Electron transport through antidot superlattices in Si/Si0.7Ge0.3 heterostructures.
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Journal Article↗
Biomedical subjects
S Kühn
Publications and source records attributed to S Kühn.
Phase-coherent electrons in a finite antidot lattice.
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Journal Article↗
Structural elements of the amino-terminal head domain of vimentin essential for intermediate filament formation in vivo and in vitro.
The biological functions of the non-alpha-helical, N- and C-terminal head and tail domains of intermediate filament (IF) proteins are still ill-defined. Previously, it has been shown that the basic, N-terminal head piece of the type III IF protein vimentin is essential for regular IF assembly and that arginine residues within the N-terminus may be involved. In order to identify particular regions within this domain essential for filament formation and stabilization, N-terminally truncated and arginine substitution forms of vimentin were constructed via site-directed in vitro mutagenesis of murine vimentin cDNA. The de novo filament assembly properties of these modified forms were compared with those of wild-type vimentin after transient expression in vimentin-free, cultured cells. In order to investigate their filament assembly competence in vitro, they were also produced in an E. coli expression system. It could be demonstrated that deletion of the first 10, 13, 17, and 32 amino acid residues, respectively, from the N-terminus of vimentin has an increasingly deleterious effect on filament assembly in vitro and network formation in vivo and that, thus, the highly conserved sequence motif, SSYRRXFGG, located in the N-terminus of various IF proteins and partially or totally removed by the above deletions plays a particularly important role in both activities. These results were confirmed and extended by arginine point mutations in the N-terminal head region, which showed that only one of the two adjacent arginine residues located within the conserved sequence motif is essential for filament assembly and stability in vitro as well as network formation in vivo. The neighboring arginine residues could be replaced by lysine residues without severe effects on the assembly properties of the respective mutant proteins. Distinction between the assembly-promoting potentials of the two arginine residues of the N-terminal doublet was considerably facilitated by a Val389-->Asp substitution toward the carboxy-end of the 2B segment of the vimentin rod domain. The synergistic effect of point mutations in this and the N-terminal region of the vimentin molecule implies the interaction of both protein domains in the process of filament assembly. Mutant vimentin proteins that were characterized by distinct incompetence to assemble into IFs caused a massive collapse of the endogenous vimentin filament system when expressed in mouse skin fibroblasts.
Amino Acid Sequence↗
Dominant transposition-deficient mutants of maize Activator (Ac) transposase.
The maize transposable element Activator (Ac) encodes a transposase (TPase) protein, whose DNA-binding domain is located in a basic region around aa 200. The N-terminal 102 aa of the TPase are not required for the transposition reaction. In transfected petunia protoplasts, we analyzed the protein levels of the N-terminally truncated TPase and mutants thereof and the corresponding transposition frequencies. The TPase protein forms large insoluble aggregates at high expression levels. There is no proportionality observed between TPase levels and transposition frequency. Twenty-one mutations (of 26), which are distributed over the whole length of the protein, inactivate the TPase completely. By coexpressing inactive mutant and active truncated TPase, it was found that several mutations have a trans-dominant inhibitory effect. Among those are two DNA-binding-deficient mutants, indicating that inhibition of the active TPase is not caused by competition for the binding sites on the transposon. Accordingly, Ac TPase acts as an oligo- or multimer formed by protein-protein interactions. Peculiarly, two mutants lacking 53 and 98 aa from the C terminus that are themselves transpositionally inactive lead to an increased excision frequency when they are coexpressed with the active truncated TPase.
Amino Acid Sequence↗
Separation and characterization of homo and hetero-oligomers of the intermediate filament proteins desmin and vimentin.
