Heat shock proteins are differentially expressed in brain and spinal cord: implications for multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by demyelination, inflammation and neurodegeneration throughout the central nervous system. Although spinal cord pathology is an important factor contributing to disease progression, few studies have examined MS lesions in the spinal cord and how they differ from brain lesions. In this study we have compared brain and spinal cord white (WM) and grey (GM) matter from MS and control tissues, focusing on small heat shock proteins (HSPB) and HSP16.2. Western blotting was used to examine protein levels of HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 in brain and spinal cord from MS and age-matched non-neurological controls. Immunohistochemistry was used to examine expression of the HSPs in MS spinal cord lesions and controls. Expression levels were quantified using ImageJ. Western blotting revealed significantly higher levels of HSPB1, HSPB6 and HSPB8 in MS and control spinal cord compared to brain tissues. No differences in HSPB5 and HSP16.2 protein levels were observed, although HSPB5 protein levels were higher in brain WM versus GM. In MS spinal cord lesions, increased HSPB1 and HSPB5 expression was observed in astrocytes, and increased neuronal expression of HSP16.2 was observed in normal-appearing GM and type 1 GM lesions. The high constitutive expression of several HSPBs in spinal cord and increased expression of HSPBs and HSP16.2 in MS illustrate differences between brain and spinal cord in health and upon demyelination. Regional differences in HSP expression may reflect differences in astrocyte cytoskeleton composition and influence inflammation, possibly affecting the effectiveness of pharmacological agents.

Lens Epithelial Cell Proliferation in Response to Ionizing Radiation.

Lens epithelial cell proliferation and differentiation are naturally well regulated and controlled, a characteristic essential for lens structure, symmetry and function. The effect of ionizing radiation on lens epithelial cell proliferation has been demonstrated in previous studies at high acute doses, but the effect of dose and dose rate on proliferation has not yet been considered. In this work, mice received single acute doses of 0.5, 1 and 2 Gy of radiation, at dose rates of 0.063 and 0.3 Gy/min. Eye lenses were isolated postirradiation at 30 min up until 14 days and flat-mounted. Then, cell proliferation rates were determined using biomarker Ki67. As expected, radiation increased cell proliferation 2 and 24 h postirradiation transiently (undetectable 14 days postirradiation) and was dose dependent (changes were very significant at 2 Gy; P = 0.008). A dose-rate effect did not reach significance in this study (P = 0.054). However, dose rate and lens epithelial cell region showed significant interactions (P < 0.001). These observations further our mechanistic understanding of how the lens responds to radiation.

The beaded filament of the eye lens: an unexpected key to intermediate filament structure and function.

In 1959, an unusual filamentous polymer, now called the beaded filament, was described in the lens of the eye. The constituent proteins, assembly properties and functions of the beaded filament have been elusive. The recent publication of the sequences for two major lens filament proteins (CP49 and filensin) and the reconstitution in vitro of structures closely resembling beaded filaments, suggests that the beaded filament is related structurally to intermediate filaments (IFs). The association of the lenticular chaperones, the alpha-crystallins, with the filament contributes to the characteristic beaded morphology, as well as giving important clues to the function of this unusual filament in the lens. These recent results have several implications for IF function and assembly.

Crystalline tubes of myosin subfragment-2 showing the coiled-coil and molecular interaction geometry.

