We describe the effect of nearest-neighbor sequence context on mismatch-dependent activation of hMSH2-hMSH6. Examination of the intrinsic sequences that occur around symmetric mismatched nucleotides suggests little if any effect of non-nearest-neighbor base pairs on hMSH2-hMSH6 mismatch recognition and ATPase activation (20), although longer-range effects have been reported (22). Although a sequence context effect is not novel in MMR (21), the underlying mechanism is unknown. Our studies have suggested that when a significant nearest-neighbor sequence context effect is manifest, 2 × 3′-purines enhanced, and 2 × 3′-pyrimidines reduced hMSH2-hMSH6 ATPase activation (kcat). A similar trend is observed for mismatch binding (KD), whereas an inverse effect was observed for the Tm of unbound mismatched oligonucleotides. Importantly, the KD and Tm do not accurately account for hMSH2-hMSH6 ATPase activation. Interestingly, the effect of sequence context on KD appears associated with alteration of ...
Specific cis-acting sequences within the carlavirus potato virus S (PVS) genomic RNA molecule appear to control gene expression at the translational level. Two sequences have been investigated, the untranslated sequence upstream from the initiation codon of the viral coat protein gene, designated VTE and the 5 untranslated leader sequence from the genomic RNA molecule (PVS 5). In vitro and in vivo, either of these sequences enhance the translation of a downstream open reading frame when provided as the untranslated leader in a transcript molecule. Translational enhancement was also detected at the transgenic plant level. Both PVS sequences were deleted in an attempt to identify the core regulatory element responsible for this translational enhancement phenomenon. Results indicate that in vitro and in vivo, the functional motif is contained within the 5 promixal portion of both sequences. When the sequences of these important regions were compared, a homologous block of nucleotides was ...
TY - JOUR. T1 - Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay. AU - Senear, D. F.. AU - Brenowitz, M.. PY - 1991/9/9. Y1 - 1991/9/9. N2 - We have investigated the question of whether the gel mobility-shift assay can provide data that are useful to the demonstration of cooperativity in the site-specific binding of proteins to DNA. Three common patterns of protein-DNA interaction were considered: (i) the cooperative binding of a protein to two sites (illustrated by the Escherichia coli Gal repressor); (ii) the cooperative binding of a bidentate protein to two sites (illustrated by the E. coli Lac repressor); and (iii) the cooperative binding of a protein to three sites (illustrated by the λcI repressor). A simple, rigorous, and easily extendable statistical mechanical approach to the derivation of the binding equations for the different patterns is presented. Both stimulated and experimental data for each case are ...
Cbf1p is a basic-helix-loop-helix-zipper protein of Saccharomyces cerevisiae required for the function of centromeres and MET gene promoters, where it binds DNA via the consensus core motif CACRTG (R = A or G). At MET genes Cbf1p appears to function in both activator recruitment and chromatin-remodeling. Cbf1p has been implicated in the regulation of other genes, and CACRTG motifs are common in potential gene regulatory DNA. A recent genome-wide location analysis showed that the majority of intergenic CACGTG palindromes are bound by Cbf1p. Here we tested whether all potential Cbf1p binding motifs in the yeast genome are likely to be bound by Cbf1p using chromatin immunoprecipitation. We also tested which of the motifs are actually functional by assaying for Cbf1p-dependent chromatin remodeling. We show that Cbf1p binding and activity is restricted to palindromic CACGTG motifs in promoter-proximal regions. Cbf1p does not function through CACGTG motifs that occur in promoter-distal locations within coding
Methods for in-depth characterization of transcriptomes and quantification of transcript levels have emerged as valuable tools for understanding cellular physiology and human disease biology, and have begun to be utilized in various clinical diagnostic applications. Today, current methods utilized by the scientific community typically require RNA to be converted to cDNA prior to comprehensive measurements. However, this cDNA conversion process has been shown to introduce many biases and artifacts that interfere with the proper characterization and quantitation of transcripts. We have developed a direct RNA sequencing (DRS) approach, in which, unlike other technologies, RNA is sequenced directly without prior conversion to cDNA. The benefits of DRS include the ability to use minute quantities (e.g. on the order of several femtomoles) of RNA with minimal sample preparation, the ability to analyze short RNAs which pose unique challenges for analysis using cDNA-based approaches, and the ability to ...
Fingerprint Dive into the research topics of Characterization of single stranded viral DNA sequences present during replication of adenovirus types 2 and 5. Together they form a unique fingerprint. ...
The full-length ORF clone contains only the coding sequence of the full-length protein, while the other full-length cDNA clones contain some untranslated sequences, such as the 5side or 3′ side non translation. It is well known that these untranslated sequences may have a negative effect on the transcription and translation processes of the encoded proteins in the host.. If it is the ORF expressed clone, it can be transfected into cells and expressed in cells.. In the general situation, the carrier of cDNA clone is not the expression vector, so it can not be directly used for transfection of cells.. The difference between ORF, cDNA and CDS:. 1.what is a full-length ORF clone?. A full-length ORF clone is a plasmid inserted into a DNA fragment encoding a full-length protein. The inserted DNA fragment only contains sequence incoding a full-length protein, and does not contain the untranslated region of 5 or 3 end (UTRs) or intron.. 2.why should the full length ORF cDNA clones are used instead ...
Fibroblast growth factor-2 (FGF-2) or basic FGF is a multifunctional protein that, through interaction with specific cell surface receptors, plays important roles in the growth and development of tissues and organs. Thus, considerable attention has focused on the control of FGF-2 gene expression, including assessments of RNA levels through blotting and the use of radiolabeled FGF-2 cDNA probes. Multiple transcripts of different sizes have been reported for FGF-2 by this approach, however, more recent evidence indicates that at least one of these RNAs of about 1.5 kb, is not an authentic FGF-2 transcript. A major band of 4.7 kb and a minor band of 6.1 kb were detected in total rat glial tumor cell RNA, using the intact rat ovarian FGF-2 cDNA as a probe at high stringency. This cDNA contains both coding and 5′-untranslated sequences. Although the 6.1 kb transcript levels were increased in RNA enriched for polyadenylated species, the levels of the 4.7 kb band were decreased and also shared a mobility
In the preceding paper we described an experiment that determined the in vivo forward mutation rate in a single replication cycle for spleen necrosis virus. In addition to substitutions, frameshifts, and hypermutations, the mutated proviruses contained two classes of deletions. One class of deletions contained short direct repeats at the deletion junctions. Another class of deletions had short stretches of sequences inserted at the deletion junctions. In this report, we describe the deletion mutations, and we present models for their generation. Detailed analysis of two deletions with insertions indicates that these mutations occurred as a result of template switching during plus-strand DNA synthesis. The analysis also indicates that fragments of viral RNA generated by the viral RNase H endonuclease are used as templates and contribute to the sequences inserted at the deletion junctions.. ...
