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Selected translocation of plasmid genes: frequency and regional specificity of translocation of the Tn3 element.

A procedure is described that selects for the insertion of transposable antibiotic resistance elements in a variety of recipient replicons. The selected translocation procedure, which employs a plasmid having a temperature-sensitive defect in replication as a donor of transposable genetic elements, was used to investigate certain characteristics of the translocation process. Our results indicate that translocation of the Tn3 element from plasmid to plasmid occurs at a 10(3)- to 10(4)-times-higher frequency than from plasmid to chromosome. In both cases, continued accumulation of Tn3 on recipient genomes is prevented by development of an apparent equilibrium when only a small fraction of molecules in the recipient population contain Tn3. An alternative method for estimation of translocation frequency has shown that the translocation process is temperature sensitive and that its frequency is unaffected by the presence of host recA mutation. Insertions of Tn3 onto the 65 X 10(6)-dalton R6-5 plasmid in Escherichia coli are clustered on EcoRI fragments 3 (8 of 23 insertions) and 9 (7 of 23 insertions), which contain 12 and 5%, respectively, of the R6-5 genome. The occurrence of multiple insertions of Tn3 within EcoRI fragment 9, which contains the IS1 element and a terminus of the Tn4 element, is consistent with earlier evidence indicating that terminal deoxyribonucleic acid sequences of already present transposable elements may provide recognition sequences for subsequent illegitimate recombinational events.

Ampicillin

An infertile male with balanced Y;19 translocation. Review of Y;autosome translocations.

Cytogenetic studies on a phenotypically normal male, presenting with infertility, revealed a balanced Y;19 translocation - 46,XY,t (Y;19) (q11; p or q13). The patient had a normal hormone profile, but semen analysis showed immature cells in the fluid. The possible mechanisms causing the infertility are discussed. An extensive review of the literature of Y ; autosome translocations indicates that there are 2 types, those in which the broken segment of the Y is translocated to the short arm or centromeric region of an acrocentric chromosome, and those in which the Y material is translocated onto a long or short arm region of a non-acrocentric chromosome. The first type is less frequently associated with infertility and hypogonadism than the second type. There is presumptive evidence that the first type is non-random.

Adult

Meiotic studies of translocations causing male sterility in the mouse. II. Double heterozygotes for Robertsonian translocations.

Unusual meiotic behavior of the XY chromosome pair was observed in sterile male mice doubly heterozygous for two Robertsonian translocations, Rb(16.17)7Bnr and Rb(8.17)1Iem. Nonrandom association between the X chromosome and the translocation configuration, ascertained from the frequencies of relevant C-band contacts, was found in 9 of 10 sterile males. Besides the nonrandom association, the XY chromosomes showed signs of impaired condensation, as judged by measurement of their lengths at diakinesis/MI of the first meiotic division. In contrast, neither nonrandom contact nor decondensation of the XY chromosomes pair was found in fertile males heterozygous for a single Robertsonian translocation, Rb1Iem or Rb7Bnr. The present observations lend indirect support to the working hypothesis advanced previously, the assumption that interference with X-chromosome inactivation is a possible cause of spermatogenic breakdown in carriers of various male-sterile chromosomal transloations. Alternative explanations of the available data, which cannot be ruled out, are briefly discussed.

Animals

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Analysis of proton translocation associated with oxidation of endogenous ubiquinol.

A study is presented of the kinetics and stoichiometry of fast proton translocation associated to aerobic oxidation of components of the mitochondrial respiratory chain. 1. Aerobic oxidation of ubiquinol and b cytochromes is accompanied in EDTA particles, obtained by sonication of beef-heart mitochondria, by synchronous proton uptake. 2. The rapid proton uptake associated to oxidation and b cytochromes is greatly stimulated by valinomycin plus K+, but is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 3. 4 gion H+ are taken up per mol ubiquinol oxidized by oxygen. This H+/2e- ratio, measured in the rapid anaerobic-aerobic transition of the particles is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 4. Intact mitochondria aerobic oxidation of oxygen-terminal electron carriers is accompanied by antimycin-insensitive synchronous proton release, oxidation of ubiquinol and reduction of b cytochromes. The amount of protons released is in excess with respect to the amount of ubiquinol oxidized. 5. It is concluded that electron flow along complex III, from ubiquinol to cytochrome c, is directly coupled to vectorial proton translocation. The present data suggest that there exist(s) between ubiquinol and cytochrome c one (or two) respiratory carrier(s), whose oxido-reduction is directly linked to effective transmembrane proton translocation.

