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Sequence Context (sequence + context)
Selected AbstractsAxoneme-dependent tubulin modifications in singlet microtubules of the Drosophila sperm tailCYTOSKELETON, Issue 4 2008Henry D. Hoyle Abstract Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different ,-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence context was also important. We compared tubulin modifications in the 9 + 9 + 2 insect sperm tail axonemes of Drosophila with the canonical 9 + 2 axonemes of sperm of the sea urchin Lytichinus pictus and the 9 + 0 motile sperm axonemes of the eel Anguilla japonica. In contrast to Drosophila sperm, L. pictus sperm have equivalent levels of modified tubulins in both doublet and central pair microtubule fractions, whereas the doublets of A. japonica sperm exhibit little glutamylation but extensive glycylation. Tubulin C-terminal modifications are a prevalent feature of motile axonemes, but there is no conserved pattern for placement or amount of these modifications. We conclude their functions are likely species-specific. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Polypyrimidine tract binding protein regulates alternative splicing of an aberrant pseudoexon in NF1FEBS JOURNAL, Issue 24 2008Michela Raponi In disease-associated genes, understanding the functional significance of deep intronic nucleotide variants represents a difficult challenge. We previously reported that an NF1 intron 30 exonization event is triggered from a single correct nomenclature is ,c.293-279 A>G' mutation [Raponi M, Upadhyaya M & Baralle D (2006) Hum Mutat 27, 294,295]. In this paper, we investigate which characteristics play a role in regulating inclusion of the aberrant pseudoexon. Our investigation shows that pseudoexon inclusion levels are strongly downregulated by polypyrimidine tract binding protein and its homologue neuronal polypyrimidine tract binding protein. In particular, we provide evidence that the functional effect of polypyrimidine tract binding protein is proportional to its concentration, and map the cis -acting elements that are principally responsible for this negative regulation. These results highlight the importance of evaluating local sequence context for diagnostic purposes, and the utility of developing therapies to turn off activated pseudoexons. [source] Deletion hotspot in the argininosuccinate lyase gene: association with topoisomerase II and DNA polymerase , sites ,HUMAN MUTATION, Issue 11 2006John Christodoulou Abstract Molecular analysis of argininosuccinate lyase (ASAL) deficiency has led to the identification of a deletion hotspot in the ASL gene. Six individuals with ASAL deficiency had alleles that led to a complete absence of exon 13 from the ASL mRNA; each had a partial deletion of exon 13 in the genomic DNA. In all six patients, the deletions begin 18 bp upstream of the 3, end of exon 13. In four cases, the deletions were 13 bp in length, and ended within exon 13, whereas in two other patients the deletions were 25 bp and extended into intron 13. The sequence at which these deletions begin overlaps both a putative topoisomerase II recognition site and a DNA polymerase , mutation/frameshift site. Moreover, the topoisomerase II cut site is situated precisely at the beginning of the deletions, which are flanked by small (2- and 3-bp) direct repeats. We note that a similar concurrence of these two putative enzyme sites can be found in a number of other deletion sites in the human genome, most notably the ,F508 deletion in the CFTR gene. These findings suggest that the joint presence of these two enzyme sites represents a DNA sequence context that may favor the occurrence of small deletions. Hum Mutat 27(11), 1065,1071, 2006. © 2006 Wiley-Liss, Inc. [source] Using Real-time PCR to Discriminate and Quantify the Closely Related Wheat Pathogens Oculimacula yallundae and Oculimacula acuformisJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2005K. Walsh Abstract Oculimacula yallundae and O. acuformis are the causal agents of eyespot disease of wheat and other cereals. The two fungi react differently to the application of fungicides, but they cannot be distinguished visually and their occurrence is often masked by other less damaging pathogens. Current methods to detect and distinguish Oculimacula species are impractical when testing large numbers of samples. A real-time polymerase chain reaction (PCR) assay, suitable for large-scale testing, was developed and used for quantitative detection and discrimination of O. yallundae and O. acuformis. As the available DNA sequences differ by only a small number of conserved nucleotide polymorphisms, three different methods were investigated to achieve the desired specificity. A combination of mutagenically separated PCR and a shortened primer gave the best specificity for O. yallundae and O. acuformis, respectively. Although the comparison illustrated the practicalities of each method, it was not possible to devise a fixed set of rules for the design of primers to discriminate two closely related sequences; it is assumed that sequence context is an overriding and difficult to predict factor, and that a range of empirical approaches may need to be taken to reach the specificity required. [source] The processing of a Benzo(a)pyrene adduct into a frameshift or a base substitution mutation requires a different set of genes in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 2 2000Nathalie Lenne-Samuel Replication through a single DNA lesion may give rise to a panel of translesion synthesis (TLS) events, which comprise error-free TLS, base substitutions and