relatively large number of bitter melon microsatellite markers have been reported; however, only few resulted in successful PCR amplification and a small fraction shown polymorphisms. This limited chance of recovering polymorphic markers makes the primer screening a cost-demanding process. To test the hypothesis that microsatellites with longer motifs as well as shorter motifs repeated substantially shall have better prospects to be polymorphic, we performed a genome-wide microsatellite mining. We selected a sample of genome-wide microsatellites with prescribed motif lengths or satisfying a target repeat number, which were considered potentially-hyper variable, for primer designing and validation. Seventy five microsatellites satisfying these criteria were identified, of which 69 were validated through successful PCR amplification. Among them, 40 (53.33% of the markers identified) were polymorphic. This result showed a significantly higher success compared to our initial results of 51 (20.64%) polymorphic markers out of the 188 amplified when 247 previously reported markers were screened. The screening of two cultivars revealed that markers were efficient to identify up to three alleles. The characterization of these 69 new markers with 247 markers previously reported showed that di-nucleotide motifs were most abundant, followed by tri- and tetra-nucleotide motifs. TC motif markers were most polymorphic (12.08%) followed by AG and CT motifs (both 9.89%). Similarly, AGA (6.59%) and TATT (3.29%) were most polymorphic among the tri- and tetra-nucleotide motifs. These 69 hypervariable microsatellite markers along with 188 markers initially validated in this study shall be useful for phylogenetic analyses, studies of linkage, QTL, and association mapping in bitter melon.

Tight DNA-protein complexes isolated from barley seedlings are rich in potential guanine quadruplex sequences.

BackgroundThe concept of chromatin domains attached to the nuclear matrix is being revisited, with nucleus described as a set of topologically associating domains. The significance of the tightly bound to DNA proteins (TBP), a protein group that remains attached to DNA after its deproteinization should be also revisited, as the existence of these interactions is in good agreement with the concept of the topologically associating domain. The work aimed to characterize the DNA component of TBP isolated from barley seedlings.MethodsThe tight DNA-protein complexes from the first leaves, coleoptiles, and roots of barley seedlings were isolated by purification with chromatography on nitrocellulose or exhaustive digestion of DNA with DNase I. Cloning and transformation were performed using pMOSBBlue Blunt Ended Cloning Kit. Inserts were amplified by PCR, and sequencing was performed on the MegaBace 1000 Sequencing System. The BLAST search was performed using sequence databases at NCBI, CR-EST, and TREP and Ensembl Plants databases. Comparison to MAR/SAR sequences was performed using http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi database. The prediction of G quadruplexes (GQ) was performed with the aid of R-studio library pqsfinder. CD spectra were recorded on a Chirascan CS/3D spectrometer.ResultsAlthough the barley genome is AT-rich (43% of GC pairs), most DNA fragments associated with TBP were GC-rich (up to 70% in some fractions). Both fractionation procedures yielded a high proportion of CT-motif sequences presented predominantly by the 16-bp CC(TCTCCC)2 TC fragment present in clones derived from the TBP-bound DNA and absent in free DNA. BLAST analysis revealed alignment with different barley repeats. Some clones, however, aligned with both nuclear and chloroplast structural genes. Alignments with MAR/SAR motifs were very few. The analysis produced by the pqsfinder program revealed numerous potential quadruplex-forming sites in the TBP-bound sequences. A set of oligonucleotides containing sites of possible GQs were designed and ordered. Three of them represented the minus strand of the CT-repeat. Two were derived from sequences of two clones of nitrocellulose retained fraction from leaves and contained GC-rich motifs different from the CT motif. Circular dichroism spectroscopy revealed profound changes in spectra when oligonucleotides were incubated with 100 mM KCl. There was either an increase of positive band in the area of 260 nm or the formation of a positive band at 290 nm. In the former case, changes are typical for parallel G-quadruplexes and, in the latter, 3 + 1 structures. DiscussionThe G-quadruplexes anchor proteins are probably involved in the maintenance of the topologically associated domain structure.

Microsatellites in palm (Arecaceae) sequences.

