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Results for “Divergent primer”

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At least 19 recordsLinked to original sources

PubMed2025

Quantitative Real-Time PCR for Circular RNA Detection and Analysis.

In eukaryotes, nearly 2% of the genome represented by the coding proteins. However, emerging evidence suggest more than 75% of the human genome referred to as noncoding part also plays a crucial role in governing major regulatory pathways. Noncoding RNAs can be categorized into several groups, such as microRNAs (miRNAs), small nuclear RNA (snRNAs), small nucleolar RNA (snoRNAs), transfer RNA (tRNA), and circular RNA (circRNAs), which contribute to this regulatory landscape. Circular RNAs (circRNAs) are identified as a new class of regulatory noncoding RNAs with gene regulatory roles by acting as miRNA or RNA binding protein sponges or interacting with proteins. Researchers employ quantitative real-time PCR methods to examine circular RNA expression utilizing divergent primers for identification and quantification.

RNA, Circular↗

PubMed1990

Mutations affecting primer RNA interaction with the replication repressor RNA I in plasmid CoIE1: potential RNA folding pathway mutants.

The control of plasmid ColE1 copy number is mediated by the kinetics of interaction of two complementary plasmid-encoded RNAs. One RNA is the primer precursor and the other is a small counter-transcript called RNA I. The interaction of these highly structured RNAs results in inhibition of formation of mature primer RNA necessary for replication initiation. We have studied several plasmid copy number mutants which have single base changes in the primer which render the primer resistant to inhibition by RNA I despite the fact that the mutations are located outside the overlap between primer and RNA I. We propose a model to account for the resistance of the mutant primers which is based on the differential folding of the nascent primer transcripts during transcription. We propose that the mutant primers diverge in structure from their wild-type counterparts during a discrete period during transcription. During this brief divergence, they are proposed to interact kinetically more slowly with RNA I than wild-type primer because a particular domain (the anti-tail) required for efficient interaction with RNA I is buried in a stem-loop structure while this same domain is predicted to be single-stranded in the wild-type. Despite substantial sequence divergence from ColE1, the primer precursors of the related plasmids CloDF13, RSF1030 and p15A also have retained the potential to expose their anti-tail in a similar manner to ColE1, suggesting that the folding pathway has been conserved in evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

Ribosomal DNA: molecular evolution and phylogenetic inference.

Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.

Animals↗

PubMed2005

SplitTester: software to identify domains responsible for functional divergence in protein family.

BACKGROUND: Many protein families have undergone functional divergence after gene duplications such that current subgroups of the family carry out overlapping but distinct biological roles. For the protein families with known functional subtypes (a functional split), we developed the software, SplitTester, to identify potential regions that are responsible for the observed distinct functional subtypes within the same protein family. RESULTS: Our software, SplitTester, takes a multiple protein sequences alignment as input, generated from protein members of two subgroups with known functional divergence. SplitTester was designed to construct the neighbor joining tree (a split cluster) from variable-sized sliding windows across the alignment in a process called split-clustering. SplitTester identifies the regions, whose split cluster is consistent with the functional split, but may be inconsistent with the phylogeny of the protein family. We hypothesize that at least some number of these identified regions, which are not following a random mutation process, are responsible for the observed functional split. To test our method, we used reverse transcriptase from a group of Pseudoviridae retrotransposons: to identify residues specific for diverged primer recognition. Candidate regions were then mapped onto the three dimensional structures of reverse transcriptase. The locations of these amino acids within the enzyme are consistent with their biological roles. CONCLUSION: SplitTester aims to identify specific domain sequences responsible for functional divergence of subgroups within a protein family. From the analysis of retroelements reverse transcriptase family, we successfully identified the regions splitting this family according to the primer specificity, implying their functions in the specific primer selection.

Algorithms↗

PubMed2003

rpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria.

Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the rpoB gene, which encodes the bacterial beta subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited <2% partial rpoB gene sequence divergence from 1 of 20 species under study and were regarded as correctly identified at the species level. Mycobacterium abscessus and Mycobacterium mucogenicum isolates were clearly distinguished from Mycobacterium chelonae; Mycobacterium mageritense isolates were clearly distinguished from "Mycobacterium houstonense." Four isolates were not identified at the species level because they exhibited >3% partial rpoB gene sequence divergence from the corresponding type strain; they belonged to three taxa related to M. mucogenicum, Mycobacterium smegmatis, and Mycobacterium porcinum. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp rpoB sequence is a rapid and accurate tool for identification of RGM.

Base Sequence↗

PubMed1995

Functional and regulatory analysis of the OmpF-like porin, OpnP, of the symbiotic bacterium Xenorhabdus nematophilus.

