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Whole genome analysis of human papillomavirus type 16 multiple infection in cervical cancer patients.
Chansaenroj, J, Theamboonlers, A, Junyangdikul, P, Swangvaree, S, Karalak, A, Poovorawan, Y
Asian Pacific journal of cancer prevention : APJCP. 2012;(2):599-606
Abstract
The characterization of the whole genome of human papillomavirus type 16 (HPV16) from cervical cancer specimens with multiple infections in comparison with single infection samples as the oncogenic potential of the virus may differ. Cervical carcinoma specimens positive for HPV16 by PCR and INNO-LiPA were randomly selected for whole genome characterization. Two HPV16 single infection and six HPV16 multiple infection specimens were subjected to whole genome analysis by using conserved primers and subsequent sequencing. All HPV16 whole genomes from single infection samples clustered in the European (E) lineage while all multiple infection specimens belonged to the non-European lineage. The variations in nucleotide sequences in E6, E7, E2, L1 and Long control region (LCR) were evaluated. In the E6 region, amino acid changes at L83V were related to increased cancer progression. An amino acid variation N29S within the E7 oncoprotein significantly associated with severity of lesion was also discovered. In all three domains of the E2 gene non synonymous mutations were found. The L1 region showed various mutations which may be related to conformation changes of viral epitopes. Some transcription factor binding sites in the LCR region correlated to virulence were shown on GRE/1, TEF- 1, YY14 and Oct-1. HPV16 European variant prone to single infection may harbor a major variation at L83V which significantly increases the risk for developing cervical carcinoma. HPV16 non-European variants prone to multiple infections may require many polymorphisms to enhance the risk of cervical cancer development.
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2.
A generic approach to identify Transcription Factor-specific operator motifs; Inferences for LacI-family mediated regulation in Lactobacillus plantarum WCFS1.
Francke, C, Kerkhoven, R, Wels, M, Siezen, RJ
BMC genomics. 2008;:145
Abstract
BACKGROUND A key problem in the sequence-based reconstruction of regulatory networks in bacteria is the lack of specificity in operator predictions. The problem is especially prominent in the identification of transcription factor (TF) specific binding sites. More in particular, homologous TFs are abundant and, as they are structurally very similar, it proves difficult to distinguish the related operators by automated means. This also holds for the LacI-family, a family of TFs that is well-studied and has many members that fulfill crucial roles in the control of carbohydrate catabolism in bacteria including catabolite repression. To overcome the specificity problem, a comprehensive footprinting approach was formulated to identify TF-specific operator motifs and was applied to the LacI-family of TFs in the model gram positive organism, Lactobacillus plantarum WCFS1. The main premise behind the approach is that only orthologous sequences that share orthologous genomic context will share equivalent regulatory sites. RESULTS When the approach was applied to the 12 LacI-family TFs of the model species, a specific operator motif was identified for each of them. With the TF-specific operator motifs, potential binding sites were found on the genome and putative minimal regulons could be defined. Moreover, specific inducers could in most cases be linked to the TFs through phylogeny, thereby unveiling the biological role of these regulons. The operator predictions indicated that the LacI-family TFs can be separated into two subfamilies with clearly distinct operator motifs. They also established that the operator related to the 'global' regulator CcpA is not inherently distinct from that of other LacI-family members, only more degenerate. Analysis of the chromosomal position of the identified putative binding sites confirmed that the LacI-family TFs are mostly auto-regulatory and relate mainly to carbohydrate uptake and catabolism. CONCLUSION Our approach to identify specific operator motifs for different TF-family members is specific and in essence generic. The data infer that, although the specific operator motifs can be used to identify minimal regulons, experimental knowledge on TF activity especially is essential to determine complete regulons as well as to estimate the overlap between TF affinities.
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3.
Comparison of p53 and the PDZ domain containing protein MAGI-3 regulation by the E6 protein from high-risk human papillomaviruses.