The Pem gene encodes an atypical homeodomain protein, distantly related to Prd/Pax family members, that we demonstrate is regulated in a complex transcriptional and post-transcriptional manner. We show that the rat Pem genomic structure includes three 5-untranslated (5-UT) exons and four coding exons, three of which encode the homeodomain. Several alternatively spliced transcripts were identified, including one that skips an internal coding exon, enabling this mRNA to express a novel form of the Pem protein. Other alternatively spliced mRNAs were characterized that possess different 5-UT regions, including a muscle-specific transcript. The different 5-UT termini present in Pem transcripts conferred different levels of translatability in vitro. Two promoters containing multiple transcription initiation sites were identified: a distal promoter (Pd) in the first 5-UT exon and a proximal promoter (Pp) located in the intron upstream of the first coding exon. The Pd was active in placenta, ovary, tumor
Gene expression is the process by which the genetic code - the nucleotide sequence - of a gene is used to direct protein synthesis and produce the structures of the cell. Genes that code for amino acid sequences are known as structural genes.. Gene control regions:. A promoter. A region a few hundred nucleotides upstream of the gene (toward the 5′ end). It is not transcribed into mRNA, but plays a role in controlling the transcription of the gene. Transcription factors bind to specific nucleotide sequences in the promoter region and assist in the binding of RNA polymerases.. Enhancers. Some transcription factors (called activators) bind to regions called enhancers that increase the rate of transcription. These sites may be thousands of nucleotides from the coding sequences or within an intron. Some enhancers are conditional and only work in the presence of other factors as well as transcription factors ...
Comparison with MarA structure.Both of the amino acids in H-T-H 1 of RhaS that make sequence-specific DNA contacts align with MarA residues also predicted to make specific DNA contacts. Arg202 of RhaS aligns with MarA Trp42. One of the base pairs contacted by each of these amino acids is a G-C base pair in the analogous position in the two binding sites (−56 at marRAB and −67 atrhaBAD), while each of these residues also makes additional, different contacts (Fig. 4). Arg206 of RhaS aligns with Arg46 of MarA. In this case, the identical amino acid contacts the identical base pair in the analogous position in the two binding sites (G-C at −56 inmarRAB and −67 in rhaBAD), and each amino acid also contacts a C-G base pair (−55 at marRAB and −65 atrhaBAD). MarA has a third amino acid in H-T-H 1 that contacts DNA (Gln45). MarA Gln45 aligns with RhaS His205, which was found to be important for DNA binding by RhaS but not specific for any base positions. It seems likely that only Arg202 and ...
The Base Position feature in the Genome Browser annotations track image shows the genomic coordinates of the displayed region and -- when zoomed in to the base level -- the base composition of the sequence underlying the annotation tracks display. To quickly zoom in to the base level, click the base zoom-in button. The base button may also be used to automatically zoom the window to maximize the base display when the image is resized. Click the arrow to the left of the base display to show the base complement. To toggle between dense and full display mode, click anywhere on the base composition line. In dense display mode, clicking anywhere on the genome coordinate line will zoom-in the display, centered on the coordinate of the click. The degree of the zoom may be configured through the Zoom configuration buttons (the default is 3X). In full display mode, the track shows three frames of translation when viewed at the base level. Methionines (including potential start codons) are displayed in ...
ProtoScript II First Strand cDNA Synthesis Kit features two optimized mixes, ProtoScript II Enzyme Mix and ProtoScript II Reaction Mix.
The invention provides in situ nucleic acid sequencing to be conducted in biological specimens that have been physically expanded. The invention leverages the techniques for expansion microscopy (ExM) to provide new methods for in situ sequencing of nucleic acids as well as new methods for fluorescent in situ sequencing (FISSEQ) in a new process referred to herein as
Symmetry Elements The concept of symmetry explains how similar objects (known as motifs) are repeated systematically in space to produce ordered structures where all objects have specific and predictable ...
Figure 1. Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach. Panel A. Reverse transcription and PCR amplification of TCR subunit mRNA sequences. First-strand cDNA synthesis is primed by the TCR dT Primer and performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5′ end of each mRNA molecule, the RT adds non-templated nucleotides to the first-strand cDNA. The SMART-Seq v4 Oligonucleotide contains a sequence that is complementary to the non-templated nucleotides added by the RT, and the oligo hybridizes to the first-strand cDNA. In the template-switching step, the RT uses the remainder of the SMART-Seq v4 Oligonucleotide as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. Full-length variable regions of TCR cDNA are selectively amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (SMART Primer 1) and the constant region(s) of TCR-α and/or TCR-β ...
Figure 1. Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach. Panel A. Reverse transcription and PCR amplification of TCR subunit mRNA sequences. First-strand cDNA synthesis is primed by the TCR dT Primer and performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5′ end of each mRNA molecule, the RT adds non-templated nucleotides to the first-strand cDNA. The SMART-Seq v4 Oligonucleotide contains a sequence that is complementary to the non-templated nucleotides added by the RT, and the oligo hybridizes to the first-strand cDNA. In the template-switching step, the RT uses the remainder of the SMART-Seq v4 Oligonucleotide as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. Full-length variable regions of TCR cDNA are selectively amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (SMART Primer 1) and the constant region(s) of TCR-α and/or TCR-β ...
P27 (Negative Regulatory Factor or Nef) - Drugs in Development, 2021 provides in depth analysis on P27 (Negative Regulatory Factor or Nef) targeted pipeline therapeutics. The report provides comprehensive information complete with Analysis by Indications, Stage of Development, Mechanism of Action (MoA), Route of Administration (RoA) and Molecule Type. The report also covers the descriptive pharmacological action of the therapeutics, its complete research and development history and latest news and press releases. Additionally, the report provides an overview of key players involved in P27 (Negative Regulatory Factor or Nef) targeted therapeutics development and features dormant and discontinued projects. The report analyses the pipeline products across relevant therapy areas under development targeting P27 (Negative Regulatory Factor or Nef).. The report helps in identifying and tracking emerging players in the market and their portfolios, enhances decision making capabilities and helps to ...
Fingerprint Dive into the research topics of A:T → G:C base pair substitutions occur at a higher rate than other substitution events in Pms2 deficient mouse cells. Together they form a unique fingerprint. ...
Dive into the research topics of Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. Together they form a unique fingerprint. ...
In the current study, we applied cDNA cloning and RNA-Seq methods to identify two alternative CYP3A4 mRNA transcripts, one of which contained partial intron-6 retention and the other with a shorter 3′-UTR.. The CYP3A4 mRNA transcript with partial intron-6 retention can potentially be translated into a novel protein with a shortened amino acid sequence as a result of a translational stop codon in intron 6. However, the absence of a heme-binding signature in the encoded polypeptide precludes a catalytically active protein. Proportion of this transcript in all examined samples is less than 2% of total CYP3A4 mRNA. Therefore, its influence on CYP3A4 function is considered to be limited; as a result, we did not investigate function of this transcript in the current study. Although RNA-Seq also showed faint peaks in intron 11, Cufflinks did not assemble a transcript with intron-11 retention. Thus, the possible existence of intron-11 retention cannot be excluded based on the current evidence, and ...
king interactions (hydrogen bonding merely provides specificity of the pairing, not stability). As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determine the strength of the association between the two strands of DNA. Long DNA helices with a high GC content have stronger-interacting strands, while short helices with high AT content have weaker-interacting strands. In biology, parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in some promoters, tend to have a high AT content, making the strands easier to pull apart. In the laboratory, the strength of this interaction can be measured by finding the temperature required to break the hydrogen bonds, their melting temperature (also called Tm value). When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules have no single common shape, but some ...