The function and novel regulation of OpnP of the symbiotic/pathogenic bacterium, Xenorhabdus nematophilus was studied. In vitro pore-function analysis of purified OpnP indicated that the single-channel-conductance values were similar to that measured for the porin protein, OmpF, of Esherichia coli. Nucleotide sequence analysis revealed that the mature OpnP protein contained 348 amino acid residues and shared 55% amino acid sequence identity with OmpF. Similar to ompF, opnP mapped between asnS and aspC. The 16 transmembrane beta-sheet structures and the internal loop 3 were highly conserved, while the remaining external loop domains were more divergent. Primer extension analysis identified the start site of transcription of opnP. A sigma 70-type promoter, a perfect 20 bp OmpR-binding site, and a binding site for the antisense molecule, micF RNA, were found in the upstream region of opnP. While the overall sequence identity of the asn-opnP-aspC region was high, the intergenic region between asnS and opnP had diverged markedly. The asnS-opnP region was 313 bp shorter than the intergenic region between asnS and ompF and lacked the OmpR-binding site that is required for ompF repression by high osmolarity in E. coli. Results from osmolarity-shift experiments indicated that OpnP was not repressed by high osmolarity. It was also found that OpnP was thermally regulated.

Amino Acid Sequence↗

PubMed1996

Stress-induced rearrangement of Fusarium retrotransposon sequences.

Rearrangement of fusarium oxysporum retrotransposon skippy was induced by growth in the presence of potassium chlorate. Three fungal strains, one sensitive to chlorate (Co60) and two resistant to chlorate and deficient for nitrate reductase (Co65 and Co94), were studied by Southern analysis of their genomic DNA. Polymorphism was detected in their hybridization banding pattern, relative to the wild type grown in the absence of chlorate, using various enzymes with or without restriction sites within the retrotransposon. Results were consistent with the assumption that three different events had occurred in strain Co60: genomic amplification of skippy yielding tandem arrays of the element, generation of new skippy sequences, and deletion of skippy sequences. Amplification of Co60 genomic DNA using the polymerase chain reaction and divergent primers derived from the retrotransposon generated a new band, corresponding to one long terminal repeat plus flanking sequences, that was not present in the wild-type strain. Molecular analysis of nitrate reductase-deficient mutants showed that generation and deletion of skippy sequences, but not genomic amplification in tandem repeats, had occurred in their genomes.

Blotting, Southern↗

PubMed1990

Control of gene expression in the temperate coliphage 186. VIII. Control of lysis and lysogeny by a transcriptional switch involving face-to-face promoters.

The lysogenic and early lytic operons of the temperate coliphage 186 are transcribed divergently. Primer extension mapping of the 5' ends of these in vivo transcripts showed that the rightward lytic promoter, pR, and the leftward lysogenic promoter, pL, are arranged face-to-face, with their transcripts overlapping by 60 bases. We examined the control of transcription from pR and pL using galK as a reporter gene. The product of the lysogenic cI gene strongly repressed pR transcription while allowing pL transcription. The product of the lytic apl gene (formerly CP75) strongly repressed pL transcription while allowing pR transcription. Thus, the cI-pR-pL-apl region functioned as a transcriptional switch, determining whether transcription was lytic or lysogenic. Also, the cI gene product was able to stimulate pL, possibly by alleviating an inhibition of pL transcription caused by convergent transcription from pR. Other consequences of the face-to-face promoter arrangement are discussed.

Base Sequence↗

PubMed1998

MAGE Xp-2: a member of the MAGE gene family isolated from an expression library using systemic lupus erythematosus sera.

Two regions of the genome contain members of the MAGE gene family; Xq27-qter and Xp21.3. We isolated a transcript, MAGE Xp-2, by screening a cDNA library from the human epithelial carcinoma cell line, HEp-2, using autoantibodies from patients with systemic lupus erythematosus (SLE). The open reading frame (ORF) of MAGE Xp-2 is entirely contained in exon 4, a signature feature of the MAGE gene family. While MAGE Xp-2 shares genomic homology with MAGE Xp-1, the predicted proteins are quite divergent. Specific primers were designed to reliably distinguish between MAGE Xp-1 and MAGE Xp-2 expression. MAGE Xp-2 is expressed in testis, but not in other normal tissues. It is also expressed strongly in two of seven melanoma cell lines and one of four breast carcinomas. MAGE gene expression may be important not only for tumor recognition and cancer therapy, but, because it is the apparent target of autoantibodies in SLE sera, it may also play a role in autoimmune diseases.

Amino Acid Sequence↗

PubMed2005

The intron 1 of HPV 16 has a suboptimal branch point at a guanosine.

