Analysis of the “Poso” wheat translocation

The two translocation chromosomes in the Poso 5B/7B translocation have been isolated in separate heterozygous aneuploid stocks (19^II+5B+T). The translocation breakpoints are in the long arm of chromosome 7B and the short arm of chromosome 5B. The translocation chromosome bearing the 5B^L pairing inhibitor was obtained as a homozygous aneuploid (19^II+T ₁ ^II ). The heterozygous aneuploid hemizygous for the pairing inhibitor (19^II+5B+T₂) was used to produce intergeneric hybrids. Only a small percentage were of the high-pairing type (17%), the majority having received chromosome 5B through the egg. This indicates a strong selection against eggs containing the translocation chromosome deficient for the pairing inhibitor.     

Distinct Effects of Fatty Acids on Translocation of γ- and ε-Subspecies of Protein Kinase C

Effects of fatty acids on translocation of the γ- and ε-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). γ-PKC–GFP and ε-PKC–GFP predominated in the cytoplasm, but only a small amount of γ-PKC–GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on γ-PKC–GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of ε-PKC–GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of ε-PKC–GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of ε-PKC–GFP than that of γ-PKC–GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of γ-PKC–GFP. Simultaneous application of arachidonic acid (25 μM) and DiC8 (10 μM) elicited a slow, irreversible translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation.These findings confirm the involvement of fatty acids in the translocation of γ- and ε-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3′-phosphate 5′-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

Construction and study of leaf rust-resistant common wheat lines with translocations of <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch. Genetic material

Genotyping was performed for the leaf rust-resistant line 73/00i (Triticum aestivum × Aegilops speltoides). Fluorescence in situ hybridization (FISH) with probes Spelt1 and pSc119.2 in combination with microsatellite analysis were used to determine the locations and sizes of the Ae. speltoides genetic fragments integrated into the line genome. Translocations were identified in the long arms of chromosomes 5B and 6B and in the short arm of chromosome 1B. The Spelt1 and pSc119.2 molecular cytological markers made it possible to rapidly establish lines with single translocation in the long arms of chromosomes 5B and 6B. The line carrying the T5BS · 5BL-5SL translocation was highly resistant to leaf rust, and the lines carrying the T6BS · 6BL-6SL translocation displayed moderate resistance. The translocations differed in chromosomal location from known leaf resistance genes transferred into common wheat from Ae. speltoides. Hence, it was assumed that new genes were introduced into the common wheat genome from Ae. speltoides. The locus that determined high resistance to leaf rust and was transferred into the common wheat genome from the long arm of Ae. speltoides chromosome 5S by the T5BS · 5BL-5SL translocation was preliminarily designated as LrAsp5.     

Uptake and translocation of griseofulvin by wheat seedlings

Summary 1. A roughly quantitative technique for studying uptake and translocation of the antibiotic griseofulvin by wheat plants has been devised. Wheat plants were grown in nutrient solutions containing griseofulvin and translocation measured by bioassay of the griseofulvin appearing in the guttation drops induced by transfer to a humid atmosphere.2. Griseofulvin was phytotoxic at concentrations of 5 µg/ml and above, the first symptoms observed being stunting and swelling of the roots.3. The concentration of griseofulvin in the guttation drops was directly related to the concentration in the nutrient solution; there was evidence of griseofulvin accumulation in the leaves, the concentration in the guttation drops being frequently higher than that in the nutrient solution.4. Atmospheric conditions favouring transpiration increased uptake and translocation of griseofulvin.5. Uptake and translocation of griseofulvin was inhibited by inclusion of respiratory enzyme inhibitors in the nutrient solution.     

IGFBP-5 Promotes Fibrosis Independently of Its Translocation to the Nucleus and Its Interaction with Nucleolin and IGF

Background

Insulin-like growth factor binding protein (IGFBP)-5 levels are increased in systemic sclerosis (SSc) skin and lung. We previously reported that IGFBP-5 is a pro-fibrotic factor that induces extracellular matrix (ECM) production and deposition. Since IGFBP-5 contains a nuclear localization signal (NLS) that facilitates its nuclear translocation, we sought to examine the role of nuclear translocation on the fibrotic activity of IGFBP-5 and identify IGFBP-5 binding partners relevant for its nuclear compartmentalization.