Affinity chromatography on single-stranded (ss)DNA-cellulose in conjunction with gel permeation chromatography in the presence of urea was employed to separate the intermediate filament (IF) protein complement of catalytically oxidized BHK-21 cell Triton cytoskeletons into disulfide-cross-linked homo- and heterodimers of desmin and vimentin and uncross-linked homodimers. The same separation was performed on a desmin-vimentin mixture under autoxidizing conditions in 6 M-urea to obtain the respective cross-linked collision complexes of both proteins. In 5 M-urea, the oxidation products were identified as dimers that were physically indistinguishable from uncross-linked homodimers, suggesting that they were in the form of partially denatured face-to face pairs. Heterodimers derived from intact IFs were identical to those derived from collision complexes. In the presence of 2-mercaptoethanol, heterodimers were unstable and transformed spontaneously into homodimers. After removal of urea, all cross-linked dimers were totally unable to polymerize into filaments; however, in the presence of 2-mercaptoethanol they showed a normal assembly competence. This inability of oxidized homo- and heterodimers to polymerize, together with the relatively low yield of cross-linked dimers obtained from cytoskeletons, is probably due to the introduction of steric strain into the dimers by disulfide bond formation. Substantial amounts of cross-linked heterodimers could also be isolated from IFs reconstituted from mixtures of desmin and vimentin in their homodimeric or tetrameric forms. Taken together, these results suggest that the cross-linked dimers isolated from cytoskeletons arise from a reaction between subfilament strands of IFs rather than from disulfide bond formation within pre-existing dimers and that the heterotypic IFs of BHK-21 cells are largely formed from homodimers and tetramers, respectively, rather than from heterodimers. The differential capacity of desmin and vimentin to interact with ssDNA has also been exploited to distinguish between homotypic and heterotypic protofilaments, the latter consisting of one homodimer of each protein species. This distinction could be made on the basis of characteristic differences in the sedimentation behavior of the respective protein-DNA complexes.
Animals↗
A novel short consensus repeat-containing molecule is related to human complement factor H.
We have identified a novel factor H-related cDNA, which was isolated from a human liver cDNA library. The DOWN16 clone is 1269 base pairs in size and hybridized to a mRNA of 1.4 kilobases. Similar to the previously described factor H-related proteins, the predicted translation product of 331 amino acids contains a hydrophobic signal sequence followed by a stretch of five short consensus repeats (SCRs). These five SCRs display homology to SCRs of factor H: SCRs1-3 (DOWN16) are homologous to SCRs6-8 of factor H, while SCRs4 and -5 are related to SCRs19 and -20. In vitro translation demonstrated that the DOWN16 cDNA encodes a primary translation product of an apparent molecular mass of 37,500 Da which is directed to the secretory pathway and is glycosylated. Thus, we propose that the protein will be present in human serum. The relatedness of structural elements between this novel gene and factor H may suggest common functions of these proteins not yet determined.
Amino Acid Sequence↗
Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581.
The gene nprM encoding the calcium-dependent extracellular proteinase from Bacillus megaterium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HB101. The DNA sequence of the cloned 3.7 kb fragment revealed only one open reading frame consisting of 1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation start site (ATG). A possible promoter sequence (TAGACG for the -35 region and TATAAT for the -10 region) was found about 69 bp upstream of the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide 'pro' sequence of 221 amino acids preceding the putative mature protein of 317 amino acid residues. Amino acid sequence comparison revealed 84.5% homology between the mature protein and that of a thermolabile neutral protease from B. cereus. It also shares 73% homology with the thermostable neutral proteases of B. thermoproteolyticus and B. stearothermophilus. The zinc-binding sites and the catalytic residues are completely conserved in all four proteases. NprM has a temperature optimum of 58 degrees C, a pH optimum of between 6.4 and 7.2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.
Amino Acid Sequence↗
Characterization of the nucleic acid-binding activities of the isolated amino-terminal head domain of the intermediate filament protein vimentin reveals its close relationship to the DNA-binding regions of some prokaryotic single-stranded DNA-binding proteins.