The branch point sequence (BPS) of intron 1 of the HPV-16 was determined via RT-PCR in a cell free system, using lariat intermediates obtained by in vitro splicing reactions. We used synthetic E6/E7 transcripts and HeLa nuclear protein extracts to obtain the splicing intermediates. Then, a divergent oligonucleotide primer set, pairing on the lariat RNA that encompassed the 2'-5' phosphodiester bond formed between the 5' end of the intron and the BPS, was used for cDNA synthesis and PCR amplification. Subsequent RT-PCR assays revealed four splicing intermediates, made up of a major intermediary corresponding to the BPS and four cryptic branched sequences. Only intermediates bound at the 5' end of the intron are probably the authentic branch point sequence, and all of them branch at guanosine 328 instead of the typical adenosine. Unusually, the BPS of intron 1 of HPV-16 is a suboptimal sequence (AGUGAGU) that differs from the eukaryotic consensus BPS, which correlates with the splicing profile observed for early transcripts of HPV-16 in tumors and tumor derived cell lines. The implications of this unusual branch point sequence for splicing of the HPV-16 pre-mRNA are discussed.

Cell Extracts↗

PubMed1994

Detection of circular excised DNA deletion elements in Tetrahymena thermophila during development.

Extensive programmed DNA deletion occurs in ciliates during development. In this study we examine the excised forms of two previously characterized deletion elements, the R- and M-element, in Tetrahymena. Using divergently oriented primers in polymerase chain reactions we have detected the junctions formed by joining the two ends of these elements, providing evidence for the presence of circular excised forms. These circular forms were detected in developing macronuclear DNA from 12-24 h after mating began, but not in micronuclear or whole cell DNA of vegetative cells. They are present at very low abundance, detectable after PCR only through hybridization with specific probes. Sequence analysis shows that the circle junctions occur at or very near the known ends of the elements. There is sequence microheterogeneity in these junctions, which does not support a simple reciprocal exchange model for DNA deletion. A model involving staggered cuts and variable mismatch repair is proposed to explain these results. This model also explains the sequence microheterogeneity previously detected among the junction sequences retained in the macronuclear chromosome.

Animals↗

PubMed2004

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon.

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in approximately 10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.

Amino Acid Sequence↗

PubMed2004

Utility of environmental primers targeting ancient enzymes: methylotroph detection in Lake Washington.

Methods have been explored for detection of methylotrophs in natural samples, using environmental primers based on genes involved in the tetrahydromethanopterin (H4MPT)-linked C1 transfer pathway. The underlying hypotheses were that the H4MPT-linked pathway is an ancient methylotrophy pathway, based on gene divergence, and that primers targeting more divergent genes will detect a broader variety of methylotrophs compared to the variety uncovered using probes and primers targeting highly conserved genes. Three groups of novel primer sets were developed targeting mch, mtdB, and fae, key genes in the H4MPT-linked pathway, and these were used to assess the variety of microorganisms possessing these genes in sediments from Lake Washington in Seattle, WA. Environmental clone libraries were constructed for each of the genes and were analyzed by RFLP, and representatives of different RFLP groups were sequenced and subjected to phylogenetic analysis. A combination of all three sets of novel primers allowed detection of the two previously characterized groups of methylotrophs in the site: methanotrophs of the (alpha- and the gamma-proteobacterial groups, belonghg to genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, Methylomicrobium, and Methylococcus. In addition to the genes belonging to known methanotroph populations, novel genes were identified, suggesting existence of previously undetected microbial groups possessing C1 transfer functions in this site. These included sequences clustering with the well-characterized methylotrophic phyla, Methylobacterium, Hyphomicrobium, and Xanthobacter. In addition, sequences divergent from those known for any groups of methylotrophs or methanogens were obtained, suggesting the presence of a yet unidentified microbial group possessing this H4MPT-linked C1 transfer pathway.

Bacteria↗

PubMed2002

A polymerase chain reaction-based method for cloning novel members of a gene family using a combination of degenerate and inhibitory primers.

We have developed a novel method for cloning gene family members by using a polymerase chain reaction technique. The method is based on the amplification of a broad range of homologous genes in combination with the specific inhibition of already cloned genes. To accomplish this, we designed degenerate primers to highly conserved regions among the gene family members, and inhibitory primers to the divergent region at the 3'-margin of each degenerate primer. The 5'-end of the inhibitory primer, the 3'-end of which was aminated, had 3-4 bases overlapping the 3'-end of the degenerate primer. The potential of this method was demonstrated by the successful cloning of a novel member of the yeast MKC7/YAP3 gene family homologue from a filamentous fungus, Aspergillus oryzae, by inhibiting amplification of an already cloned homologue, opsB.

Amino Acid Sequence↗

PubMed1997

Swine centromeric DNA repeats revealed by primed in situ (PRINS) labeling.