We have produced crystalline tubes of chicken breast myosin long subfragment-2 that show order to resolutions better than 2 nm. The tubes were formed from a thin sheet in which the myosin long subfragment-2 molecules were arranged on an approximately rectangular crystalline lattice with a = 14.1 +/- 0.2 nm and b = 3.9 +/- 0.1 nm in projection. Shadowing indicated that the tube wall was approximately 7 nm thick and that the sheets from which it was formed followed a right-handed helix. Superposition of the lattices from the top and bottom of the tube produced a moire pattern in negatively stained material, but images of single sheets were easily obtained by computer image processing. Although several molecules were superimposed perpendicular to the plane of the sheet, the modulation in density due to the coiled-coil envelope was clear, indicating that the coiled-coils in these molecules were in register (or staggered by an even number of quarter pitches). In projection the coiled-coil had an apparent pitch of 14.1 nm (the axial repeat of the unit cell), but the small number of molecules (probably four) superimposed perpendicular to the plane of the sheet meant that pitches within approximately 1 nm of this value could have shown a modulation. Therefore, a more precise determination of the coiled-coil pitch must await determination of the sheet's three-dimensional structure. The coiled-coils of adjacent molecules within the plane of the sheet were staggered by an odd number of quarter pitches. This arrangement was similar to that between paramyosin molecules in molluscan thick filaments and may have features in common with other coiled-coil protein assemblies, such as intermediate filaments. Each molecule in the crystal had two types of neighbor: one staggered by an odd number of quarter pitches and the other by an even number of quarter pitches, as has been proposed for the general packing of coiled-coils (Longley, W., 1975, J. Mol. Biol., 93:111-115). We propose a model for the detailed packing within the sheet whereby molecules are inclined slightly to the plane of the sheet so that its thickness is determined by the molecular length.

Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure.

We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.

Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage.

PURPOSE: Epidemiological evidence regarding the radiosensitivity of the lens of the eye and radiation cataract development has led to changes in the EU Basic Safety Standards for protection of the lens against ionizing radiation. However, mechanistic details of lens radiation response pathways and their significance for cataractogenesis remain unclear. Radiation-induced DNA damage and the potential impairment of repair pathways within the lens epithelium, a cell monolayer that covers the anterior hemisphere of the lens, are likely to be involved. MATERIALS AND METHODS: In this work, the lens epithelium has been analyzed for its DNA double-strand break (DSB) repair response to ionizing radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to at the very periphery of the monolayer (germinative and transitional zones). Described here are the different responses in the two regions and across four strains (C57BL/6, 129S2, BALB/c and CBA/Ca) over a low dose (0-25 mGy) in-vivo whole body X-irradiation range up to 24 hours post exposure. RESULTS: DNA damage and repair as visualized through 53BP1 staining was present across the lens epithelium, although repair kinetics appeared non-uniform. Epithelial cells in the central region have significantly more 53BP1 foci. The sensitivities of different mouse strains have also been compared. CONCLUSIONS: 129S2 and BALB/c showed higher levels of DNA damage, with BALB/c showing significantly less inter-individual variability and appearing to be a more robust model for future DNA damage and repair studies. As a result of this study, BALB/c was identified as a suitable radiosensitive lens strain to detect and quantify early low dose ionizing radiation DNA damage effects in the mouse eye lens specifically, as an indicator of cataract formation.

Molecular chaperones: small heat shock proteins in the limelight.

Small heat shock proteins have been the Cinderellas of the molecular chaperone world, but now the crystal structure of a small heat shock protein has been solved and mutation of two human homologues implicated in genetic disease. Intermediate filaments appear to be one of the key targets of their chaperone activity.

THE IMPACT OF CIRCADIAN RHYTHMS ON MEDICAL IMAGING AND RADIOTHERAPY REGIMES FOR THE PAEDIATRIC PATIENT.

Daily rhythmic changes are found in cellular events in cell cycle, DNA repair, apoptosis and angiogenesis in both normal and tumour tissue, as well as in enzymatic activity and drug metabolism. In this paper, we hypothesize that circadian rhythms need to be considered in radiation protection and optimization in personalized medicine, especially for paediatric care. The sensitivity of the eye lens to ionizing radiation makes the case for limiting damage to the lens epithelium by planning medical radio-imaging procedures for the afternoon, rather than the morning. Equally, the tumour and normal tissue response to radiotherapy is also subject to diurnal variation enabling optimization of time of treatment.