frameshift mutations. In order to determine the genetic control of the various TLS events induced by a single lesion, we have chosen the major N2-dG adduct of (+)- anti -Benzo(a)pyrene diol epoxide [(+)- anti -BPDE] adduct located within a short run of guanines as a model lesion. Within this sequence context, in addition to the major event, i.e. error-free TLS, the adduct also induces base substitutions (mostly G , T transversions) and ,1 frameshift mutations. The pathway leading to G , T base substitution mutagenesis appears to be SOS independent, suggesting that TLS is most probably performed by the replicative Pol III holoenzyme itself. In contrast, both error-free and frameshift TLS pathways are dependent upon SOS-encoded functions that belong to the pool of inducible DNA polymerases specialized in TLS (translesional DNA polymerases), namely umuDC (Pol V) and dinB (Pol IV). It is likely that, given the diversity of conformations that can be adopted by lesion-containing replication intermediates, cells use one or several translesional DNA polymerases to achieve TLS. [source] Genomic mutation rates: what high-throughput methods can tell usBIOESSAYS, Issue 9 2009Koodali T. Nishant Abstract High-throughput DNA analyses are increasingly being used to detect rare mutations in moderately sized genomes. These methods have yielded genome mutation rates that are markedly higher than those obtained using pre-genomic strategies. Recent work in a variety of organisms has shown that mutation rate is strongly affected by sequence context and genome position. These observations suggest that high-throughput DNA analyses will ultimately allow researchers to identify trans -acting factors and cis sequences that underlie mutation rate variation. Such work should provide insights on how mutation rate variability can impact genome organization and disease progression. [source] Structure of a human telomeric DNA sequence stabilized by 8-bromoguanosine substitutions, as determined by NMR in a K+ solutionFEBS JOURNAL, Issue 14 2007Akimasa Matsugami The structure of human telomeric DNA is controversial; it depends upon the sequence contexts and the methodologies used to determine it. The solution structure in the presence of K+ is particularly interesting, but the structure is yet to be elucidated, due to possible conformational heterogeneity. Here, a unique strategy is applied to stabilize one such structure in a K+ solution by substituting guanosines with 8-bromoguanosines at proper positions. The resulting spectra are cleaner and led to determination of the structure at a high atomic resolution. This demonstrates that the application of 8-bromoguanosine is a powerful tool to overcome the difficulty of nucleic acid structure determination arising from conformational heterogeneity. The obtained structure is a mixed-parallel/antiparallel quadruplex. The structure of telomeric DNA was recently reported in another study, in which stabilization was brought about by mutation and resultant additional interactions [Luu KN, Phan AT, Kuryavyi V, Lacroix L & Patel DJ (2006) Structure of the human telomere in K+ solution: an intramolecular (3+1) G-quadruplex scaffold. J Am Chem Soc 128, 9963,9970]. The structure of the guanine tracts was similar between the two. However, a difference was seen for loops connecting guanine tracts, which may play a role in the higher order arrangement of telomeres. Our structure can be utilized to design a small molecule which stabilizes the quadruplex. This type of molecule is supposed to inhibit a telomerase and thus is expected to be a candidate anticancer drug. [source] Somatic microindels: analysis in mouse soma and comparison with the human germline,,HUMAN MUTATION, Issue 1 2007Kelly D. Gonzalez Abstract Microindels, defined as mutations that result in a colocalized microinsertion and microdeletion with a net gain or loss of between 1 and 50 nucleotides, may be an important contributor to cancer. We report the first comprehensive analysis of somatic microindels. Our large database of mutations in the lacI transgene of Big Blue® mice contains 0.5% microindels, 2.8% pure microinsertions, and 11.5% pure microdeletions. There appears to be no age, gender, or tissue-type specificity in the frequency of microindels. Of the independent somatic mutations that result in a net in-frame insertion or deletion, microindels are responsible for 13% of protein expansions and 6% of protein contractions. These in-frame microindels may play a crucial role in oncogenesis and evolution via "protein tinkering" (i.e., modest expansion or contraction of proteins). Four characteristics suggest that microindels are caused by unique mechanisms, not just simple combinations of the same mechanisms that cause pure microinsertions and pure microdeletions. First, microinsertions and microdeletions commonly occur at hotspots, but none of the 30 microindels are recurrent. Second, the sizes of the deletions and insertions in microindels are larger and more varied than in pure microdeletions and pure microinsertions. Third, microinsertions overwhelmingly repeat the adjacent base (97%) while the insertions in microindels do so only infrequently (17%). Fourth, analysis of the sequence contexts of microindels is consistent with unique mechanisms including recruitment of translesion DNA synthesis polymerases. The mouse somatic microindels have characteristics similar to those of human germline microindels, consistent with similar causative mechanisms in mouse and human, and in soma and germline. Hum Mutat 28(1), 69,80, 2007. Published 2006 Wiley-Liss, Inc. 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