Methods

We generated functional wild type IGFBP-5 and IGFBP-5 with a mutated NLS or a mutated IGF binding site. Abrogation of nuclear translocation in the NLS mutant was confirmed using immunofluorescence and immunoblotting of nuclear and cytoplasmic cellular extracts. Abrogation of IGF binding was confirmed using western ligand blot. The fibrotic activity of wild type and mutant IGFBP-5 was examined in vitro in primary human fibroblasts and ex vivo in human skin. We identified IGFBP-5 binding partners using immunoprecipitation and mass spectrometry. We examined the effect of nucleolin on IGFBP-5 localization and function via sequence-specific silencing in primary human fibroblasts.

Results

Our results show that IGFBP-5-induced ECM production in vitro in primary human fibroblasts is independent of its nuclear translocation. The NLS-mutant also induced fibrosis ex vivo in human skin, thus confirming and extending the in vitro findings. Similar findings were obtained with the IGF-binding mutant. Nucleolin, a nucleolar protein that can serve as a nuclear receptor, was identified as an IGFBP-5 binding partner. Silencing nucleolin reduced IGFBP-5 translocation to the nucleus but did not block the ability of IGFBP-5 to induce ECM production and a fibrotic phenotype.

Conclusions

IGFBP-5 transport to the nucleus requires an intact NLS and nucleolin. However, nuclear translocation is not necessary for IGFBP-5 fibrotic activity; neither is IGF binding. Our data provide further insights into the role of cellular compartmentalization in IGFBP-5-induced fibrosis.     

Results of estimation of mutation rates for translocation trisomy 21

Among 1332 cases of trisomy 21 born within 1979-1999 in St. Petersburg, 76(5.7%) were carriers of a translocation between chromosome 21 and other acrocentrics. Among 43 Dq; 21q translocations, 17 were inherited from the mother, and one was inherited from the father, 16 were of sporadic occurrence, and in 9 cases the mode of inheritance was not established. Out of 31 cases displaying Gq;21 translocation, 23 were mutants and 8 of unknown origin. One case of non-Robertsonian translocation 21;22 was maternal in origin. It was assumed that the proportion of sporadic cases among translocations of unknown origin is the same as that among translocations of the known origin. However, it is conceivable that the parents of a child with a sporadic anomaly, previously having an uncomplicated reproductive history and healthy children, tend to avoid cytogenetic examination more often than the carriers of translocation. Hence, the reported proportion of de novo cases (-0.6) might be underestimated. The analysis of pregnancy outcomes in mothers of children with Down syndrome, who inherited translocation (n = 12), sporadic translocation (n = 12) and translocation of unknown origin (n = 8), supports this suggestion. Analysis of the data from 8 reports, where the origin of Dq;21 was specified, revealed that in those samples, where the origin was traced in almost all families, the proportion of de novo cases (0.75-0.82) was higher than in samples where an appreciable part of families was not examined (0.46-0.73). Therefore, with the aim of correct determination of mutation rate for Dq;21 translocation, the true proportions in D;21 cases merit evaluation. Meanwhile, using average estimation from all the above mentioned reports (0.67), the mutation rate for translocations Dq;21 in St. Petersburg was calculated to be 1.2 x 10(-5) and 0.8 x 10(-5) in 1980-1989 and 1990-1999, respectively. For Gq;21 translocations/isochromosomes, the corresponding figures were 1.6 x 10(-5) and 1.5 x 10(-5).     