Animals

Ionophore-mediated cation translocation in artificial systems. I. A23187-mediated calcium translocation.

The inophore A23187 stimulates the translocation of calcium from an aqueous Hepes buffer into an organic immiscible phase. At saturating calcium concentrations, 2 molecules of ionophore seem to complex each atom of calcium. Consistent with such a stoichiometric behaviour, the apparent ratio of calcium-ionophore association to dissociation rate constants increases as the concentration of ionophore is raised. As a result, at low calcium concentrations, the amount of translocated calcium increases as a power function of A23187 concentration. When allowance is made for such a phenomenon, the relation between calcium translocation and concentration is characterized by usual substrate-receptor binding kinetics.

Anti-Bacterial Agents

Cytogenetic Diversity of Variant Philadelphia Translocations in Chronic Myeloid Leukemia.

INTRODUCTION: Chronic myeloid leukemia (CML) is a disease characterized by Philadelphia (Ph) translocations. These translocations can be classical or variant. The structural features and diagnostic implications of variant Philadelphia translocations remain incompletely defined, and they display considerable cytogenetic heterogeneity. METHODS: In this retrospective study, variant Ph translocations identified by conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) were systematically classified among 639 patients diagnosed with CML. A total of 35 patients with variant Ph translocations were included in the analysis. Molecular follow-up data, when available, were assessed using RT-qPCR analyses in a subset of patients. RESULTS: Chromosome analysis revealed 2 simple and 33 complex variant Ph translocations. FISH analysis, performed in 20 patients, identified deletions involving BCR, ABL1, or both in a limited number of cases. Additional chromosomal abnormalities and secondary translocations accompanied variant Ph translocations in four patients. The partner chromosomes involved in variant Ph translocations showed marked diversity, involving multiple chromosomal loci. CONCLUSION: Variant Philadelphia chromosome translocations in CML exhibit substantial cytogenetic diversity, reflecting the complexity of their underlying genomic architecture. The rarity and heterogeneity of these rearrangements complicate their classification and interpretation in routine diagnostic practice. Descriptive reporting of variant Ph translocations may contribute to a better understanding of their diagnostic complexity and support more accurate cytogenetic interpretation in CML.

Humans

Ribosomal translocation assayed by the matrix-bound poly(uridylic acid) column technique.

The system of translation of cellulose-bound poly(uridylic acid) by Escherichia coli ribosomes has been used for preparation of pre-translocation state ribosomes in columns. Translocation has been induced by passing the elongation factor G (EF-G) with GTP or its non-cleavable analog (guanosine 5'-[beta, gamma-methylene]triphosphate) through the column. A method for quantitative comparison of translocation rates, and thus of effectiveness of translocation-inducing factors, has been proposed. The method is based on an analysis of the profile of deacylated tRNA elution resulting from translocation in the column. The determination of the rate and amount of translocation has been done under different ionic conditions. It has been found that the Mg2+ concentration is a decisive factor of translocation in vitro: at high Mg2+ (30 mM) EF-G cannot induce translocation, and lowering the Mg2+ concentration (to 10 mM) is required for EF-G to become effective. Sufficiently low Mg2+ (3 mM) itself has proved to induce fast and complete translocation, without EF-G.

Chromatography

[Sequence of events in the process of factor-promoted translocation in the ribosome].

A system of translation of matrix-bound poly(U) by purified Escherichia coli ribosomes was used to obtain pre-translocation state ribosomes in columns and then to induce translocation under controlled conditions by passing the elongation factor G (EF-G) with the non-cleavable GTP analog (guanylyl-methylene diphosphonate). It has been shown that translocation in the ribosome, checked by the release of deacylated tRNA, as well as by the puromycin reaction, is induced by the attachment of EF-G (with the non-cleavable GTP analog) to the ribosome and not by its detachment. In accordance with this, the ionic conditions under which the affinity of EF-G with the GTP analog to the ribosome is increased (NH4Cl instead of KCl, a lowered ionic strength) have been also found to be more effective for translocation. On the other hand, it has been shown that the detachment (removal) of EF-G is a strict pre-requisite for the appearance of competence to bind the next aminoacyl-tRNA, and thus for a continuation of the elongation cycle. A conclusion is made that the mechanical shifts of products and substrates, such as peptidyl-tRNA and deacylated tRNA, within the ribosome in the process of translocation are promoted only by the affinity of EF-G to the ribosome and does not depend on the cleavage of GTP. On the basis of the results obtained, the following sequence of events is deduced for the process of EF-G-promoted translocation: 1) interaction of EF-G.GTP with the pre-translocative ribosome, 2) translocation displacements of products and substrates, including the release of deacylated tRNA (probably conjugated with the shift of mRNA), 3) GTP hydrolysis, 4) release of EF-G and GTP from the post-translocated ribosome.