Not so many people are aware that extensions method are possible to implemented also on JavaScript code. Here you have quick example how to achieve that:1. Lets create class definition function MyClass(imput){ this.myProperty = input; } 2. Lets create object of our class var myObject = new MyClass(test); 3. Now, we are ready to add…
It transports the genetic information into the cytoplasm, where the ribosomes use it as a template to produce a specific protein (translation). TGC-TTA. mRNA that is transcribed is normally a copy of the sense strand, however, it is the antisense strand that is transcribed. The RNA product is complementary to the template strand of DNA and is almost identical to the nontemplate DNA strand, or the sense strand. As shown schematically above, messenger RNA is synthesized complementary and antiparallel to the template strand (anticodons) of DNA, so the resulting mRNA consists of codons corresponding to those in the coding strand of DNA. cDNA is often used to clone eukaryotic genes in prokaryotes. Use the RNA base-pairing rules. The type of amino acid is determined by the anticodon sequence of the transferRNA. The only difference is that in RNA, all of the T nucleotides are replaced with U nucleotides; during RNA synthesis, U is incorporated when there is an A in the complementary antisense strand. ...
Hx-amides are fluorescent hybrids of imidazole (1)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx-I/P-I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and Delta T-M studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T-A/T; Hx/IP prefers C-A/T; Hx/PI prefers A/T-C; and Hx/II prefers C-C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts. (C) 2013 Elsevier Ltd. All rights reserved.
Luciferase Assay Reagents. OVERVIEW Control of gene expression in eukaryotic cells is regulated on transcriptional and post-transcriptional levels. Transcription factors are important regulators of transcription rates, and microRNAs are key mediators of mRNA stability and translation efficiency.. Regulation of transcription. DNA-encoded elements like promoters interact with transcription factors and other regulatory proteins to determine when, where, and how much of a genes mRNA product gets made. Reporter assays are a powerful technique for measuring the activity of promoters in living cells. Promoter GoClone Reporter Constructs are created by isolating a promoter from the human genome and cloning it upstream of the RenSP luciferase reporter gene on a plasmid. Once this plasmid is transfected into a living cell, a change in promoter activity causes a change in reporter signal (light output).. SwitchGear Promoter GoClone Reporter Assays can be used to monitor the activity of any promoter in a ...
cloning of non-coding region for protein expression - posted in Protein Expression and Purification: Hi! I am trying to see the function of my non-coding region in protein expression. I have clone my gene and its non-coding region upstream of 5UTR to find if non-coding region plays any role in expression of cloned protein. I have other clones with no non-coding region just 5UTR and coding region. Has anybody cloned these type construct in mammalian expression vector and does...
A simplified method for isolating primer extension products and generating them in a form appropriate for electrophoresis is disclosed. The method is compatible with automated DNA sequencing procedures.
Gel shift assay: (aka gel mobility shift assay (GMSA), band shift assay (BSA), electrophoretic mobility shift assay (EMSA)) A method by which one can determine whether a particular protein preparation contains factors which bind to a particular DNA fragment. When a radiolabeled DNA fragment is run on a gel, it shows a characteristic mobility. If it is first incubated with a cellular extract of proteins (or with purified protein), any protein-DNA complexes will migrate slower than the naked DNA - a shifted band.. Gene: A unit of DNA which performs one function. Usually, this is equated with the production of one RNA or one protein. A gene contains coding regions, introns, untranslated regions and control regions.. Genome: The total DNA contained in each cell of an organism. Mammalian genomic DNA (including that of humans) contains 6x109 base pairs of DNA per diploid cell. There are somewhere in the order of a hundred thousand genes, including coding regions, 5 and 3 untranslated regions, ...
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ChiP (Chromosome Immunoprecipitation) is a technique where DNA binding proteins, like transcription factors, can be localized to regions of a DNA molecule. We can use this method to identify which DNA sequences control expression and regulation for diverse genes. In the ChIP procedure, cells are treated with a reversible cross-linking agent to fix proteins to other proteins that are nearby, as well as the chromosomal DNA where theyre bound. The DNA is then purified and broken into smaller chunks by digestion or shearing and antibodies are used to precipitate any protein-DNA complexes that contain their target antigen. After the immunoprecipitation step, unbound DNA fragments are washed away, the bound DNA fragments are released, and their sequences are analyzed to determine the DNA sequences that the proteins were bound to. Only few years ago, this procedure was much more complicated than it is today, for example, the fragments had to be cloned before they could be sequenced. When microarrays ...
View Notes - BIOL 112 - Practice Qs from 2008 from BIOL 112 at UBC. sheet. 71. The initiator sequence and promoter sequences must exhibit a large degree of overlap. 72. If the lac operon was designed
A novel method for detecting and isolating DNA sequences commonly held by different DNA preparations or repeated or amplified within a complex genome has been provided. The DNA preparations of interest are digested with the same restriction enzyme and a portion of at least one preparation is labeled with 32 P. The labeled and unlabeled DNA preparations are combined and electrophoresed in an agarose gel. Following electrophoresis, the DNA is denatured in situ and allowed to reanneal within the gel so that homologous DNA sequences present within restriction fragments of the same size can reanneal. After reannealing, unhybridized single-stranded DNA is digested in situ followed by detection of the reannealed DNA by autoradiography. When labeled and unlabeled DNAs are derived from different DNA preparations, only the restriction fragments commonly held by these two preparations are detected. When a restriction digest of total eukaryotic DNA is reassociated in the gel by this procedure, repeated restriction
Bottom line: What are the sequences of phage polymerase termination signals? Yes, I am still trying to get good in vitro transcription of a particular sequence for use in RNase protection analysis. Basically I can only really use one region and this is next to a T7 promoter so I am fiddling with the conditions. The transcript I get is mainly full length but the yield is awful. I tried 15degC for 3 h. It was worse then 37degC for 1 h. I tried the Maslak & Martin buffer and this may have improved things but the salt/detergent in the buffer screw up the gel purification of the probe (I am working on this). I tried beefing up the amount of enzyme or the amount of limiting nucleotide (32P-CTP) but this didnt really help. I have yet to try T4g32 protein or its equivalent. In the back of the Ambion MAXIscript guide their is a vague reference to accidental incorporation of phage RNA polymerase termination signals in a template being a reason for poor yield. No information is given as to what these ...
An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5 end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5 and 3 ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing ...
DNA. Computer artwork of a DNA (deoxyribonucleic acid) double helix seen against autoradiograms of genetic sequences (upper right). The spiralling strands of the DNA helix (blue) are composed of complex chemical groups called nucleotides, which consist of a sugar phosphate and a base group. Complementary pairing between the base groups of nucleotides on opposite strands holds the helix together. The sequence of the 4 base groups (adenine, cytosine, guanine and thymine; coloured rods) along the DNA helix is unique for every individual and is known as the genetic code. The autoradiograms depict DNA base sequences, each band (dark blue) representing an individual base. - Stock Image G110/0642
Knowledge of the sequence of a DNA segment has many uses, and some examples follow. First, it can be used to find genes, segments of DNA that code for a specific protein or phenotype. If a region of DNA has been sequenced, it can be screened for characteristic features of genes. For example, open reading frames (ORFs)-long sequences that begin with a start codon (three adjacent nucleotides; the sequence of a codon dictates amino acid production) and are uninterrupted by stop codons (except for one at their termination)-suggest a protein-coding region. Also, human genes are generally adjacent to so-called CpG islands-clusters of cytosine and guanine, two of the nucleotides that make up DNA. If a gene with a known phenotype (such as a disease gene in humans) is known to be in the chromosomal region sequenced, then unassigned genes in the region will become candidates for that function. Second, homologous DNA sequences of different organisms can be compared in order to plot evolutionary ...
