119,835 research outputs found
Protein translocation across mitochondrial membranes
No full textProtein translocation across biological membranes is of fundamental importance for the biogenesis of organelles and in protein secretion. We will give an overview of the recent achievements in the understanding of protein translocation across mitochondrial membranes(1-5). In particular we will focus on recently identified components of the mitochondrial import apparatus
A novel three-colour fluorescence in situ hybridization approach for the detection of t(7;12)(q36;p13) in acute myeloid leukaemia reveals new cryptic three way translocation t(7;12;16)
No full text© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The t(7;12)(q36;p13) translocation is a recurrent chromosome abnormality that involves the ETV6 gene on chromosome 12 and has been identified in 20–30% of infant patients with acute myeloid leukaemia (AML). The detection of t(7;12) rearrangements relies on the use of fluorescence in situ hybridization (FISH) because this translocation is hardly visible by chromosome banding methods. Furthermore, a fusion transcript HLXB9-ETV6 is found in approximately 50% of t(7;12) cases, making the reverse transcription PCR approach not an ideal screening method. Considering the report of few cases of variant translocations harbouring a cryptic t(7;12) rearrangement, we believe that the actual incidence of this abnormality is higher than reported to date. The clinical outcome of t(7;12) patients is believed to be poor, therefore an early and accurate diagnosis is important in the clinical management and treatment. In this study, we have designed and tested a novel three-colour FISH approach that enabled us not only to confirm the presence of the t(7;12) in a number of patients studied previously, but also to identify a cryptic t(7;12) as part of a complex rearrangement. This new approach has proven to be an efficient and reliable method to be used in the diagnostic setting
Construction of Balanced Translocation t(1;11)(q42.1;q14.3) Probe and Screening Application in Genomic Samples in Taiwan
No full textThe disrupted-in-schizophrenia 1 (DISC1) gene is a candidate gene in schizophrenia. The balanced t(1;11)(q42.1;q14.3) translocation with a breakpoint between exons 8 and 9 of DISC1 has been found to be co- segregated with psychosis in a Scottish family. To examine whether the t(1 ;11)(q42.1;q14.3 ) translocation exists in Taiwanese samples, we constructed a plasmid probe that carried the two DNA fragments of chromosome 1 (738 bp) and chromosome 11 (719 bp) that covered the breakpoint. This probe was validated using a derived DNA gift from the translocation carrier of the Scottish family. We screened genomic DNA samples from 619 subjects (507 cases and 112 controls). None of the subjects showed the designed polymerase chain reaction (PCR) product detected by the probe. We concluded that the significant association between schizophrenia and the DISC1 gene in the Taiwanese sample was not caused by balance translocation, but rather by polymorphic variations of the gene to be detected
Linkage map construction involving a reciprocal translocation
No full textThis paper is concerned with a novel statistical–genetic approach for the construction of linkage maps in populations obtained from reciprocal translocation heterozygotes of barley (Hordeum vulgare L.). Using standard linkage analysis, translocations usually lead to ‘pseudo-linkage’: the mixing up of markers from the chromosomes involved in the translocation into a single linkage group. Close to the translocation breakpoints recombination is severely suppressed and, as a consequence, ordering markers in those regions is not feasible. The novel strategy presented in this paper is based on (1) disentangling the “pseudo-linkage” using principal coordinate analysis, (2) separating individuals into translocated types and normal types and (3) separating markers into those close to and those more distant from the translocation breakpoints. The methods make use of a consensus map of the species involved. The final product consists of integrated linkage maps of the distal parts of the chromosomes involved in the translocation
Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex
No full textThe bacterial ATPase SecA and protein channel complex SecYEG form the core of an essential protein translocation machinery. The nature of the conformational changes induced by each stage of the hydrolytic cycle of ATP and how they are coupled to protein translocation are not well understood. The structure of the SecA-SecYEG complex revealed a 2-helix-finger (2HF) of SecA in an ideal position to contact the substrate protein and push it through the membrane. Surprisingly, immobilization of this finger at the edge of the protein channel had no effect on translocation, whereas its imposition inside the channel blocked transport. This analysis resolves the stoichiometry of the active complex, demonstrating that after the initiation process translocation requires only one copy each of SecA and SecYEG. The results also have important implications on the mechanism of energy transduction and the power stroke driving transport. Evidently, the 2HF is not a highly mobile transducing element of polypeptide translocation
The protein translocation systems in plants - composition and variability on the example of Solanum lycopersicum
No full textBackground: Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species.
Results: The literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identified (co-)orthologues in plants, which in combination yielded 148 and 143 orthologues in A. thaliana and S. lycopersicum, respectively. Interestingly, we traced 82% overlap in findings from both approaches though we did not find any orthologues for 27% of the factors by either procedure. In turn, 29% of the factors displayed the presence of more than one (co-)orthologue in tomato. Moreover, our analysis revealed that the genomic composition of the translocation machineries in the bryophyte Physcomitrella patens resemble more to higher plants than to single celled green algae. The monocots (Z. mays and O. sativa) follow more or less a similar conservation pattern for encoding the translocon components. In contrast, a diverse pattern was observed in different eudicots.
Conclusions: The orthologue search shows in most cases a clear conservation of components of the translocation pathways/machineries. Only the Get-dependent integration of tail-anchored proteins seems to be distinct. Further, the complexity of the translocation pathway in terms of existing orthologues seems to vary among plant species. This might be the consequence of palaeoploidisation during evolution in plants; lineage specific whole genome duplications in Arabidopsis thaliana and triplications in Solanum lycopersicum
Assembly and mechanism of bacterial twin-arginine translocation systems
No full textThe bacterial twin-arginine translocation (Tat) pathway is able to export pre-folded
cofactor containing proteins across the cytoplasmic membrane. Tat substrates bear
cleavable N-terminal signal peptides that are characterized by the presence of a
critical and highly conserved twin-arginine motif which lends the Tat pathway its
name.