Acylation

Translocation of proteins across membranes: the signal hypothesis and beyond.

Proteins are translocated across membranes either coupled to translation (co-translationally) or after translation (post-translationally). The information for both modes of translocation is encoded in the protein in the form of a short-lived sequence extension (signal sequence). Additional information resides in the ribosome in the case of co-translational translocation, which proceeds via a ribosome--membrane junction. Translocation is mediated by specific receptors (ribosome and/or signal receptors) which are restricted in their location to distinct cellular membranes. In most cases the signal sequence is removed by a signal peptidase operating in an endoproteolytic mode. Membranes endowed with receptors for co-translational translocation are: the rough endoplasmic reticulum (RER) including the outer nuclear envelope membrane, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts, in eukaryotic cells; and the plasma membrane in prokaryotic cells. Each of these membranes presumably contains a single distinctive signal receptor, ribosome receptor and signal peptidase. Membranes endowed with one distinct receptor each for post-translational translocation are both mitochondrial membranes, the chloroplast envelope membrane and the peroxisomal membrane. A signal sequence for co-translational translocation across the RER membrane that is identical in its secondary structure is shared by secretory, lysosomal and certain bitopic integral membrane proteins. Some integral membrane proteins presumably share another common sequence--referred to as stop-transfer sequence--which serves to interrupt translocation and thereby to orient the polypeptide chain in the lipid bilayer. Furthermore, the existence of a few specific 'sorting' sequences is postulated. These would be common to many proteins and would serve to route them to their final destination following translocation across or orientation within the membrane. Thus, the topological information which determines the intracellular pathway and the final location of a great number of proteins appears to reside in a small repertoire of specific sequences which are either a transient or a permanent part of the protein.

Animals

Cytogenetics and reproduction of sheep with multiple centric fusions (Robertsonian translocations).

The significance of centric fusions (Robertsonian translocations) in domestic animals, with special reference to sheep, is reviewed. The mating is described of a further 856 ewes with either a normal chromosome number 2n = 54 or carrying one or more of the three different translocations (centric fusions) t1, t2 and t3 in various heterozygous and homozygous arrangements. Rams which were used in the matings were homozygous for one of the translocation chromosomes (2n = 52), double heterozygotes (2n = 52), triple heterozygotes (2n = 51) or were carriers of 4 translocation chromosomes (2n = 50) and 5 translocation chromosomes (2n = 49). A remarkably even distribution of segregation products was recorded in the progeny of all combinations of translocation ewes x translocation rams in those groups in which sufficient animals were available for statistical analysis. Forty-eight chromosomally different groups of animals were mated. Further, the overall fertility of the translocation sheep, measured by conception rate to first service, lambing percentage and number of ewes which did not breed a lamb, was not significantly different from New Zealand national sheep breeding data. In some groups the poorer reproductive performance could be explained by the age structure of the flock and inbreeding depression, which probably affected the performance of some animals. Sheep with progressively decreasing chromosome numbers, due to centric fusion, 2n = 50, 2n = 49 and 2n = 48, are reported. The 2n = 48 category represents a triple homozygous ewe and a triple homozygous ram and is the first report of the viable evolution of such domestic animals. Less than 1% of phenotypically abnormal lambs were recorded in a total of 1995 progeny born over 10 years. It is now considered that there is little or no evidence to suggest that centric fusions in a variety of combinations affect the total productive fitness of domestic sheep. It is suggested that future research should be more actively directed to understanding their genetic significance.

Animals

A novel mechanism for group translocation: substrate-product reutilization by gamma-glutamyl transpeptidase in peptide and amino acid transport.

Gamma-glutamyl transpeptidase (gamma-GTP) is suggested to act as a carrier in the group translocation of oligopeptides and possibly some amino acids across cellular membranes. It is proposed that the process may involve the repetitive transfer of gamma-glutamyl groups to acceptor peptides which are being translocated from the exterior of the cell to its interior. After group translocation of the peptides has occurred with concomitant formation of gamma-glutamyl peptide products, it is suggested that the products might then be utilized as substrate for the enzyme in order to permit the translocation of other peptides from the exterior. The system is economical and requires only that it be primed with an appropriate source of gamma-glutamyl peptides, such as glutathione. In contrast to most group translocation systems previously described, substrate-product reutilization by gamma-GTP would not be expected to accumulate peptides against a concentration gradient. Mechanisms for maintaining low intracellular concentrations of the translocated peptides are described. Studies on acceptor substrate specificity of gamma-GTP from bovine choroid plexus and rat kidney show some glycyl peptides are much better substrates than free amino acids in accord with the proposal that gamma-GTP might be primarily involved in peptide translocation. Both kinetic and topological evidence support the suggestion that repetitive transfer of gamma-glutamyl moieties by gamma-GTP could occur during group translocation of peptides and possibly some amino acids.