The kinetic theory of replication has been extended to include dual mechanisms for conversion of self-annealed single-strand RNA to double-strand molecules, which do not replicate. An analysis of experimental results established that the replicate-template annealing reaction during transcription significantly retarded replication in vitro among three RNA variants copied by Qβ replicase. Annealing between complementary RNA strands free in solution had far less significance. The finding that an RNA variant can be replicated in a multiple hairpin configuration, but not as its single, long hairpin conformer, the correlation between stability of strand secondary structure and replicative fitness, and a lack of homology in the internal sequence of RNA variants copied by Qβ replicase support the conclusion that template competence depends on strand configuration, independent of most of the underlying base sequence. Occurrence of self-annealed strands in the Qβ replicase system was attributed to its ...
Oxford Nanopore’s MinION is a small sensing device which can sequence DNA and RNA directly, without the need to perform an enzymatic synthesis reaction. The device is portable and is po
However, in cases 5 and 6 we seem to come to a radical discontinuity. In both of these cases, there can be an analogical (and therefore meaningful) relationship between the nucleotide sequence ACA in DNA and the corresponding amino acid sequence in a translated polypeptide, either in vitro or in a cell. What makes this difference possible (and what may make it necessary) is the analogical relationship between the nucleotide sequence and the corresponding amino acid sequence (if one exists). If the DNA sequence ACA is located in the template strand of an actively transcribed DNA sequence (i.e. a DNA sequence beginning with a promoter to which RNA polymerase can bind) and furthermore its complementary RNA analog is located in an mRNA molecule following the start codon AUG but not following a stop codon (either UAA, UAG, or UGA, assuming a three-base reading frame), then that the DNA sequence does indeed contain meaningful information: it is encoded in one medium, is translated into another ...
However, in cases 5 and 6 we seem to come to a radical discontinuity. In both of these cases, there can be an analogical (and therefore meaningful) relationship between the nucleotide sequence ACA in DNA and the corresponding amino acid sequence in a translated polypeptide, either in vitro or in a cell. What makes this difference possible (and what may make it necessary) is the analogical relationship between the nucleotide sequence and the corresponding amino acid sequence (if one exists). If the DNA sequence ACA is located in the template strand of an actively transcribed DNA sequence (i.e. a DNA sequence beginning with a promoter to which RNA polymerase can bind) and furthermore its complementary RNA analog is located in an mRNA molecule following the start codon AUG but not following a stop codon (either UAA, UAG, or UGA, assuming a three-base reading frame), then that the DNA sequence does indeed contain meaningful information: it is encoded in one medium, is translated into another ...
The human genome is densely populated with transposons and transposon-like repetitive elements. Although the impact of these transposons and elements on human genome evolution is recognized, the significance of subtle variations in their sequence remains mostly unexplored. This study reports homozygosity mapping of an infantile neurodegenerative disease locus in a genetic isolate. Complete DNA sequencing of the 400-kb linkage locus revealed a point mutation in a primate-specific retrotransposon that was transcribed as part of a unique noncoding RNA, which was expressed in the brain. In vitro knockdown of this RNA increased neuronal apoptosis, consistent with the inappropriate dosage of this RNA in vivo and with the phenotype. Moreover, structural analysis of the sequence revealed a small RNA-like hairpin that was consistent with the putative gain of a functional site when mutated. This study show here that a mutation in a unique transposable element-containing RNA is associated with lethal ...
resistens in its natural habitat, probably the histidine-rich inguinal and perineal areas of the human body. The ability of C. resistens to utilize l-histidine as a sole source of nitrogen was demonstrated by growth assays. in synthetic minimal media. Reverse transcriptase PCRs revealed enhanced transcript levels of the hut genes in C. resistens cells grown in the presence of l-histidine. Promoter-probe assays showed that the hut genes are organized in three transcription units: hutHUI,hutR, and hutG. The respective transcriptional start sites were mapped by 5′ RACE-PCR to detected putative promoter regions. DNA band shift assays with purified HutR protein identified Talazoparib order the 14-bp DNA sequence TCTGwwATwCCAGA located upstream of the mapped promoters. This GSK J4 supplier DNA motif includes a 4-bp terminal palindrome, which turned out. to be essential for HutR binding in vitro. These data add a new physiological function to the large IclR family of transcriptional regulators. ...
The invention relates to methods for the use of the polymerase chain reaction to amplify a segment of a cloned gene of interest in such a way as to allow a simplified introduction of alterations such as deletions, insertions, repetitions (both direct and inverted) and substitutions into the cloned gene in a specific and precise manner. The unique, amplified segment of the cloned gene so amplified is a common substrate for each of the different approaches to introducing the various alterations into the gene. Choice of the primer sites within the amplified segment coupled with choice of the orientation of the molecule once ligated to itself results in the various resulting embodiments of the invention.
92118DNAArtificial SequenceA synthetic DNA fragment 1aaggagcgat cgccatgn 18210DNAArtificial SequenceA synthetic DNA fragment, wherein nnn is the first codon which is 3 to the start codon followed by the remainder of an open reading frame 2cgccatgnnn 10312DNAArtificial SequenceA synthetic DNA fragment 3nnnnnngtct tc 12410DNAArtificial SequenceA synthetic DNA fragment 4nnnngaagag 10513DNAArtificial SequenceA synthetic DNA fragment 5gcagcnnnnn nnn 13611DNAArtificial SequenceA synthetic DNA fragment 6nnnnngagac g 11711DNAArtificial SequenceA synthetic DNA fragment 7gccnnnnngg c 11814DNAArtificial SequenceA synthetic DNA fragment 8ggatgnnnnn nnnn 14911DNAArtificial SequenceA synthetic DNA fragment 9nnnnngagac c 111010DNAArtificial SequenceA synthetic DNA fragment 10gacgcnnnnn 101111DNAArtificial SequenceA synthetic DNA fragment 11ccnnnnnnng g 111211DNAArtificial SequenceA synthetic DNA fragment 12gcnnnnnnng c 111310DNAArtificial SequenceA synthetic DNA fragment 13nnnnngagac 101411DNAArtificial ...
1) A low GC content, preferably between about 30-52%.(2) At least 2, preferably at least 3 A or U bases at positions 15-19 of the siRNA on the sense strand.(3) An A base at position 19 of the sense strand.(4) An A base at position 3 of the sense strand.(5) A U base at position 10 of the sense strand.(6) An A base at position 14 of the sense strand.(7) A base other than C at position 19 of the sense strand.(8) A base other than G at position 13 of the sense strand.(9) A Tm, which refers to the character of the internal repeat that results in inter- or intramolecular structures for one strand of the duplex, that is preferably not stable at greater than 50° C., more preferably not stable at greater than 37° C., even more preferably not stable at greater than 30° C. and most preferably not stable at greater than 20° C.(10) A base other than U at position 5 of the sense strand.(11) A base other than A at position 11 of the sense strand.(12) A base other than an A at position 1 of the sense ...