Translocation of the C terminus of a tail-anchored protein across the endoplasmic reticulum membrane in yeast mutants defective in signal peptide-driven translocation

C-tail-anchored proteins are defined by an N-terminal cytosolic domain followed by a transmembrane anchor close to the C terminus. Their extreme C-terminal polar residues are translocated across membranes by poorly understood post-translational mechanism(s). Here we have used the yeast system to study translocation of the C terminus of a tagged form of mammalian cytochrome b(5), carrying an N-glycosylation site in its C-terminal domain (b(5)-Nglyc). Utilization of this site was adopted as a rigorous criterion for translocation across the ER membrane of yeast wild-type and mutant cells. The C terminus of b(5)-Nglyc was rapidly glycosylated in mutants where Sec61p was defective and incapable of translocating carboxypeptidase Y, a well known substrate for post-translational translocation. Likewise, inactivation of several other components of the translocon machinery had no effect on b(5)-Nglyc translocation. The kinetics of translocation were faster for b(5)-Nglyc than for a signal peptide-containing reporter. Depletion of the cellular ATP pool to a level that retarded Sec61p-dependent post-translational translocation still allowed translocation of b(5)-Nglyc. Similarly, only low ATP concentrations (below 1 microm), in addition to cytosolic protein(s), were required for in vitro translocation of b(5)-Nglyc into mammalian microsomes. Thus, translocation of tail-anchored b(5)-Nglyc proceeds by a mechanism different from that of signal peptide-driven post-translational translocation.     

Effect of temperature on translocation frequency of the Tn3 element.

The effect of temperature on the translocation frequency of the Tn3 element was investigated. The temperature optimum for translocation of Tn3 was in the range from 26 to 30 degrees C. At temperatures above 30 degrees C, the translocation frequency decreased rapidly and linearly; at 36 degrees C it was only 5% of the frequency observed at 30 degrees C. The duration and reversibility of the temperature effect were utilized to demonstrate a requirement for protein synthesis in the translocation process.     

Cytogenetic and molecular characterization of eight new reciprocal translocations in the pig species. Estimation of their incidence in French populations

Eight new cases of reciprocal translocation in the domestic pig are described. All the rearrangements were highlighted using GTG banding techniques. Chromosome painting experiments were also carried out to confirm the proposed hypotheses and to accurately locate the breakpoints. Three translocations, rcp(4;6)(q21;p14), rcp(2;6)(p17;q27) and rcp(5;17)(p12;q13) were found in boars siring small litters (8.3 and 7.4 piglets born alive per litter, on average, for translocations 2/6 and 5/17, respectively). The remaining five, rcp(5;8)(p12;q21), rcp(15;17)(q24;q21), rcp(7;8)(q24;p21), rcp(5;8)(p11;p23) and rcp(3;15)(q27;q13) were identified in young boars controlled before entering reproduction. A decrease in prolificacy of 22% was estimated for the 3/15 translocation after reproduction of the boar carrier. A parental origin by inheritance of the translocation was established for the (5;8)(p11;p23) translocation. The overall incidence of reciprocal translocations in the French pig populations over the 2000/2001 period was estimated (0.34%).     

Determination of RNA orientation during translocation through a biological nanopore

We investigate single-molecule electrophoretic translocation of A(50), C(50), A(25)C(50), and C(50)A(25) RNA molecules through the alpha-hemolysin transmembrane protein pore. We observe pronounced bilevel current blockages during translocation of A(25)C(50) and C(50)A(25) molecules. The two current levels observed during these bilevel blockages are very similar to the characteristic current levels observed during A(50) and C(50) translocation. From the temporal ordering of the two levels within the bilevel current blockages, we infer whether individual A(25)C(50) and C(50)A(25) molecules pass through the pore in a 3'-->5' or 5'-->3' orientation. Correlation between the level of current obstruction and the inferred A(25)C(50) or C(50)A(25) orientation indicates that 3'-->5' translocation of a poly C segment causes a significantly deeper current obstruction than 5'-->3' translocation. Our analysis also suggests that the 3' ends of C(50) and A(25)C(50) RNA molecules are more likely to initiate translocation than the 5' ends. Orientation dependent differences in a smaller current blockage that immediately precedes many translocation events suggest that this blockage also contains information about RNA orientation during translocation. These findings emphasize that the directionality of polynucleotide molecules is an important factor in translocation and demonstrate how structure within ionic current signals can give new insights into the translocation process.     