In order to demonstrate that the nucleic acid-binding activities of vimentin are dictated by its Arg-rich N-terminal head domain, this was cut off at position Lys96 with lysine-specific endoproteinase and analysed for its capacity to associate with a variety of synthetic and naturally occurring nucleic acids. The isolated polypeptide (vim NT) showed a preference for single-stranded (ss) polynucleotides, particularly for ssDNAs of high G-content. A comparison of the sequence and predicted secondary structure of vim NT with that of two prokaryotic ssDNA-binding proteins, G5P and G32P of bacteriophages fd and T4, respectively, revealed that the nucleic acid-binding region of all three polypeptides is almost entirely in the beta-conformation and characterized by a very similar distribution of aromatic amino acid residues. A partial sequence of vim NT can be folded into the same beta-loop structure as the DNA-binding wing of G5P of bacteriophage fd and related viruses. As in the case of G5P, nitration of the Tyr residues with tetranitromethane was blocked by single-stranded nucleic acids. This and spectroscopic data indicate intercalation of the Tyr aromatic ring systems between the bases of the nucleic acids and thus the contribution of a stacking component to the binding reaction. The binding was accompanied by significant changes in the ultraviolet absorption spectra of both vim NT and single-stranded nucleic acids. Upon mixing of vim NT with nucleic acids, massive precipitation of the reactants occurred, followed by the quick rearrangement of the aggregates with the formation of specific and soluble association products. Even at very high ionic strengths, at which no electrostatic reaction should be expected, a distinct fraction of vim NT incorporated naturally occurring ssRNAs and ssDNAs into fast sedimenting complexes, suggesting co-operative interaction of the polypeptide with the nucleic acids. In electron microscopy, the complexes obtained from 28 S rRNA appeared as networks of extended nucleic acid strands densely covered with vim NT, in contrast to the compact random coils of uncomplexed RNA. The networks produced from fd DNA were heterogeneous in appearance and their nucleoprotein strands in rare cases were very similar to the rod-like structures of G5P-fd DNA complexes.
Amino Acid Sequence↗
Preparation of monoclonal antibodies against a poly(U), poly(C) specific ribonuclease prepared from the insect Ceratitis capitata.
A poly(U), poly(C) specific RNase of apparent MW 34 kDa has recently been purified from 6 day old larvae of the insect Ceratitis capitata. Two monoclonal antibodies were obtained by immunizing mice with this protein. Immunoblot analysis of the RNase revealed that both antibodies recognize the 34 kDa protein. Furthermore, immunoprecipitation experiments show that both antibodies were capable of precipitating the ribonuclease without affecting its catalytic activity.
Animals↗
Coding sequence and flanking regions of the mouse vimentin gene.
Using a polyclonal antibody, a cDNA clone coding for part of mouse vimentin was identified in a lambda gt11 expression library. DNA from this clone was used to screen a genomic library from Ehrlich Ascites Tumor cells for the mouse vimentin gene. A clone was found which contained the whole coding sequence and a large part of the 5'- and 3'-untranslated sequences. It was used to prepare a construct equivalent to a full-length cDNA clone. Extensive homologies to the vimentin sequence from other species were found for the coding and 3'-untranslated sequences and the promoter region.
Animals↗
Control of pulsed field gel electrophoresis at short switching intervals by a microcomputer.
Pulsed field gel electrophoresis allows not only the separation of very large DNA molecules (up to 10 megabase pairs) but also gives an enhanced resolution in separations of DNA in the size range of 10-100 kilobase pairs (kbp). For this application, rapid alternation of the electrical field polarity is required. Here we describe equipment for the delivery of short switching pulses that is easy and inexpensive to build and is controlled by a standard microcomputer. It has proved to be useful in the separation of lambda DNA and its fragments. Parameters for enhanced separation of 23- and 48-kbp DNA molecules at high voltage gradients (15 V/cm) are presented and shown to provide superior resolution when compared to those for conventional electrophoresis at both high and low voltage gradients.
DNA, Viral↗
Interaction in vitro of non-epithelial intermediate filament proteins with supercoiled plasmid DNA.