We have characterized the 5′ region of the human alpha 1(V) collagen gene (COL5A1). The transcriptional promoter is shown to have a number of features characteristic of the promoters of housekeeping and growth-control-related genes. It lacks obvious TATA and CAAT boxes, has multiple transcription start sites, has a high GC content, lies within a well-defined CpG island and has a number of consensus sites for the potential binding of transcription factor Sp1. This type of promoter structure, while unusual for a collagen gene, is consistent with the broad distribution of expression of COL5A1 and is reminiscent of the promoter structures of the genes encoding type VI collagen, which has a similarly broad distribution of expression. Stepwise deletion of COL5A1 5′ sequences, placed upstream of a heterologous reporter gene, yielded a gradual decrease in promoter activity, indicating that the COL5A1 promoter is composed of an array of cis-acting elements. A minimal promoter region contained ...
TY - JOUR. T1 - Inhibition of sequence-specific protein-DNA interaction and restriction endonuclease cleavage via triplex stabilization by poly(L-lysine)-graft-dextran copolymer. AU - Ferdous, Anwarul. AU - Akaike, Toshihiro. AU - Maruyama, Atsushi. PY - 2000/6. Y1 - 2000/6. N2 - Triplex stabilization by poly(L-lysine)-graft-dextran copolymer within a mammalian gene promoter inhibits the DNA binding activity of nuclear proteins from HeLa cells as well as restriction endonuclease cleavage at physiological pH and ionic conditions in vitro. Electrophoretic mobility shift assays using a 30-mer hornopurine-homopyrimidine stretch (located between -170 and -141 bp) of rat α1 (I) collagen gene promoter reveal that the copolymer, at its wide range of charge ratio with DNA, stabilizes triplex DNA and enhances triplex-specific inhibition of the protein - DNA interaction. When the triplex-forming region (located between -165 and -146 bp) of the promoter is engineered at the Bam H1 and Pst 1 sites of a ...
In cells productively infected with adenovirus type 5, transcription is not terminated between the E1a gene and the adjacent downstream E1b gene. Insertion of the mouse beta(maj)-globin transcription termination sequence (GGT) into the E1a coding region dramatically reduces early, but not late, E1b expression (E. Falck-Pedersen, J. Logan, T. Shenk, and J. E. Darnell, Jr., Cell 40:897-905, 1985). In the study described herein, we showed that base substitution mutations in the globin DNA that specifically relieved transcription termination also restored early E1b promoter activity in cis, establishing that maximal early E1b expression requires readthrough transcription originating from the adjacent upstream gene. To identify potential targets of readthrough activation, a series of recombinant viruses with double mutations was constructed. Each double-mutant virus strain had the transcription termination sequences in the first exon of E1a and a deletion within the transcription control region of ...
The major transcription initiation site (cap site) of PCNA is localized 89 bp upstream from the ATG codon. Neither a TATA box nor a CAAT box is found within the 600-bp region upstream of the cap site. Clusters of 10 bp of sequence, similar to the binding sites for Drosophila proteins containing homeodomains, were found in the region from -127 to -413. DNase I footprint analysis reveals that the Drosophila homeodomain proteins coded by even-skipped and zerknullt genes can specifically bind to these sites. There are two sequences, starting at -52 and -39, of which 8 and 7 (respectively) of 10 nucleotides match the consensus sequence for HeLa cell transcription factor Sp 1 binding. These results suggest that the expression of the PCNA gene is under the control of genes coding for homeodomain proteins (Yamaguchi, 1990). The proliferating-cell nuclear antigen (PCNA) promoter function resides within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a ...
Invitrogen™ Cloned AMV First-Strand cDNA Synthesis Kit 100 reactions Invitrogen™ Cloned AMV First-Strand cDNA Synthesis Kit RT-PCR Kits
Over the last 20 years several observations at the peptide level have indicated the possible existence of additional members of the vasopressin (VP)-oxytocin (OT) gene family in mammals. In this study, the human genome was analyzed for the existence of genes structurally related to the VP and OT genes. Human genomic blots probed under low stringency conditions with exon B of the human OT gene, that codes for the conserved constant region of neurophysin, revealed the presence of two distinct bands in addition to the known VP and OT gene fragments. Five clones were obtained from a library of genomic EcoRI fragments ranging from 4-8 kb, that comprised both low stringency signals, by low stringency hybridization with the OT exon B probe. One clone of 7 kb hybridized at high stringency conditions to bands of the same size as previously detected with OT exon B on a human genomic blot. However, no similarity was observed between the open reading frames of this clone and the neurophysin portion of the ...
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The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5 LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the
The target site specificity of Tos17 was revealed clearly by large-scale analysis of target sequences. The insertion site consensus sequence is the palindrome ANGTT-TSD-AACNT. Interestingly, in vitro studies of retroviral integrase activity revealed that palindromic sequences that form cruciform structures are preferred targets for retroviral integrase (Katz et al., 1998). Indeed, for retroviral integrase, a cruciform structure is a more important determinant of target preference than the specific sequence of the site. The fact that the Tos17 target site consensus sequence is a palindrome suggests that the integrase encoded by Tos17 also may have a preference for cruciform structures. However, because the nucleotide sequence is well conserved at the target sites of Tos17, it is likely that the sequence itself also is a determinant of target specificity for this retrotransposon. The conserved sequence may act as a recognition sequence for Tos17 integrase. Interestingly, a small-scale analysis of ...
Transcription from the Rous sarcoma virus (RSV) Long terminal repeat (LTR) in untransformed rat 3Yl fibroblasts is dependent on the presence of serum. Within an hour of addition of serum to a serum-deprived culture there is a 5 fold stimulation in the level of transcripts initiated at the LTR. This stimulation does not require synthesis of new proteins. Mutations in the RSV LTR revealed that serum-stimulated transcription was mostly dependent on two CCAAT boxes in the LTR, though other upstream sequences may play a secondary role. Serum caused the rapid appearance of a nuclear protein that binds to the two CCAAT boxes. This serum-induced CCAAT factor was also bound by CCAAT sequences from other promoters, e.g. those of human heat shock protein 70, human c-Ha-ras, human histone 1 etc, but not by the adenovirus origin of replication, or the SV40 enhancer core sequence. This data suggests that the serum induced CCAAT factor is related to CPl or CP2 rather than the NFl or CjEBP types of CCAAT binding
The gene spans approximately 23 kilobases and is composed of 21 exons interrupted by 20 introns. Exon sizes range from 52 bases (exon 7) to over 1200 bases (exon 21), intron sizes from 68 bases (intron L) to 10.8 kilobases (intron A). The splice sites for donor and acceptor were in agreement with the canonical GT/AG rule. Functional regions of beta ARK are described with respect to their location within the exon-intron organization of the gene. Primer extension and RNase protection assays suggest a major transcription start site approximately 246 bases upstream of the start ATG. Sequence analysis of the 5-flanking/promoter region reveals many features characteristic of mammalian housekeeping genes, i.e. the lack of a TATA box, an absent or nonstandard positioned CAAT box, high GC content, and the presence of Sp1-binding sites. The extraordinarily high GC content of the 5-flanking region (, 80%) helps define this region as a CpG island that may be a principal regulator of beta ARK ...