Antiischemic effects of SB203580 are mediated through the inhibition of p38alpha mitogen-activated protein kinase: Evidence from ectopic expression of an inhibition-resistant kinase.

The aim of the present study was to determine whether the attenuation of myocardial ischemic injury by SB203580 is due to the inhibition of p38 mitogen-activated protein kinase (MAPK) or to other documented nonspecific effects of the drug. We made adenoviral vectors encoding the alpha isoform of p38 MAPK with or without site-directed mutations to prevent SB203580 binding and inhibition. In embryonal rat heart-derived cells and adult rat cardiocytes expressing wild-type p38alpha MAPK, injury was reduced significantly by SB203580 present during simulated ischemia. In contrast, SB203580 did not protect cells expressing the SB203580-resistant form of p38alpha MAPK. These observations suggest that SB203580-mediated protection depends on the inhibition of p38alpha MAPK.

Antagonistic action of Six3 and Prox1 at the gamma-crystallin promoter.

Gamma-crystallin genes are specifically expressed in the eye lens. Their promoters constitute excellent models to analyse tissue-specific gene expression. We investigated murine CRYGE/f promoters of different length in lens epithelial cell lines. The most active fragment extends from position -219 to +37. Computer analysis predicts homeodomain and paired-domain binding sites for all rodent CRYGD/e/f core promoters. As examples, we analysed the effects of Prox1 and Six3, which are considered important transcription factors involved in lens development. Because of endogenous Prox1 expression in N/N1003A cells, a weak stimulation of CRYGE/f promoter activity was found for PROX1. In contrast, PROX1 stimulated the CRYGF promoter 10-fold in CD5A cells without endogenous PROX1. In both cell lines Six3 repressed the CRYGF promoter to 10% of its basal activity. Our cell transfection experiments indicated that CRYG expression increases as Six3 expression decreases. Prox1 and Six3 act antagonistically on regulation of the CRYGD/e/f promoters. Functional assays using randomly mutated gammaF-crystallin promoter fragments define a Six3-responsive element between -101 and -123 and a Prox1-responsive element between -151 and -174. Since Prox1 and Six3 are present at the beginning of lens development, expression of CRYGD/e/f is predicted to remain low at this time. It increases as Six3 expression decreases during ongoing lens development.

Structural differences between blood-platelet tubulin and other mammalian tubulins.

Carboxymethylated pig blood-platelet tubulin subunits have been characterized by comparing their electrophoretic mobilities and two-dimensional tryptic peptide maps with those of tubulin subunits isolated from four other mammalian tissues. These were pig brain, pig lung, pig sperm and bovine kidney. In addition to the major blood-platelet alpha- and beta-tubulin subunits, minor alpha- and beta-components could be resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The major blood-platelet beta-tubulin, as well as the minor brain beta 2-tubulin subunit, were clearly tissue specific. In both cases, their two-dimensional peptide maps contained unique heavily labelled peptides. Distinct peptide maps were also observed for the major blood-platelet alpha-tubulin subunit as well as for sperm tail alpha-tubulin, both of which were missing peptides (different in each case) that were present in all of the other alpha-tubulin maps. Under our conditions, a protein with a molecular weight of 70,000 and an isoelectric point of pH 6.4 co-purified with blood-platelet microtubules.

Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells.