In Escherichia coli and many other Gram-negative bacteria, three integral membrane
proteins: TatA, TatB and TatC are essential for Tat-dependent translocation. In
contrast Bacillus subtilis possesses a simpler TatAC system which lacks the TatB
component. In E. coli the TatA protein assembles into homo-oligomeric complexes
that vary considerably in size. The TatA proteins found in B. subtilis do not exhibit
the same degree of heterogeneity and this suggested mechanistic differences between
the Tat pathways of Gram-negative and Gram-positive bacteria. How the Tat system
works is still poorly understood, and the work presented in this thesis sought to gain
insights into the assembly and mechanism of E. coli and B. subtilis Tat pathways.
This work focused on the study of two previously uncharacterized components: the
E. coli TatA paralog TatE subunit and B. subtilis TatAc subunit.
In this thesis the purification and characterization of E. coli TatE complexes is
reported. Using analytical gel filtration chromatography, blue-native gelelectrophoresis
(BN-PAGE) and single-particle analysis of purified TatE complexes,
it was found that the TatE complexes are more discrete than the highly
heterogeneous TatA complexes. This finding, together with the ability of TatE to
support the translocation of the 90-kDa TorA protein, suggested alternative
translocation models in which single TatE complexes do not contribute the bulk of
the translocation channel, similar to the B. subtilis model.
In addition, co-purification and BN-PAGE experiments demonstrated for the first
time that TatE interacts with TatA to form TatAE mixed complexes in the
membrane, and reveals a completely novel form of Tat complex that might be
functionally significant.
A soluble population of TatE was also identified in E. coli cell extracts, and phase
separation experiments using Triton X-114 suggested it may be mis-localized.
In a separate set of studies, the ability of the B. subtilis TatAc protein to form active
translocases in combination with the B. subtilis TatCd or TatCy proteins was
investigated for the first time. The TatAcCd and TatAcCy mixed translocases were
able to translocate several E. coli Tat substrates including, TorA, AmiA and AmiC.
Finally BN-PAGE and gel filtration chromatography showed that the TatAcCd and
TatAcCy complexes were significantly smaller than the previously described E. coli
TatABC substrate-binding complex
Driven translocation of a polymer: Fluctuations at work
No full textThe impact of thermal fluctuations on the translocation dynamics of a polymer chain driven through a narrow pore has been investigated theoretically and by means of extensive molecular dynamics (MD) simulation. The theoretical consideration is based on the so-called velocity Langevin (V-Langevin) equation which determines the progress of the translocation in terms of the number of polymer segments, s(t), that have passed through the pore at time t due to a driving force f. The formalism is based only on the assumption that, due to thermal fluctuations, the translocation velocity v=s(t) is a Gaussian random process as suggested by our MD data. With this in mind we have derived the corresponding Fokker-Planck equation (FPE) which has a nonlinear drift term and diffusion term with a time-dependent diffusion coefficient D(t). Our MD simulation reveals that the driven translocation process follows a super diffusive law with a running diffusion coefficient D(t)?t? where ?<1. This finding is then used in the numerical solution of the FPE which yields an important result: For comparatively small driving forces fluctuations facilitate the translocation dynamics. As a consequence, the exponent ? which describes the scaling of the mean translocation time ??? with the length N of the polymer, ????N? is found to diminish. Thus, taking thermal fluctuations into account, one can explain the systematic discrepancy between theoretically predicted duration of a driven translocation process, considered usually as a deterministic event, and measurements in computer simulations. In the nondriven case, f=0, the translocation is slightly subdiffusive and can be treated within the framework of fractional Brownian motion (fBm).Delft Institute of Applied MathematicsElectrical Engineering, Mathematics and Computer Scienc
A family case of fertile human 45,X,psu dic(15;Y) males
No full textWe report on a familial case including four male probands from three generations with a 45,X,psu dic(15;Y)(p11.2;q12) karyotype. 45,X is usually associated with a female phenotype and only rarely with maleness, due to translocation of small Y chromosomal fragments to autosomes. These male patients are commonly infertile because of missing azoospermia factor regions from the Y long arm. In our familial case we found a pseudodicentric translocation chromosome, that contains almost the entire chromosomes 15 and Y. The translocation took place in an unknown male ancestor of our probands and has no apparent effect on fertility and phenotype of the carrier. FISH analysis demonstrated the deletion of the pseudoautosomal region 2 (PAR2) from the Y chromosome and the loss of the nucleolus organizing region (NOR) from chromosome 15. The formation of the psu dic(15;Y) chromosome is a reciprocal event to the formation of the satellited Y chromosome (Yqs). Statistically, the formation of 45,X,psu dic(15;Y) (p11.2;q12) is as likely as the formation of Yqs. Nevertheless, it has not been described yet. This can be explained by the dicentricity of this translocation chromosome that usually leads to mitotic instability and meiotic imbalances. A second event, a stable inactivation of one of the two centromeres is obligatory to enable the transmission of the translocation chromosome and thus a stably reduced chromosome number from father to every son in this family
Protein folding causes an arrest of preprotein translocation into mitochondria in vivo
No full textWith vital yeast cells, a hybrid protein consisting of the amino- terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria. Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain. Thereby, three salient features of mitochondrial protein uptake in vivo were demonstrated: its posttranslational character; the requirement for unfolding of precursors; and import through translocation contact sites. The permanent occupation of translocation sites by the fusion protein inhibited the import of other precursors; it did, however, not lead to leakage of mitochondrial ions, implying the existence of a channel that is sealed around the membrane spanning polypeptide segment
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