Transcriptional analysis of the human pseudogene psi alpha globin has revealed the following features: (1) The promoter with a 23 bp deletion between the CCAAT and ATA boxes is functional both in vitro and in vivo, 3 fold and 10 fold less efficient, respectively, than alpha. (2) Both the psi alpha and alpha globin gene promoters are active in the absence of transcriptional enhancers, either a gene-encoded or viral enhancer. (3) The mutated poly(A) addition signal in psi alpha (AATGAA) appears to be completely nonfunctional. This result provides an explanation for the absence of psi alpha transcripts in human erythroid cells.
Alternative splicing of transcripts from a single gene is often used as a mechanism for generating protein variants with diverse functions (reviewed by McKeown, 1992). In the case of transcription factor genes, alternative splicing frequently gives rise to protein isoforms with distinct or even opposing transcriptional activities (reviewed by Foulkes and Sassone‐Corsi, 1992). However, few cases are known where the DNA sequence specificity of a transcription factor is altered by alternative splicing. Pax6 belongs to this class of genes which normally code for transcription factors with modular DNA‐binding domains such as the mammalian WT‐1, the Drosophila Tramtrack and CF2 zinc finger proteins (Bickmore et al., 1992; Gogos et al., 1992; Read and Manley, 1992). Here we have demonstrated that a second Pax gene, Pax8, also codes for alternative splice products with drastically different DNA‐binding specificities.. The paired domain is a bipartite DNA‐binding region consisting of an ...
Transcription factors (TFs) regulate gene transcription and play pivotal roles in various biological processes such as development, cell cycle progression, cell differentiation and tumor suppression. Identifying cis-regulatory elements associated with TF-encoding genes is a crucial step in understanding gene regulatory networks. To this end, we have used a comparative genomics approach to identify putative cis-regulatory elements associated with TF-encoding genes in vertebrates. We have created a database named TFCONES (T ranscription F actor Genes & Associated CO nserved N oncoding E lementS) ( http://tfcones.fugu-sg.org ) which contains all human, mouse and fugu TF-encoding genes and conserved noncoding elements (CNEs) associated with them. The CNEs were identified by gene-by-gene alignments of orthologous TF-encoding gene loci using MLAGAN. We also predicted putative transcription factor binding sites within the CNEs. A significant proportion
Fibulin-1 is a multifunctional extracellular protein involved in diverse biological processes including cardiovascular development, haemostasis and cancer. To investigate the transcriptional regulation of the gene encoding fibulin-1 we cloned and analysed about 4.0kb of the 5′-flanking regions of both the human and mouse fibulin-1 genes. The human and mouse fibulin-1 promoters share little sequence similarity except for a short region of approx. 150-170bp immediately upstream of the translation start site. The conserved region contains a TATA-like sequence (ATAATT) and multiple consensus binding sites for Sp1 and activator protein 2 (AP-2). That the short conserved region in each gene confers basal promoter activity is demonstrated by transient transfections of promoter deletion constructs for both the human and mouse genes into cells that express fibulin-1 constitutively. Co-transfections of promoter constructs with expression plasmids for Sp1, Sp3 and Sp4 into Drosophila SL2 cells indicate ...
We report the isolation and complete sequence of the gene encoding the rabbit erythroid-cell-specific 15-lipoxygenase (RBC 15-LOX), containing 14 exons spanning 8.0 kb. The transcription start point was mapped by S1 nuclease-protection experiments and comparison with the sequence of the RBC 15-LOX mRNA, as defined previously by primer extension experiments. The promoter contains a TATA-like motif, but no CCAAT motif in the canonical position, and lies within a CpG-rich island. Functional analysis of the immediate 5-flanking DNA by transfection experiments shows that a 150 nucleotide (nt) 5 fragment linked to the chloramphenicol acetyltransferase gene acts as a functional promoter in both erythroid and nonerythroid cell lines and responds in an erythroid-specific manner to the enhancer from the Friend murine leukaemia virus long terminal repeat, whereas a 40-nt fragment is inactive. Intron 7 contains eight copies of a 54-nt repeat containing a region with homology to the simian virus ...
Today, there are several algorithms and tools available for aligning nucleotide sequences locally. In this master thesis is especially MegaBlast, bu also other tools in the BLAST family, used in the construction of two different applications. What these applications have in common, is that they aim to find all perfect matches when aligning short nucleotide sequences to a large nucleotide database. Both accuracy and time efficiency is emphasized in the applications. UniquePrimers, one of the applications, is constructed in the context of verifying potential primers for a polymerase chain reactions. The application uses the primers as input, and returns all sequences that include the primers within a specified distance between the primers. This means that the application verifies whether or not a pair of primers are unique for only one specific database sequence. A search takes approximately from 15 seconds up to 5 minutes to execute. By using MegaBlast and word length (W) 12, it is guaranteed ...
The present invention relates to a method for detecting gene polymorphism by PCR, using, as a primer, an oligonucleotide, wherein the third nucleotide from the 3′-end thereof is a 2′-O,4′-C-ethylene nucleotide (ENA) unit, the other oligonucleotides are natural oligonucleotides, the 3′-end position thereof is a nucleotide complementary to the nucleotide of the reference sequence of a polymorphic sequence of a target gene, and the other positions are nucleotides complementary to the nucleotide sequence of the target gene, or an oligonucleotide, wherein the 3′-end of the nucleotide sequence thereof is a polymorphic position, the second nucleotide from the 3′-end thereof is a nucleotide having a base that is not complementary to a gene to be detected, and the third nucleotide from the 3′-end thereof is a 2′-O,4′-C-ethylene nucleotide (ENA) unit; oligonucleotides used in detection of gene polymorphism; and a kit for detecting gene polymorphism, comprising the above oligonucleotides.
Transcription initiation is an important step in the process of gene regulation in prokaryotes. Promoters are stretches of DNA sequence that are present in the upstream region of transcription start sites (TSSs), where RNA polymerase and other transcription factors bind to initiate transcription. Recent advancement in sequencing technologies has resulted in huge amount of raw data in the form of whole genome sequences. This sequence data has to be annotated, in order to identify coding, non-coding and regulatory regions. Computational tools are useful for a quick and fairly reliable annotation of many genome sequences. Promoter prediction is an important step in genome annotation process which is needed, not only for the validation of predicted genes, but also for the identification of novel genes, especially those coding for non-coding RNA, which are missed by gene prediction programs. DNA sequence dependent structural properties such as DNA duplex stability, bendability and intrinsic ...
Fingerprint Dive into the research topics of Multiple positive and negative 5 regulatory elements control the cell-type-specific expression of the embryonic skeletal myosin heavy-chain gene.. Together they form a unique fingerprint. ...
We have isolated recombinant lambda (A) phages which contain a part of the rat choline acetyltransferase (ChAT) gene. Restriction and Southern blot analyses using synthetic oligonucleotides indicate that these clones overlap one another and contain at least four exons which reside in 16.4 kb of sequence encoding from the middle to the 3 end, but not the 5-region, of the rat ChAT gene. Partial sequence analyses revealed that the clones contain an exon whose nucleotide sequence corresponds to a highly conserved region of ChAT during evolution. RNase protection mapping experiments show that sequences represented by this exon are expressed at high levels in the spinal cord of adult rats and at low but detectable levels in PC12 cells. By using the genomic sequences, including the exon, as a hybridization probe, we have detected ChAT mRNAs in situ in rat tissues. In situ hybridization experiments using radioactive and non-radioactive probes revealed that cholinergic motoneurons in the spinal cord, ...