Microsatellites are the most promising co-dominant markers, widely distributed throughout the genome. Identification of these repeating genomic subsets is a tedious and iterative process making computational approaches highly useful for solving this biological problem. Here 38,083 microsatellites were localized in palm sequences. A total of 2, 97,023 sequences retrieved from public domains were used for this study. The sequences were unstained using the tool Seqclean and consequently clustered using CAP3. SSRs are located in the sequences using the microsatellite search tool, MISA. Repeats were detected in 33,309 sequences and more than one SSR had appeared in 3,943 sequences. In the present study, dinucleotide repeats (49%) were found to be more abundant followed by mononucleotide (30%) and trinucleotide (19%). Also among the dinucleotides, AG/GA/TC/CT motifs (55.8%) are predominantly repeating within the palm sequences. Thus in future this study will lead to the development of specific algorithm for mining SSRs exclusively for palms.

Identification and characterization of new variants of three associated SNPs and a microsatellite in the TSH receptor gene which are useful for genetic studies

The purpose of the present work was to characterize g.IVS5 − 69C > T, g.IVS6 + 13C > T and c.561C > T SNPs and the [CT] n microsatellite in the TSHR gene for genetic analysis. Exons 6 and 7 of the TSHR gene, including the flanking intronic sequences, were screened for the presence of g.IVS5 − 69C > T, g.IVS6 + 13C > T and c.561C > T SNPs by SSCP. We found genetic association between the three SNPs and a total of three different haplotypes were observed. Two were homozygous blocks, g.IVS5 − 69T/g.IVS6 + 13G/c.561C (Haplotype TGC, 3.3%) and g.IVS5 − 69C/g.IVS6 + 13A/c.561T (Haplotype CAT, 75%). Every individual who was heterozygous for g.IVS5 − 69C > T was equally heterozygous for g.IVS6 + 13A > G and c.561T > C (Haplotype CAT/TGC, 21.7%). The [CT] n microsatellite, localized in intron 7 of the TSHR gene was amplified by PCR and the labeled products were separated in a polyacrylamide denaturing sequencing gel. Three variable numbers of CT motif were identified, two previously reported ([CT] 6 and [CT] 8) and one previously unreported ([CT] 9). The construction and expression of the hybrid minigenes using pSPL3 and α-globin-fibronectin EDB (pTB) vectors showed that the [CT] n microsatellite itself does not interfere with exon 8 definition and processing in vitro. In conclusion, g.IVS5 − 69C > T/g.IVS6 + 13C > T/c.561C > T haplotypes and [CT] n microsatellite are informative polymorphic markers and can be used in linkage studies in families with germ line TSHR mutations or autoimmunity thyroid diseases.

Improvement of the pBI121 plant expression vector by leader replacement with a sequence combining a poly(CAA) and a CT motif

To improve expression levels of recombinant proteins in plants, a new leader sequence was designed. Several elements known to enhance gene translation and/or transcription were considered, including the CaMV 35S Inr site, a CT-rich motif often shared by highly expressed plant genes and a poly(CAA) region widespread in tobamovirus and plant leaders. The effect of the synthetic leader on gusA expression was evaluated in genetically modified tobacco plants by measuring the beta-glucuronidase activity and the mRNA level. When compared to the gusA leader of pBI121, the new sequence determined a 8.6-fold and a 12.5-fold increase of enzyme concentration taking into account the whole plant population or the above-average expressors, respectively. Since most pCAMBIA vectors harbour a very short 5'-UTR, identical to a fragment of the pBI121 leader, leader replacement with the sequence herein described is strongly suggested.

Polymorphism in ornamental and common carp strains (Cyprinus carpio L.) as revealed by AFLP analysis and a new set of microsatellite markers.