Controlled introgression to wheat of genes from rye chromosome arm 1RS by induction of allosyndesis

Summary Chromosome pairing between rye chromosome arm 1RS, present in two wheat-rye translocation stocks, and its wheat homoeologues was induced by introducing the translocations into either a ph1bph1b or a nullisomic 5B background. This rye arm carries a gene conferring resistance to wheat stem rust, but lines carrying the translocation produce a poor quality dough unsuitable for breadmaking. Storage protein markers were utilised along with stem rust reaction to screen for allosyndetic recombinants. From a 1DL-1RS translocation, three lines involving wheat-rye recombination were recovered, along with thirteen lines derived from wheat-wheat homoeologous recombination. From a 1BL-1RS translocation, an additional three allosyndetic recombinants were recovered. Nullisomy for chromosome 5B was as efficacious as the ph1b mutant for induction of allosyndesis, and the former stock is easier to manipulate due to the presence of a 5BL-encoded endosperm protein. The novel wheat-rye chromosomes present in the recombinant lines may enable the rye disease resistance to be exploited without the associated dough quality defect.     

Genetic characterization of a reciprocal translocation present in a widely grown barley variety

Artificially induced translocation stocks have been used to physically map the barley genome; however, natural translocations are extremely uncommon in cultivated genotypes. Albacete is a barley variety widely grown in recent decades in Spain and carrying a reciprocal translocation which obviously does not affect its agronomical fitness. This translocation has been characterized by a combination of cytological and molecular genetic approaches. Firstly, recombination frequencies between markers on chromosomes 1H and 3H were estimated to determine the boundaries of the reciprocal interchange. Secondly, 1H-3H wheat barley telosome addition lines were used to assign selected markers to chromosome arms. Thirdly, fluorescence in situ hybridization (FISH) with rDNA probes (5S and 18S-5.8S-26S) and microsatellite probes [(ACT)(5), (AAG)(5) and (CAG)(5)] was used to determine the locations of the translocation breakpoints more precisely. Fourthly, fine-mapping of the regions around the translocation breakpoints was used to increase the marker density for comparative genomics. The results obtained in this study indicate that the translocation is quite large with breakpoints located on the long arms of chromosomes 1H and 3H, between the pericentromeric (AAG)(5) bands and above the (ACT)(5) interstitial distal bands, resulting in the reciprocal translocation 1HS.1HL-3HL and 3HS.3HL-1HL. The gene content around the translocation breakpoints could be inferred from syntenic relationships observed among different species from the grass family Poaceae (rice, Sorghum and Brachypodium) and was estimated at approximately 1,100 and 710 gene models for 1H and 3H, respectively. Duplicated segments between chromosomes Os01 and Os05 in rice derived from ancestral duplications within the grass family overlap with the translocation breakpoints on chromosomes 1H and 3H in the barley variety Albacete.     

PEX5 and Ubiquitin Dynamics on Mammalian Peroxisome Membranes

Peroxisomes are membrane-bound organelles within eukaryotic cells that post-translationally import folded proteins into their matrix. Matrix protein import requires a shuttle receptor protein, usually PEX5, that cycles through docking with the peroxisomal membrane, ubiquitination, and export back into the cytosol followed by deubiquitination. Matrix proteins associate with PEX5 in the cytosol and are translocated into the peroxisome lumen during the PEX5 cycle. This cargo translocation step is not well understood, and its energetics remain controversial. We use stochastic computational models to explore different ways the AAA ATPase driven removal of PEX5 may couple with cargo translocation in peroxisomal importers of mammalian cells. The first model considered is uncoupled, in which translocation is spontaneous, and does not immediately depend on PEX5 removal. The second is directly coupled, in which cargo translocation only occurs when its PEX5 is removed from the peroxisomal membrane. The third, novel, model is cooperatively coupled and requires two PEX5 on a given importomer for cargo translocation — one PEX5 with associated cargo and one with ubiquitin. We measure both the PEX5 and the ubiquitin levels on the peroxisomes as we vary the matrix protein cargo addition rate into the cytosol. We find that both uncoupled and directly coupled translocation behave identically with respect to PEX5 and ubiquitin, and the peroxisomal ubiquitin signal increases as the matrix protein traffic increases. In contrast, cooperatively coupled translocation behaves dramatically differently, with a ubiquitin signal that decreases with increasing matrix protein traffic. Recent work has shown that ubiquitin on mammalian peroxisome membranes can lead to selective degradation by autophagy, or ‘pexophagy.’ Therefore, the high ubiquitin level for low matrix cargo traffic with cooperatively coupled protein translocation could be used as a disuse signal to mediate pexophagy. This mechanism may be one way that cells could regulate peroxisome numbers.     