Sucrose gradient analysis of reaction products obtained from non-epithelial intermediate filament (IF) subunit proteins and a mixture of supercoiled, relaxed and linearized plasmid pBR322 DNA at low ionic strength revealed that limited amounts of these polypeptides interacted exclusively with the supercoiled form of the plasmid DNA. These results were corroborated by electron-microscopic analysis of the reaction products, which showed that only circles of supercoiled pBR322 DNA were completely and smoothly covered with vimentin. IFs reconstituted from pure vimentin reacted with supercoiled pBR322 DNA only through their physical ends. The reaction of an aged preparation of vimentin with supercoiled pBR322 DNA produced large aggregates consisting of a central, axially oriented protein scaffold to which individual loops of DNA were attached at their bases in a halo-like arrangement. The electron-microscopic appearance of such complexes was very reminiscent of that of histone-depleted metaphase chromosomes. Together with the previous observations that non-epithelial IF proteins have high affinities for single-stranded DNA and core histones and that they are structurally and functionally closely related to the nuclear lamins, these results were used to advance a novel hypothesis on the biological role of IF proteins in eukaryotic cells.
Animals↗
Probing of the structural stability of vimentin and desmin-type intermediate filaments with Ca2+-activated proteinase, thrombin and lysine-specific endoproteinase Lys-C.
Intermediate filaments (IFs) reconstituted from purified, delipidated vimentin and desmin as well as respective protofilaments were subjected to degradation by Ca2+-activated neutral thiol proteinase, thrombin and lysine-specific endoproteinase Lys-C, respectively. The breakdown products were analyzed by SDS-polyacrylamide gel electrophoresis and negative stain electron microscopy. While Ca2+-activated proteinase and thrombin caused rapid and complete degradation of IFs with kinetics not significantly different from those of the degradation of protofilaments, lysine-specific endoproteinase did not exert any electron microscopically detectable effect on filament structure. Although both types of subunit proteins were truncated at their non-alpha-helical, C-terminal polypeptides by this proteinase, they were still able to assemble into 10 nm filaments. Closer electron microscopic inspection of IFs treated with Ca2+-activated proteinase revealed numerous ruptures along the filaments already at very early stages of digestion. SDS-polyacrylamide gel electrophoresis of the processed filaments in conjunction with previous biochemical characterizations of the breakdown of protofilaments by Ca2+-activated proteinase showed that these inhomogeneities primarily arose from degradation of the arginine-rich, non-alpha-helical N-termini of the filament proteins. These findings demonstrate that, although the N-terminus of vimentin and desmin is essential for filament stability, it is still highly susceptible to proteolytic attack in particular and very likely to posttranslational modification in general. Such structural modifications of the N-termini of IF proteins might exert great influences on the intracellular distribution and molecular organization of IFs in various physiological and pathological conditions.
Animals↗
Efficient interaction of nonpolar lipids with intermediate filaments of the vimentin type.
Based on the finding that vimentin isolated and purified from cultured mammalian cells is heavily contaminated by neutral lipids, the binding of a series of radioactively labeled nonpolar lipids to pure, delipidated vimentin was investigated. Employing gel permeation chromatography of the complexes on Sephacryl S-300, cholesterol, cholesteryl fatty acid esters and mono-, di- and triglycerides were found to efficiently associate with vimentin. These compounds also showed a strong tendency to bind to vimentin filaments. While the non-alpha-helical head piece of vimentin did not interact with neutral lipids under the above assay conditions, the alpha-helical rod domain was highly active. When cholesterol or 1,2-dioleoyl-glycerol was incorporated into phospholipid vesicles, the affinity of the liposomes for vimentin filaments was considerably increased. However, in sucrose density gradient equilibrium centrifugation the filament-vesicle adducts were only stable when the liposomes contained negatively charged phospholipids. These results suggest that the association of intermediate filaments with lipid vesicles is initiated by interaction of the arginine-rich N-termini of their subunit proteins with the negatively charged vesicle surface and stabilized by partial insertion of the protein molecules into the lipid bilayer, particularly at those sites where immiscible, nonpolar lipids create defects in phospholipid packing. Very likely, nonpolar lipids play a significant role in the interaction of intermediate filaments with natural membrane systems.