In the present study, we sought to examine the molecular basis for the differential regulation of several members of the IFN-α/β gene family (IFNA and IFNB) by IRF-3 and IRF-7. The IFNB, IFNA1, IFNA2, and RANTES promoters were activated by coexpression of either IRF-3 or IRF-7, whereas the IFNA4, IFNA7, and IFNA14 promoters were exclusively activated by IRF-7 and not by IRF-3. Analysis of protein-DNA interactions revealed that recombinant IRF-3 and IRF-7 selectively bound to different regions of the IFNB promoter; IRF-3 bound preferentially to the PRDIII domain of the IFNB promoter, while IRF-7 interacted exclusively with the PRDI domain. PCR-mediated DNA binding site selection results demonstrated that IRF-3 recognized the IRF consensus element 5′-GAAANNGAAANN-3′. Replacement of a single nucleotide within the GAAA core half-site was sufficient to preclude IRF-3 DNA binding. IRF-7 bound to a related sequence motif but with greater flexibility than IRF-3; a single nucleotide replacement did ...
Comparison of cDNA sequences of TaMSH7 gene and Triticum aestivum 3B chromosome revealed that TaMSH7 containі 15 introns and is localized in the position from 119371926 to 119373011 on 3B chromosome. Analysis of nine MSH7 5UTR derived from different plants species, including cereals Oryza sativa and Zea mays, indicates that the cDNA sequence of TaMSH7 is probably shortened from 5-end. Considering these data, a possible translation initiation codon ATG is located at 433-435 nucleotides upstream to the available cDNA gene sequence. Based on the analysis of the predicted promoter region of TaMH7 gene, a large number of CpG methylation points were determined. The methylation level of such points can reveal the features of gene expression regulation in the genomes of introgressive lines. An 816 bp sequence upstream to predicted TaMSH7 translation initiation site contains 75 CpG methylation sites and becomes a target for bisulfite sequencing to establish the predicted promoter region methylation ...
The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different
In this study, we developed a new strategy to detect DNA palindromes by coupling fast annealing genomic DNA treated by S1 nuclease (GAPF) with high-throughput sequencing (GAP-Seq) and recovery of novel palindrome junctions. We chose to use the MCF-7 breast cancer cell line for this initial proof-of-principle study because it has been extensively analyzed at the genomic level, allowing us to determine if our approach could generate novel data. In fact, none of our palindrome junctions had been identified by either sequence analysis or novel breakpoint analyses of MCF-7 [22, 25, 26]. This difference may be a result of either or both of two constraints presented by the characteristics of palindromes: 1) the breakpoint analysis was done from BAC clones, where palindromes are not stable during E.coli propagation, and 2) most of novel breakpoints identified here are located in or near to repeat-masked regions and would not be recovered by mapping of high-throughput sequencing data without knowing more ...
Functional RNAs (fRNAs) are being recognized as an important regulatory component in biological processes. Interestingly, recent computational studies suggest that the number and biological significance of functional RNAs within coding regions (coding fRNAs) may have been underestimated. We hypothesized that such coding fRNAs will impose additional constraint on sequence evolution because the DNA primary sequence has to simultaneously code for functional RNA secondary structures on the messenger RNA in addition to the amino acid codons for the protein sequence. To test this prediction, we first utilized computational methods to predict conserved fRNA secondary structures within multiple species alignments of Saccharomyces sensu strico genomes. We predict that as much as 5% of the genes in the yeast genome contain at least one functional RNA secondary structure within their protein-coding region. We then analyzed the impact of coding fRNAs on the evolutionary rate of protein-coding genes because a
Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with normal variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (∼10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect ...
Gene conversion-like events between rearranged IGHV3-23*01 gene sequences and germline VH sequences superimposed by somatic hypermutation (SHM). IGHV3-23*01 mut
The regulation of gene expression is a fundamental process within every cell that often allows exquisite control over a genes activity (for review see [1]). Altering transcription rates is an effective strategy for regulating gene activity. It is well established that transcription of a given gene is dependent upon a promoter sequence located within a few hundred base pairs of the transcriptional start site. Promoter activity is modulated by sequence-specific transcription factors that physically interact either with the protein complexes that make up the core transcriptional machinery or with the promoter sequence itself.. In eukaryotes, the activity of a promoter can be modified by transcription factors binding to DNA sequences (frequently termed cis-regulatory modules or enhancers) that are located from hundreds to hundreds of thousands of base pairs away from the promoter. These regulatory modules can either increase or decrease the rate of transcription for a target gene, depending on the ...
Pjevac, P.; Schauberger, C.; Poghosyan, L.; Herbold, C.W.; Kessel, M.A.H.J. van; Daebeler, A.; Steinberger, M.; Jetten, M.S.M. ; Lücker, S.; Wagner, M.; Daims, H. ...
Various enhancer lengths and compositions result in similar levels of virus propagation.To characterize the synthetic enhancer sequences acquired by enhancerless SV40, viral DNA was isolated from cell lysates and subcloned. After a sequencing step, selected clones were reanalyzed by retransfecting cells to identify opportunistic genomes, which had replicated without having incorporated a functional enhancer of their own. In the case of HCMV oligonucleotides, we found all four enhancer motifs in various combinations and orientations in different isolates (Fig. 1C), which upon retesting were similarly infective (first signs of cytopathic effects [CPE] at around day 10 and full infection at around day 14 after transfection). Thus, although the lengths and compositions of the isolated enhancers were quite variable, our experimental setup apparently selected for viral enhancers with similar activities. Of note, the 18-bp repeat, which harbors a previously described binding site for NF-κB (23), was ...
Considerable insights into important cis regulatory elements in a gene can be gleaned from the identification of sequence homologies among different species. To extend and optimize the sequence comparison between human and mouse erythropoietin (Epo) genes, we have obtained new human sequence from 5,547 to 385 bp upstream of the cap site and extended the 3 flank by 489 bp. In addition, we have obtained new sequence information on the mouse Epo gene extending from within the 3 untranslated region (UTR) to 1,001 bp downstream of the polyadenylation site. Analysis of these additional sequences shows considerable homology between human and mouse Epo genes as far as 4 kb (human) or 3 kb (mouse) upstream of the cap sites, as well as far more homology at the 3 end than was previously realized. In addition, both species were found to have a high frequency of short interspersed (SINE) repetitive sequences that interrupt homologies in both the 5 flank and within the transcription unit.
mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5′ splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts],3′ splice sites (3′SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3′SS, we constructed a luciferase-β-galactosidase double-reporter system. By testing ∼90 sequences, we demonstrated that the optimum poly(Y) tract length is ∼25 nucleotides. Interspersing a purely undine-containing poly(Y) tract with cytidine resulted in increased ironssplicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3′SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in irons splicing. However, ...