Cytoskeletal residues obtained after extraction of rat liver and cultured rat hepatoma cells (line MH1C1) were used to isolate cytokeratin subunit complexes by solubilization in low salt buffer containing 4 M-urea. Alternatively, the complexes were prepared by solubilization of total cytoskeletal proteins in 9.5 M-urea or 6 M-guanidinium hydrochloride (Gu . HCl), followed by separation using reversed phase high pressure liquid chromatography and dialysis first against either 9.5 M-urea or 6 M-Gu . HCl and then against buffers containing either 4 M-urea or 2 M-Gu . HCl, respectively. The complexes contained only two cytokeratin polypeptides in a 1 : 1 ratio as demonstrated by electrophoresis and isoelectric focusing, i.e. components A (Mr 55,000; isoelectric point in 9.5 M-urea, pH 6.4) and D (Mr 49,000; isoelectric point, pH 5.38) which were separated from each other at urea concentrations higher than 7 M. The complex had a sedimentation coefficient S25,w of 4.96 S in 2 M-Gu . HCl. Sedimentation equilibrium analysis gave an average Mr value of 207,000 which was interpreted as a tetramer containing two chains each of A and D. This complex was also directly demonstrated by gel electrophoresis under non-dissociating conditions. Using dimethyl suberimidate to cross-link the complex in solution of 4 M-urea or 2 M-Gu . HCl, we identified covalently linked heterodimers of A and D, and a tetrameric unit containing equal amounts of A and D which was the largest cross-link product obtained. This complex was similar to the tetrameric complex of rat and human vimentin formed under the same conditions. The constituents of the cross-linked products were identified by two-dimensional ("diagonal") gel electrophoresis, involving the cleavage of the bis(amidine) cross-links after the initial separation in the first dimension. Identical cross-link products were recognized when cytokeratin filaments were used. By electron microscopy the complexes appeared as threads of 2 to 3 nm diameter with a mean length of approximately 48 nm. On dialysis to low salt buffer, the complexes formed 2 to 3 nm protofilaments, intertwisted 3 to 4 nm protofilaments and typical 7 to 11 nm intermediate-sized filaments. Complexes formed from equivalent cytokeratins of other species such as man and cow, as well as heterologous recombinations such as human component A mixed with bovine component D and vice versa, showed the same characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of dimer subunits of intermediate filament proteins.

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of alpha-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous alpha-helical coiled-coil proteins. Using buffers containing 3 M-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 ("40K"), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins from discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.

The lens of the eye: exposures in the UK medical sector and mechanistic studies of radiation effects.

The recommendation from the International Commission on Radiological Protection that the occupational equivalent dose limit for the lens of the eye should be reduced to 20 mSv year(-1), averaged over 5 years with no year exceeding 50 mSv, has stimulated a discussion on the practicalities of implementation of this revised dose limit, and the most appropriate risk and protection framework to adopt. This brief paper provides an overview of some of the drivers behind the move to a lower recommended dose limit. The issue of implementation in the medical sector in the UK has been addressed through a small-scale survey of doses to the lens of the eye amongst interventional cardiologists and radiologists. In addition, a mechanistic study of early and late post-irradiation changes in the lens of the eye in in-vivo-exposed mice is outlined. Surveys and studies such as those described can contribute to a deeper understanding of fundamental and practical issues, and therefore contribute to a robust evidence base for ensuring adequate protection of the eye while avoiding undesirable restrictions to working practices.

Identification and characterization of axopodial tubulins from Echinosphaerium nucleofilum.

Isolated microtubule protein from axopodia of the heliozoan Echinosphaerium nucleofilum, consisting of two major bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), has been compared to axonemal and cytoplasmic tubulins from both animal and non-animal sources. The upper E. nucleofilum protein band migrated faster than the alpha-tubulins of bovine brain and sea anemone sperm tails but with approximately the same electrophoretic mobility as the axonemal alpha-tubulins of Tetrahymena pyriformis and the alga Chlorogonium elongatum and cytoplasmic alpha-tubulin from the slime mold Physarum polycephalum. The lower E. nucleofilum protein band, however, had a higher electrophoretic mobility than all the beta-tubulins which we have so far examined. It was, nevertheless, a true beta-tubulin as shown by its migration on two-dimensional gel electrophoresis and the general resemblance of its one- and two-dimensional peptide maps to those of other beta-tubulins. The Staphylococcus aureus protease cleavage pattern of the upper axopodial protein band was similar to those of other non-animal alpha-tubulins but quite different from those of the animal alpha-tubulins. In contrast, the two-dimensional tryptic peptide map of axopodial alpha-tubulin was distinct from all of them. For example, a characteristic constellation of peptides common to the peptide maps of the other alpha-tubulins was absent from that of E. nucleofilum. In contrast to Physarum and metazoan tubulins but similar to Tetrahymena tubulin, the axopodial alpha-tubulin had a more basic isoelectric point than the beta-subunit as shown by two dimensional gel electrophoresis. Some of the unusual characteristics of E. nucleofilum axopodial tubulin may not only reflect phylogenetic variation, but also the different functional requirements of axopodial microtubules.