In swine, distinct centromeric satellite DNA families have been described that correspond to either all the metacentric chromosomes except the Y (Mc1) or all the acrocentric chromosomes (Ac2). Using primed in situ (PRINS) labeling, we show here that primers derived from various sequences specifically label the centromeres of different subgroups of chromosomes. Among five primers derived from centromeric sequences of acrocentric chromosomes reported to be very homogeneous, four recognize all the acrocentric chromosomes, whereas one labels prominently chromosome 17. For the metacentric chromosomes, six primers have been derived from several divergent sequences. Among these primers, two recognize all the metacentric chromosomes except 5, 10, and 12. Three other primers label small subsets of metacentric chromosomes, including the X and one or two additional chromosomes. The last primer is specific to chromosome 1. These preliminary results suggest that it should be possible to define specific primers for almost every swine chromosome. Already, some of the primers reported here permit a distinction between swine chromosomes difficult to differentiate without banding, such as the X chromosome and chromosome 9. Therefore, the PRINS technique using centromeric motifs constitutes an additional tool for cytogenetic studies in swine.

Animals↗

PubMed2005

Intron-flanking EST-PCR markers: from genetic marker development to gene structure analysis in Rhododendron.

With a long-term goal of constructing a linkage map of Rhododendron enriched with gene-specific markers, we utilized Rhododendron catawbiense ESTs for the development of high-efficiency (in terms of generating polymorphism frequency) PCR-based markers. Using the gene-sequence alignment between Rhododendron ESTs and the genomic sequences of Arabidopsis homologs, we developed 'intron-flanking' EST-PCR-based primers that would anneal in conserved exon regions and amplify across the more highly diverged introns. These primers resulted in increased efficiency (61% vs. 13%; 4.7-fold) of polymorphism-detection compared with conventional EST-PCR methods, supporting the assumption that intron regions are more diverged than exons. Significantly, this study demonstrates that Arabidopsis genome database can be useful in developing gene-specific PCR-based markers for other non-model plant species for which the EST data are available but genomic sequences are not. The comparative analysis of intron sizes between Rhododendron and Arabidopsis (made possible in this study by aligning of Rhododendron ESTs with Arabidopsis genomic sequences and the sequencing of Rhododendron genomic PCR products) provides the first insight into the gene structure of Rhododendron.

Arabidopsis↗

PubMed2006

Conspecific and interspecific interactions between the FEM-2 and the FEM-3 sex-determining proteins despite rapid sequence divergence.

Using degenerate oligonucleotide primers, we isolated the Caenorhabditis remanei orthologue of the C. elegans sex-determining phosphatase gene fem-2 as well as two other protein phosphatase homologues. Despite the significant sequence divergence between C. elegans and C. remanei FEM-2, we used RNAi-mediated gene knockdown to demonstrate that at least some aspects of male development require FEM-2 function in C. remanei. Consistent with this functional conservation, the conspecific interaction between the FEM-2 and the FEM-3 proteins observed in C. elegans also occurs in C. remanei. To further explore whether the rapid evolution of FEM-2 and FEM-3 affects their molecular interactions, we tested for cross-species interactions between the proteins from C. elegans, C. briggsae, and C. remanei. Although all FEM-2/FEM-3 pairs from a single species interact, only two out of six interspecific pairs bind each other, showing that FEM-2 and FEM-3 are coevolving. Both interspecific interactions involved C. briggsae FEM-3. We constructed chimeric versions of FEM-2 consisting of various combinations of the C. elegans and C. remanei proteins. C. briggsae FEM-3 interacted with all the chimeras, even those that did not interact with either C. elegans or C. remanei FEM-3. We hypothesize that the promiscuity of C. briggsae FEM-3 reflects an increased reliance on evolutionarily constrained regions of FEM-2 for binding. If so, our data support the notion that the coevolution of two interacting proteins sometimes involves a shift in the domains that contribute to binding.

Amino Acid Sequence↗

PubMed2006

Development of species-specific PCR primer sets for the detection of Leptospira.

Spirochetes of the genus Leptospira infect animals and humans and are the causative agents for the emerging infectious disease leptospirosis. Rapid and simple assays for the identification of individual Leptospira species are currently not available. For identification of individual Leptospira species, PCR primers that detect the ompL1 gene sequence for the majority of pathogenic leptospires were developed in this study. The primer pairs detect Leptospira interrogans, Leptospira borgpetersenii, Leptospira kirschneri, Leptospira santarosai, Leptospira weilii and Leptospira noguchii, without cross-reacting with other Leptospira species. The development of the primers revealed a divergence of the ompL1 gene within L. interrogans, splitting this species into two separate groups. The species-specific primers will be especially useful in epidemiological studies and disease outbreak investigations for the detection of Leptospira species in human, animal and environmental samples.

Bacterial Outer Membrane Proteins↗