A sizing catheter and method of measuring a preselected internal opening within a patient to provide a rapid and precise determination of a stretched diameter of the preselected internal opening. The sizing catheter includes a dilation balloon constructed of a thin expandable plastic which is inflatable and is utilized to determine a size of the preselected opening. The dilation balloon is inflated to an inflation threshold, wherein the dilation balloon deforms about the preselected opening and the size of the dilation balloon adjacent the preselected opening approximates a stretched diameter of the preselected opening. The sizing catheter and method may be utilized to determine an appropriate sized occluding device to thereby occlude the preselected opening.
Author(s): Afik, Shaked David | Advisor(s): Yosef, Nir | Abstract: Short DNA sequences play an important role in the immune response to pathogens. As part of the non-coding regions of the genome, short DNA sequence motifs regulate cell activation and maturation by binding chromatin modifiers and transcription factors. They also determine the ability of each cell in the adaptive immune system to respond to a specific pathogen by forming the antigen-recognizing region of their receptors. This dissertation outlines computational tools I developed for utilizing and integrating high-throughput sequencing data to study the functions of short DNA sequences in the human immune system. I focus on two main aspects of short DNA sequences: (1) As components of the regulatory landscape that control the activation of dendritic cells (DCs) in response to lipopolysaccharide (LPS), and (2) as the determinants of the specificity of T cells and B cells.The first part of my dissertation investigates the regulatory
Complementary base definition, either of the nucleotide bases linked by a hydrogen bond on opposite strands of DNA or double-stranded RNA: guanine is the complementary base of cytosine, and adenine is the complementary base of thymine in DNA and of uracil in RNA. See more.
According to the IncF replicon sequence typing (RST) scheme, which demonstrates a higher discriminatory power than PBRT (13), blaCTX-M-15-carrying plasmids pCA14 and pCA28 are assigned to FII, FIA, FIB (FAB) formula F31:A4:B1, whereas the blaCTX-M-14-carrying plasmid pCA08 is assigned to F2:A2:B20. These findings are consistent with the BLAST analysis, which indicates that pCA14 is closely related to pCA28, with 99% to 100% identity and 92% coverage, including the conserved backbone region and MRRs. pCA14 shares all backbone genes with pCA28, including genes for replication, plasmid maintenance, and conjugal transfer, as well as modules associated with virulence and biochemical pathways. A total of 20 nucleotide differences are observed between their backbone regions. A one-nucleotide difference and a two-nucleotide difference are found in the genes traE and iucC, respectively, with the remainder observed in genes of hypothetical proteins or intergenic regions. In addition, pCA14 shares all ...
An accurate identification of gene promoters remains an important challenge. Computational approaches for this problem rely on promoter sequence attributes that are believed to be critical for transcription initiation. Here we report a probabilistic model that captures two important properties of promoters, not used by previous methods, viz., the location preference and co-occurrence of promoter elements. Additionally, we found that many of the position-specific DNA elements are strongly linked with the function of the gene product. For instance, a highly conserved motif CCTTT at -1 position is strongly associated with protein synthesis, cellular and tissue development. Our comparative analysis of promoter classes reveals that the promoters devoid of CpG islands are more conserved and have fewer alternative transcription start sites. The discovered links between promoter elements and gene function allows us to infer genetic networks from promoter elements. The web server for the PSPA promoter ...
In general, primers are designed to identify specific locations within a long region of DNA, either plasmid or genomic. Primer binding sites are ideally unique within the range of DNA found in the reaction tube. Single primers are used to amplify and label DNA fragments for sequencing reactions, or as probes for Southern blots. Pairs of primers are used to delimit the range of DNA amplified during a PCR reaction. Because biological enzymes selectively add bases on the 3 end of primed double stranded DNA, the binding of the 3 end of the primer is especially important, while the 5 end of the primer can either bind or not, with relatively little effect on most uses of the primer. The binding of the 4-e 3 to the 5 end, the opposite from the DNA synthesis direction found in biological systems. Synthesis starts with a glass bead filled column containing a specific 3 base bound to the glass. Chemically activated nucleotides, termed phosphoramidites, are linked in 3 to 5 order to fabricate the ...
In general, primers are designed to identify specific locations within a long region of DNA, either plasmid or genomic. Primer binding sites are ideally unique within the range of DNA found in the reaction tube. Single primers are used to amplify and label DNA fragments for sequencing reactions, or as probes for Southern blots. Pairs of primers are used to delimit the range of DNA amplified during a PCR reaction. Because biological enzymes selectively add bases on the 3 end of primed double stranded DNA, the binding of the 3 end of the primer is especially important, while the 5 end of the primer can either bind or not, with relatively little effect on most uses of the primer. The binding of the 4-e 3 to the 5 end, the opposite from the DNA synthesis direction found in biological systems. Synthesis starts with a glass bead filled column containing a specific 3 base bound to the glass. Chemically activated nucleotides, termed phosphoramidites, are linked in 3 to 5 order to fabricate the ...
Macro-array differential hybridization of a collection of 5058 human gene transcripts represented in an IMAGE infant brain cDNA library has led to the identification of transcripts displaying preferential or specific expression in brain (Genome Res. 9 (1999) 195; http://idefix.upr420.vjf.cnrs.fr/IMAGE). Most of these genes correspond to as yet undescribed functions. Detailed characterization of the expression, sequence, and genome assignment of one of these genes named C3orf4, is reported here. The full-length sequence of the transcript was obtained by 5′ extension RT-PCR. The gene transcript (2.8 kb) encodes a 253 amino acid long protein, with four transmembrane domains. The position of the C3orf4 gene was determined at 3q12.1 thanks to the draft sequence of the human genome. It is composed of five exons spanning more than 7 kb. No TATAA box but a CpG island was found upstream of the beginning of the gene. Northern blot analysis and in situ hybridization revealed a predominant expression in
Large and systematic mRNA library design disentangles the complex sequence determinants of translation efficiency in bacteria. Comparative analyses of natural and mutated sequences have been used to probe mechanisms of gene expression, but small sample sizes may produce biased outcomes. We applied an unbiased design-of-experiments approach to disentangle factors suspected to affect translation efficiency in E. coli. We precisely designed 244,000 DNA sequences implementing 56 replicates of a full factorial design to evaluate nucleotide, secondary structure, codon and amino acid properties in combination. For each sequence, we measured reporter transcript abundance and decay, polysome profiles, protein production and growth rates. Associations between designed sequences properties and these consequent phenotypes were dominated by secondary structures and their interactions within transcripts. We confirmed that transcript structure generally limits translation initiation and demonstrated its physiological
Oligonucleotides are short sequences of nucleotides (RNA or DNA), typically with twenty or fewer bases. Automated synthesizers allow the synthesis of oligonucleotides up to 160 to 200 bases. The length of a synthesized base is usually denoted by mer (from Greek meros part). For example, a fragment of 25 bases would be called a 25-mer. Oligonucleotides are often used as probes for detecting complementary DNA or RNA because they bind readily to their complements. Examples of procedures that use oligonucleotides are DNA microarrays, Southern blots, fluorescent in situ hybridization (FISH), and the synthesis of artificial genes. Oligonucleotides composed of DNA (deoxyoligonucleotides) are often used in the polymerase chain reaction (PCR), a procedure that can be employed to amplify almost any piece of DNA. In this instance, the oligonucleotide is often referred to as a primer, or a short piece of DNA that binds to its complementary target sequence. This generates a place for a polymerase to ...