Ainsworth, J, Thomas, M, Banks, L, Coutlee, F, Matlashewski, G
Virology journal. 2008;:67
Abstract
Central to cellular transformation caused by human papillomaviruses (HPVs) is the ability of E6 proteins to target cellular p53 and proteins containing PDZ domains, including MAGI-3, for degradation. The aim of this study was to compare E6-mediated degradation of p53 and MAGI-3 under parallel experimental conditions and further with respect to the involvement of proteasomes and ubiquitination. We also compared the degradation of p53 and MAGI-3 by E6 from several HPV types including different variants from HPV-33. All of the E6 genes from different HPV types displayed similar abilities to mediate the degradation of both p53 and MAGI-3 although there may be subtle differences observed with the different 33E6 variants. There were however differences in E6 mediated degradation of p53 and MAGI-3. Proteasome inhibition assays partially protected p53 from E6 mediated degradation, but did not protect MAGI-3. In addition, under conditions where p53 was ubiquitinated by E6 and MDM2 in vivo, ubiquitination of MAGI-3 was not detected. These results imply that although both p53 and MAGI-3 represent effective targets for oncogenic E6, the mechanisms by which E6 mediates p53 and MAGI-3 degradation are distinct with respect to the involvement of ubiquitination prior to proteasomal degradation.
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4.
The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint.
De Antoni, A, Pearson, CG, Cimini, D, Canman, JC, Sala, V, Nezi, L, Mapelli, M, Sironi, L, Faretta, M, Salmon, ED, et al
Current biology : CB. 2005;(3):214-25
Abstract
BACKGROUND The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands. RESULTS We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC. CONCLUSIONS We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.
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5.
NMR structures of salt-refolded forms of the 434-repressor DNA-binding domain in 6 M urea.
Pervushin, K, Wider, G, Iwai, H, Wüthrich, K
Biochemistry. 2004;(44):13937-43
Abstract
The N-terminal 63-residue fragment of the phage 434-repressor, 434(1-63), has a well-defined globular fold in H(2)O solution, and is unfolded in 6 M urea at pH 7.5. In this study, 434(1-63) has been refolded by adding either 1.7 M NaCl or 0.47 M NaTFA to the solution in 6 M urea, and the NMR structures of both refolded forms have been determined. The two refolded forms have similar free energies of unfolding and are approximately 16 kJ/mol less stable than the protein in H(2)O solution. 434(1-63) refolded with NaCl exhibits NMR chemical shifts very similar to and a three-dimensional structure nearly identical to those of 434(1-63) in H(2)O solution. The protein refolded with NaTFA also has a similar global fold, but it shows local differences near Phe44, of which two different orientations of the aromatic ring are compatible with the experimental data. This local conformational polymorphism attracted our interest because hydrophobic contacts between two subdomains of residues 1-36 and 45-63 are mediated by the Phe44 side chain. Anion binding experiments suggest that this polymorphism is caused by binding of TFA(-) anions to a cluster of positively charged Arg and Lys residues located in the loop connecting the two subdomains, with apparent binding constants for TFA(-) (K(app)) on the order of 30 mM(-1).
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Comparative genomics of the KdgR regulon in Erwinia chrysanthemi 3937 and other gamma-proteobacteria.
Rodionov, DA, Gelfand, MS, Hugouvieux-Cotte-Pattat, N
Microbiology (Reading, England). 2004;(Pt 11):3571-3590
Abstract
In the plant-pathogenic enterobacterium Erwinia chrysanthemi, almost all known genes involved in pectin catabolism are controlled by the transcriptional regulator KdgR. In this study, the comparative genomics approach was used to analyse the KdgR regulon in completely sequenced genomes of eight enterobacteria, including Erw. chrysanthemi, and two Vibrio species. Application of a signal recognition procedure complemented by operon structure and protein sequence analysis allowed identification of new candidate genes of the KdgR regulon. Most of these genes were found to be controlled by the cAMP-receptor protein, a global regulator of catabolic genes. At the next step, regulation of these genes in Erw. chrysanthemi was experimentally verified using in vivo transcriptional fusions and an attempt was made to clarify the functional role of the predicted genes in pectin catabolism. Interestingly, it was found that the KdgR protein, previously known as a repressor, positively regulates expression of two new members of the regulon, phosphoenolpyruvate synthase gene ppsA and an adjacent gene, ydiA, of unknown function. Other predicted regulon members, namely chmX, dhfX, gntB, pykF, spiX, sotA, tpfX, yeeO and yjgK, were found to be subject to classical negative regulation by KdgR. Possible roles of newly identified members of the Erw. chrysanthemi KdgR regulon, chmX, dhfX, gntDBMNAC, spiX, tpfX, ydiA, yeeO, ygjV and yjgK, in pectin catabolism are discussed. Finally, complete reconstruction of the KdgR regulons in various gamma-proteobacteria yielded a metabolic map reflecting a globally conserved pathway for the catabolism of pectin and its derivatives with variability in transport and enzymic capabilities among species. In particular, possible non-orthologous substitutes of isomerase KduI and a new oligogalacturonide transporter in the Vibrio species were detected.