Fixation of translocation 2A·4B infers the monophyletic origin of Ethiopian tetraploid wheat

Analysis of structural chromosomal polymorphism revealed the presence of a previously reported 2A·4B translocation common to all 15 strains of Ethiopian tetraploid wheat examined. Using the C-banding technique, we found two new translocations,T1B·6B and T5B·6B, and a pericentric inversion of chromosome 5A. The C-banding pattern indicated that in all three translocations the breakpoint was located in the centromeric region. Sequential N-banding and genomic in situ hybridization (GISH) confirmed the location of the breakpoint of translocation 2A·4B, and revealed that the breakpoint of another known translocation, 2A-2B, was in the proximal region of 2BL. The fixation of the 2A·4B translocation indicates the monophyletic origin of Ethiopian tetraploid wheat and the presence of a very severe bottleneck effect during its dispersal. Received: 29 June 1999 / Accepted: 18 February 2000     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3'-phosphate 5'-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

Influence of temperature and light intensity on absorption, translocation, and phytotoxicity of fenoxaprop-ethyl and imazamethabenz-methyl in Avena fatua

The absorption and translocation of fenoxaprop-ethyl and imazamethabenz-methyl were investigated in wild oat (Avena fatua L.) plants grown under different temperature and light intensity conditions by using ¹⁴C tracer techniques. The phytotoxicity of both herbicides, applied as individual droplets, was also determined under similar environments. The absorption of fenoxaprop-ethyl and imazamethabenz-methyl was increased by high temperature (30/20°C) and to a lesser extent by 70% shading; low temperature (10/5°C) had limited effect on the absorption. The basipetal translocation of fenoxaprop-ethyl was not affected by high temperature, and the increase in imazamethabenz-methyl translocation at high temperature was likely the result of the increased absorption. Low temperature decreased total translocation and translocation efficiency in both fenoxaprop-ethyl and imazamethabenz-methyl. Low light intensity tended to reduce the efficiency of basipetal translocation of both herbicides. Fenoxaprop-ethyl phytotoxicity was reduced by high temperature but not by low temperature. Temperature had little effect on imazamethabenz-methyl effectiveness. Under 70% shading, the phytotoxicity of both herbicides was enhanced.Abbreviation S.E.D. standard errors of difference     

Pure partial trisomy 5q33-->5q35 resulting from the adjacent-1 segregation of a paternal (5;14)(q33;p12) translocation.

A 14-year-old male was referred for evaluation of mental retardation with short stature and dysmorphic features. His karyotype was 46,XY,der(14)t(5;14)(q33;p12)pat, resulting in a pure partial 5q33-q35 trisomy due to the adjacent-1 segregation of a paternal balanced translocation. Paternal blood karyotype revealed a balanced translocation t(5;14)(q33;p12) retaining Ag-Nors. To date, only two cases of pure partial 5q trisomies spanning this region have been reported. Analysis of these cases and the one we report does not allow the delineation of a specific phenotype.     

Mating between two balanced translocation carriers in two unrelated families

Partial trisomy 9p and a 13/14 translocation occurred in the daughter of a t(5;9)(p15;p12) mother and a t(13;14)(p11;q11) father. Two additional offspring displayed a normal karyotype and a translocation trisomy 13 respectively. Two first cousins, selected for chromosome analysis because of a spontaneous abortion, were found to have an identical translocation t(14;21)(p11;q11). Their second pregnancy was monitored by midtrimester amniocentesis and disclosed a balanced fetus. The different zygotic chromosome constitutions and the counselling problems in the marriages between two balanced translocation carriers are discussed.