Forty-seven new microsatellite markers were generated and applied, together with the AFLP (Amplified Fragment Length Polymorphism) technique using two different enzyme combinations, to the genetic analysis of two carp species, Cyprinus carpio L. and Ctenopharyngodon idella. The extent of polymorphism and the genetic relationships between nine carp populations were studied. The incidence of microsatellites containing CA and CT motifs was estimated to be one every 17.4 and one every 126.3 kb, respectively, and their average allele numbers were four and five, respectively. Across populations, the average proportion of individuals that were heterozygous for microsatellite markers was 44.2% and the average allele number was 4.02. The EcoRI/TaqI combination generated more analyzable AFLP bands than the EcoRI/MseI pair, making the former preferable for the analysis of carp populations. The proportion of polymorphic AFLP bands within populations ranged from 6.7% in grass carp to 59.9% in Kohaku strain (Koi) of the ornamental carp. The fixation index (FST) for microsatellites in these populations was estimated to be 0.37, and for AFLP markers the value was 0.39. Genetic distance matrices derived from microsatellites and from two AFLP analyses were positively correlated. Grass carp showed fewer AFLP bands than other populations and was genotyped by only half of the microsatellite markers. These findings agree with genetic distance estimates in suggesting that the grass carp is phylogenetically quite remote from all the other populations examined.

Transcription factor Sp3 antagonizes activation of the ornithine decarboxylase promoter by Sp1.

Ornithine decarboxylase (ODC) expression is important for proliferation and is elevated in many tumor cells. We previously showed that Sp1 is a major positive regulator of ODC transcription. In this paper we have investigated transcriptional regulation of rat ODC by the closely related factor Sp3. While over-expression of Sp1 caused a dramatic activation of the ODC promoter, over-expression of Sp3 caused little or no activation in either Drosophila SL2 cells (lacking endogenous Sp1 or Sp3) or in H35 rat hepatoma cells. Furthermore, co-transfection studies demonstrated that Sp3 abolished trans -activation of the ODC promoter by Sp1. DNase I footprint studies and electrophoretic mobility shift assays demonstrated that both recombinant Sp1 and Sp3 bind specifically to several sites within the ODC promoter also protected by nuclear extracts, including overlapping GC and CT motifs located between -116 and -104. This CT element is a site of negative ODC regulation. Mutation of either element reduced binding, but mutation of both sites was required to eliminate binding of either Sp1 or Sp3. These results demonstrate that ODC is positively regulated by Sp1 and negatively regulated by Sp3, suggesting that the ratio of these transcription factors may be an important determinant of ODC expression during development or transformation.

Genomic Cloning, Characterization, and Functional Analysis of the Major Surface Adhesin WI-1 on Blastomyces dermatitidis Yeasts

WI-1 is a 120-kDa surface protein adhesin on Blastomyces dermatitidis yeasts that binds CD18 and CD14 receptors on human macrophages. We isolated and analyzed a clone of genomic WI-1 to characterize this key adherence mechanism of the yeast. The 9.3-kilobase insert contains an open reading frame of 3438 nucleotides and no introns. The amino acid sequence of native WI-1 matches the deduced sequence of genomic WI-1 at positions 757-769, 901-913, and 1119-1138, demonstrating the cloned gene is authentic WI-1. The complete coding sequence has 30 highly conserved repeats of 24 amino acids arrayed in tandem in two noncontiguous regions of the protein. The repeat sequence is homologous to the Yersiniae adhesin invasin, the C terminus displays an epidermal growth factor-like domain, and the N terminus has a short hydrophobic sequence that may be a membrane-spanning domain. The tandem repeats are predicted to be at the exposed surface of the protein, thereby explaining the adhesive properties of WI-1. The WI-1 promoter contains a CAAT box (nucleotide positions 2287-2290), TATA box (2380-2385), and CT motif (2399-2508). Transcription is initiated within the CT motif at nucleotide 2431. A 5.5-kilobase subclone containing the full coding sequence of WI-1 was expressed as a histidine-tagged fusion protein in Escherichia coli. Recombinant WI-1 has the expected molecular mass of 120 kDa, is strongly recognized in Western blots by rabbit anti-WI-1 antiserum, and binds human macrophage receptors in the same manner as native WI-1. This work clarifies a key adherence mechanism of B. dermatitidis and will permit further analysis of WI-1-mediated attachment to host cells, receptors, and extracellular matrix.