Amino Acids

Heritable translocation test on random-bred mice after prolonged triethylenemelamine treatment.

Heritable translocation and dominant lethal tests were conducted with random-bred Swiss albino male mice. The animals were provided drinking water containing triethylenemelamine (TEM) for 4 weeks, and were then mated for 3 successive weeks for analysis of dominant lethality and production of F1 progeny. Potential translocation carriers among F1 males were selected after two breedings and confirmed by cytogenetic analysis. Translocation heterozygotes were obtained in offspring of the TEM-treated groups, but not in the control groups. In F1 males produced from the first week of mating, the frequencies of translocations were 0, 1.78 6.2 and 10.0% for the control group and groups receiving TEM at 0.0125, 0.025 and 0.050 mg/kg/day, respectively, and in those produced from the third week of mating, the values were 0 and 2.1%, respectively, for the control group and the group receiving TEM at 0.050 mg/kg/day. F1 males from the second week of mating were not studied for the induction of heritable translocations. TEM-induced dominant lethality and heritable translocations were most prominent in the first week of mating after 4 weeks of treatment. In addition, heritable translocations appeared to be a more sensitive endpoint than dominant lethal mutations for the measurement of mutagenic effects of TEM.

Animals

The relation between reaction kinetics and mutagenic action of mono-functional alkylating agents in higher eukaryotic systems. I. Recessive lethal mutations and translocations in Drosophila.

The relationship in Drosophila males between chemical reaction pattern of mono-functional alkylating agents (AA), described in terms of primary alkylation pattern with DNA and proteins as well as the Swain--Scott s factor, and their biological effectiveness were investigated. The agents chosen for comparative analysis were the nitrosamides ENU and MNU, the methanesulfonic esters iPMS, EMS and MMS, the dialkylsulfate DMS, and the nitrosamines DEN and DMN. Parameters of their biological activity were mortality (LC50) of treated adult males, induction in post-meiotic stages of X-chromosomal recessive lethal mutations and 2--3 translocations after either adult feeding or injection. Induced frequencies of recessive lethals, determined for each AA with a range of concentrations, served as biological dosimeter for interaction with target DNA in the germ line. The results are interpreted as indicating for these AA a causal connection between the pattern of primary alkylation of DNA and the quality of genetic damage observed. 1. The agent with the lowest s value, ENU, and its pendant DEN, failed to produce translocations at mutation frequencies that reached 44% for ENU. The highest chromosome-breaking activity was attributed to AA with high s, MMS and DMS. For MMS, the proportions of translocations (T) to mutations (M) approximately reached a 1 : 1 ratio in stored spermatozoa, at a recessive-lethal frequency of 14%. Ability to break chromosomes, as indicated by the T : M ratios, decreased in the sequence MMS greater than or equal to DMS, MNU greater than DMN greater than EMS greater than iPMS greater than ENU = DEN. 2. Nearly the reversed sequence in relative mutagenci effectivenss was obtained when the (directly acting) AA were arranged on the basis of their CM4/LC50 ratios (CM4, the exposure condition producing 4% recessive lethals after injection): ENU greater than EMS greater than iPMS, MNU greater than MMS = DMS. 3. Among the AA, EMS had a somewhat unique position, in that it was slightly less effective in the translocation test, and also less cytotoxic but more mutagenic in the recessive-lethal test than one would expect from its s value. This is taken as an indication of the influence on biological effectiveness of factors other than the s value, e.g. methylation versus ethylation and the lipid/water partition ratio. An example of the latter was also provided by DMS which, although having the same s as MMS, with its 5-fold higher lipid/water partition ratio, was more toxic than MMS. 4. For those AA that were clearly active in the translocation tests--MMS, DMS, MNU, DMN and EMS--delayed formation of exchanges was observed. Only in 17 out of 555 translocation tests with positive response translocations were already found in progeny from unstored spermatozoa. Consequently, it was concluded that performance of storage experiments in Drosophila is an absolute necessity for the detection of this type of rearrangement by AA. 5...

Alkylating Agents