Changes in the nucleolar and coiled body compartments precede lamina and chromatin reorganization during fibre cell denucleation in the bovine lens.

Nuclear elimination accompanies differentiation in such specialized cell types such as erthyrocytes and lens fibre cells. It also accompanies apoptosis which has suggested that similar processes could operate in both. Denucleation occurs in the lens in order to reduce light scatter and this process is often disrupted in cataract. Using the adult bovine lens as a model system, nuclear changes accompanying denucleation are described with particular emphasis on the lamina, nucleolar and coiled body compartments in lens nuclei. Nuclear shape, chromatin reorganization and chromatin breakdown were also monitored to correlate the timing of events. Rearrangement of both A- and B-type nuclear lamins occurred in parallel with chromatin condensation and preceded changes in nuclear shape. The earliest changes detected in this study occurred in the coiled body and nucleolar compartments using coilin and fibrillarin antibodies respectively, suggesting that a shutdown in transcription is an early event in denucleation. Fibrillarin redistributed from an open floret pattern to several condensed spots which gradually decreased in intensity and eventually disappeared. Coilin, however, was localized in several microfoci prior to being reorganized into fewer larger foci. Prior to chromatin condensation, coilin redistributed to the nucleolar compartment and was absent from nuclei where chromatin had begun to condense. Such nuclei were positive by TUNEL staining. In contrast to the nucleus, mitochondrial degradation in lens fibre cells was a rapid process and involved a relatively sharp transition between positive and negative fibre cells for two mitochondrial specific markers, BAP 37 and prohibitin. A link between the changes in the nuclear lamina and chromatin with the initiation of mitochondrial fragmentation was also observed. Therefore, it is possible that the signal for the initiation of denucleation could originate from the mitochondria as proposed for apoptosis. Differences between apoptosis and lens fibre cell denucleation were noted and included the timescale of nuclear changes as well as the persistence of a nuclear remnant. These studies suggest that transcriptional shutdown precedes lamina reorganization and chromatin breakdown during lens fibre cell denucleation.

Filensin is proteolytically processed during lens fiber cell differentiation by multiple independent pathways.

Filensin is a lens-specific intermediate filament protein, expressed in the lens fiber cells but not the lens epithelium. Using antibodies to filensin and the other lens intermediate filament proteins, vimentin and CP49, the codistribution of filensin with CP49 and independence of this network from the vimentin network was confirmed. Monoclonal and polyclonal antibodies to peptides and specific subdomains of filensin were used to follow changes in the subcellular distribution of filensin during bovine lens fiber cell differentiation. Filensin is shown to be extensively processed during lens fiber cell differentiation to give protein fragments derived from distinct protein domains, one corresponding to the N-terminal non-alpha-helical/and rod domain and the other to the C-terminal non-alpha-helical tail domain. Immunoblotting analysis using anti-filensin peptide polyclonal antibodies suggested that the two fragment sets arose separately. Residues 331 to 430 in filensin have been identified as an important region in the processing pathway(s). Our results clarify previous confusion in the literature regarding the processing of filensin which arose because of the similar relative electrophoretic mobilities by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the different fragment sets. The predicted secondary structure characteristics of the different domains of filensin suggests different functions for the two fragment sets to give filensin a dual role in the lens. This suggestion is supported by the subtly different subcellular distributions in the peripheral and mature fiber cells of the two filensin fragment sets.