Heterologous transformation of Agrocybe aegerita with a bacterial neomycin-resistance gene fused to a fungal promoter-like DNA sequence

DNA sequences of the basidiomycete Agrocybe aegerita were cloned in E. coli based on their ability to drive the expression of the bacterial promoterless tetracycline (Tc)-resistance gene. A 0.48% frequency of the cloned sequences promoted antibiotic-resistance. The sequence conferring the highest Tc resistance (40 μg/ml) was selected to drive the expression in E. coli of two other promoterless genes encoding chloramphenicol and neomycin resistance. One of the derivative vectors, pN13-A2, carrying a chimeric neomycin-resistance gene, was used to transform an A. aegerita neomycin-sensitive strain by protoplast electroporation. Transformation frequencies ranged from 1 to 2.8 transformants per μg of DNA per 10(3) viable cells, in a relatively high background of spontaneous-resistant colonies (2% of the surviving protoplasts). Molecular analyses showed that transformation had occurred by the integration of pN13-A2 sequences, either ectopically or at the resident locus carrying the A. aegerita promoter-like sequence, with probable molecular rearrangements. The nucleotide sequence of the promoter-like fragment revealed the presence of a CT motif that is known to be involved in a promoter function in some highly expressed genes of filamentous fungi.

Chronic exposure of HIT cells to high glucose concentrations paradoxically decreases insulin gene transcription and alters binding of insulin gene regulatory protein.

Chronically culturing HIT-T15 cells in media containing high glucose concentrations leads to decreased insulin mRNA levels, insulin content, and insulin secretion. These changes can be prevented by culturing the cells in media containing lower glucose levels (Robertson, R. P., H.-J. Zhang, K. L. Pyzdrowski, and T. F. Walseth. 1992. J. Clin. Invest. 90:320-325). The mechanism of this seemingly paradoxical phenomenon was examined by transiently transfecting HIT cells with a chloramphenicol acetyl transferase (CAT) reporter gene controlled by the 5'-regulatory domain of the human insulin gene (INSCAT). Early passages of HIT cells readily expressed INSCAT, whereas late passages of cells chronically cultured in 11.1 mM glucose expressed only 28.7 +/- 2.3% (mean +/- SEM) of the CAT activity expressed in early passages. In contrast, late passages of HIT cells chronically cultured in 0.8 mM glucose retained the ability to express the INSCAT reporter gene to 69.6 +/- 10.0% of the CAT activity observed in early passages. The decrease in INSCAT expression in late passages of cells serially cultured in 11.1 mM glucose was associated with the inability to form a specific nuclear protein-DNA complex with the CT motifs of the human insulin promoter. Formation of this specific protein-DNA complex was preserved in late passages of HIT cells when serially cultured in 0.8 mM glucose. Mutations of the CT motifs caused markedly diminished CAT activity in all passages examined. These data indicate that chronic exposure of the beta cell to high glucose concentrations can paradoxically decrease insulin gene transcription, in part, by altering the ability of a regulatory protein (GSTF) to interact with the insulin gene promoter. This provides a potential mechanism for glucotoxic effects on the beta cell at the level of the insulin gene.

Molecular analysis of the Septoria nodorum β-tubulin gene and characterization of a benomyl-resistance mutation

The complete nucleotide sequence of a benomyl-resistant allele of the Septoria nodorum beta-tubulin gene (tubAR) has been determined including 745 and 1024 nucleotides 5' and 3' to the tubAR coding region, respectively. tubAR encodes a 447 amino acid polypeptide which shows a high degree of homology with other fungal beta-tubulins. The gene contains three introns at codons 4, 12 and 53, uses 48 of the possible 61 sense codons and has a GC content of 59.1% in its coding region. S1 nuclease mapping has identified two transcriptional start sites 80 bp and 83 bp upstream of the translation start, and a transcriptional termination site 192 bp downstream of the stop codon. The two transcriptional start sites lie just 8 bp and 5 bp downstream of a CT motif consisting of 18 pyrimidine nucleotides interrupted by a single adenine. The wild-type allele tubA+ has been cloned using the polymerase chain reaction and the mutation producing the benomyl-resistant phenotype of tubAR mapped to a C to T transition at the first position of codon 6, resulting in a histidine to tyrosine amino acid substitution.