Animals↗
An electron microscopic study of the interaction in vitro of vimentin intermediate filaments with vesicles prepared from Ehrlich ascites tumor cell lipids.
The interaction of intermediate filaments prepared from pure, delipidated vimentin with vesicles obtained from Ehrlich ascites tumor (EAT) cell lipids was studied employing sucrose density gradient centrifugation in combination with electron microscopy. In negative stain electron microscopy, preformed vimentin filaments were seen in lateral association with lipid vesicles; end-on contacts of filaments with liposomes were rarely detected. When the reaction of filaments with vesicles was carried out at 0 degree C, sucrose density gradient equilibrium centrifugation of the reaction products led to the banding of relatively light filament-vesicle meshworks in clear separation from free filaments and free vesicles. With certain vimentin and lipid preparations, occasionally partial breakdown of the filaments during centrifugation and banding of vesicle-free fragments in denser regions of the sucrose gradients was observed. However, when the reaction mixtures were incubated at 37 degrees C prior to sucrose gradient analysis, all filaments were released from vesicles and totally fragmented during centrifugation. Electron microscopy showed unraveling of the filament fragments into subfilament strands. Employing lipid vesicles labeled with [3H]cholesterol, a low but significant amount of radioactivity was found to be associated with the fragments in a non-vesicular form. Filament reconstitution experiments performed in the presence of EAT cell lipids revealed an inhibitory effect of vesicles on filament assembly, particularly at lower temperatures. The mechanical labilization of the filament structure by lipid vesicles might play a role in the redistribution of intermediate filaments in the course of certain cellular processes involving turnover and fragmentation of intracellular membrane systems.
Animals↗
The interaction in vitro of the intermediate filament protein vimentin with naturally occurring RNAs and DNAs.
The binding of the intermediate filament protein vimentin to a variety of naturally occurring RNAs and DNAs was studied. The relative capacities of the various nucleic acids to associate with pure [3H]vimentin were determined in competition experiments with 28 S rRNA from Ehrlich ascites tumor cells. The reaction products were analyzed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Under these ionic conditions, vimentin reacted preferentially with single-stranded nucleic acids, particularly with those of high (G + C) content. The vimentin binding potentials of single-stranded RNAs and DNAs were largely comparable. However, when the concentrations of mono- and divalent cations were raised to physiological and higher values, only single-stranded DNA retained its vimentin binding capacity. With increasing KCl concentrations at 0 to 1 mM Mg2+, increasing amounts of vimentin were detected in complexes which sedimented considerably faster than the bulk of the DNA, suggesting cooperative binding of vimentin. The salt optimum of this cooperativity was at 200 mM KCl. Thus, the capability of vimentin to discriminate between single-stranded RNA and DNA under physiological ionic conditions points to specificity of the interaction of vimentin with nucleic acids.
Animals↗
The sequence of IS4.
IS-elements are devoid of easily recognizable transacting functions and exert their visible effects in the position cis only (recent reviews Calos and Miller 1980; Starlinger 1980). It has been a matter of debate, whether these elements encode functions for their own transposition. In the case of the E. coli IS-elements this could not easily be determined by genetic methods, because most of these elements are present in several copies (Saedler and Heiss 1973; Deonier et al. 1979). In the case of the IS-elements flanking transposons, evidence has recently been brought forward that these carry the transposition specificity (Rothstein et al. 1980; Kleckner 1980; Grindley 1981). IS4 is present in one copy only in several E. coli K12 strains and should, therefore, be suitable for genetic and physiological studies (Chadwell et al. 1979). It has been cloned from several sites on the E. coli chromosome in pBR322 (Klaer and Starlinger 1980). Here we report the DNA sequence of IS4 which contains an open reading frame for 442 amino acids, and of the junctions of this element with surrounding DNA at three different sites in the E. coli chromosome.
Base Sequence↗
Studies on transposition mechanisms and specificity of IS4.
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Base Sequence↗