Gene structure and sequence comparisons of the eye lens specific protein, filensin, from rat and mouse: implications for protein classification and assembly.

The full length cDNA sequences of rat and mouse filensin are presented, as well as the structure of the rat filensin gene. This gene spanned 31 kb and included seven introns. The first six introns were conserved in position and phase with those found in the intermediate filament (IF) protein genes of the type II (type II keratin), type III (vimentin) and type V (lamin). The last intron of the filensin was unique. As none of the filensin intron positions coincided with those unique to type I, II or IV genes, it appears that filensin is most similar to type III genes. Comparison of the deduced amino acid sequences for rat and mouse filensin with those of cow and chick, and with other species of IF proteins, indicated the C-terminal non-alpha-helical tail domain of filensin to be one of the most divergent yet found in the vertebrate IF family. The tail domain had three conserved regions which are interrupted with two regions with lower identity. Two motifs, (1) PGDVPDGxxISKAF; and (2) KVEVVESIEKxxxxxIQTYEETxxIVET, were identified as sequences which were particularly highly conserved across species. Coassembly studies using CP49 and a physiologically derived 53 kDa-fragment of filensin showed the motif (2) was not required for filament assembly in vitro. These data strengthen the view that the C-terminal non-alpha-helical domain of filensin contributes in more than one way to filensin function in the lens.

Identification and functional analysis of the mouse lens filensin gene promoter.

Filensin (also called CP94; CP95; CP97; 115kDa protein) is a component of the lens-specific beaded filament which is believed to be functionally important in lens fiber cell differentiation and in maintaining lens fiber cell conformation and transparency. A 17.2kb fragment containing the 5'-upstream sequence of the filensin gene was isolated. S1-mapping analysis determined the transcription start point (tsp; +1) which locates at 94base pairs upstream from the initiating ATG on the filensin gene. In addition to a major tsp, a minor tsp (-136) was observed. DNA sequence of the fragment around the tsp (-2144 to +155) was identified. Analysis of the DNA sequence of the promoter region around tsp revealed two motifs with sequence homology to Sox2 and Maf recognition sequences in addition to one GATA-1 site, two Sp1 binding sites, and three AP-2 binding motifs. No TATA-box or CCAAT-motif was found around the tsp region. A series of sequentially deleted fragments of (-2144 to +40) were fused to firefly luciferase reporter plasmid pGL2 and tested for activity in chicken embryonic lens explants. A minimal promoter region for mouse filensin of (-70 to +40) was identified. The lens-specific promoter activity was detected using lens explants cultured within 12h after dissection. The activity was remarkably enhanced by culture in the presence of 5ng/ml of basic fibroblast growth factor. Each one of the Sp1 and AP-2 binding motifs was localized to the fragment of (-27 to +40) using electrophoretic mobility shift assays. These are the first data to identify the basic elements to the 5'-upstream sequences of the filensin gene, namely the tsp and the minimal filensin promoter.

Expression and characterization of human lamin C.

We have expressed human lamin C cDNA in E. coli using a modification of the pLcII vector system. Protein produced in this way had seven additional amino acids at its N-terminus, but retained key lamin structural and assembly properties. The modified vector we produced may prove useful when difficulties are encountered in removal of the cII fusion peptide by factor X cleavage in the pLcII system. Shadowed preparations of expressed lamin C showed the presence of 50-nm rod-like particles that closely resembled those observed for native material. Isolated molecules had two globular domains at one end, indicating that they were constructed from two parallel polypeptide chains. The expressed material also formed paracrystals with a characteristic 22.5 nm axial repeat, indicating that its assembly properties had also been retained. We also used site-specific mutagenesis to engineer a lamin fragment that lacked the C-terminal non-helical domain of the molecule. This material formed paracrystals similar to those obtained with the intact molecule, indicating that the large C-terminal non-helical domain did not contain information vital for lamin assembly.