The strongly conserved carboxyl-terminus glycine-methionine motif of the Escherichia coli GroEL chaperonin is dispensable

The universally distributed heat-shock proteins (HSPs) are divided into classes based on molecular weight and sequence conservation. The members of at least two of these classes, the HSP60s and the HSP70S, have chaperone activity. Most HSP60s and many HSP70s feature a striking motif at or near the carboxyl terminus which consists of a string of repeated glycine and methionine residues. We have altered the groEL gene (encoding the essential Escherichia coli HSP60 chaperonin) so that the protein produced lacks its 16 final (including nine gly, and five met) residues. This truncated product behaves like the intact protein in several in vitro tests, the only discernible difference between the two proteins being in the rate at which ATP is hydrolysed. GroEL_tr can substitute for GroEL in vivo although cells dependent for survival on the truncated protein survive slightly less well during the stationary phase of growth. Elevated levels of the wild-type protein can suppress a number of temperature-sensitive mutations; the truncated protein lacks this ability.     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic dnaA mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in dnaA mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or dnaA mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of dnaA mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or dnaA membrane preparations. Thus, the dnaA mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

Expression of antifreeze proteins in transgenic plants

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. Theafa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.     

The PucC protein of Rhodobacter capsulatus mitigates an inhibitory effect of light-harvesting 2 α and β proteins on light-harvesting complex 1

Rhodobacter capsulatus contains lhaA and pucC genes that have been implicated in light-harvesting complex 1 and 2 (LH1 and LH2) assembly. The proteins encoded by these genes, and homologues in other photosynthetic organisms, have been classified as the bacteriochlorophyll delivery (BCD) family of the major facilitator superfamily. A new BCD family phylogenetic tree reveals that several PucC, LhaA and Orf428-related sequences each form separate clusters, while plant and cyanobacterial homologues cluster more distantly. The PucC protein is encoded in the pucBACDE superoperon which also codes for LH2 α (PucA) and β (PucB) proteins. PucC was previously shown to be necessary for formation of LH2. This article gives evidence indicating that PucC has a shepherding activity that keeps the homologous α and β proteins of LH1 and LH2 apart, allowing LH1 to assemble properly. This shepherding function was indicated by a 62% reduction in LH1 levels in ΔLHII strains carrying plasmids encoding pucBA along with a C-terminally truncated pucC gene. More severe reductions in LH1 were seen when the truncated pucC gene was co-expressed in the presence of C-terminal PucC::PhoA fusion proteins. It appears that interaction between truncated PucC::PhoA fusion proteins and the truncated PucC protein disrupts LH1 assembly, pointing towards a PucC dimeric or multimeric functional unit.     

Obtaining recombinant forms of the outer membrane protein F of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and assessment of their immunogenic properties

Two recombinant forms of the outer membrane protein F (OprF) of Pseudomonas aeruginosa were obtained, the full-length protein OprF and the C-terminal part of the OprF protein (aa 192–342). As a result of double immunizations, these recombinant proteins provided mice with resistance to experimental intraperitoneal challenge with P. aeruginosa. The best protective effects were observed at a dose of 25 μg for OprF and 50 μg for the truncated OprF variant (indices of efficiency were 3.3 and 2.8, respectively). Rabbit antisera immune to the recombinant proteins were also able to protect mice from the experimental infection with P. aeruginosa. Indices of efficiency were 6.4 for OprF and 6.0 for the OprF C-terminal part; these values were approximately two times as high as the effect of sera from intact test animals (3.2).     

伯氏疟原虫Pb22截短蛋白免疫血清的传播阻断能力的研究

为探讨伯氏疟原虫(Plasmodium berghei)Pb22蛋白免疫血清的传播阻断能力,应用原核表达系统高效表达Pb22截短蛋白免疫小鼠后获得免疫血清。IFA实验证明Pb22截短蛋白免疫血清均可与疟原虫天然抗原结合。通过传播阻断实验,比较了Pb22截短蛋白和全长蛋白免疫血清的传播阻断效果,证明截短蛋白和全长蛋白对雄配子体出丝均有抑制作用,结果表明全长蛋白免疫小鼠的雄配子体出丝与对照组相比显著下降,截短蛋白免疫小鼠的雄性配子体出丝具有下降趋势但无统计学差异。全长蛋白和截短蛋白免疫小鼠的动合子形成数量较对照组均显著下降。以上结果表明抗Pb22截短蛋白免疫血清具有传播阻断能力,但阻断效果不如全长蛋白免疫血清。     

Radiation inactivation of membrane proteins: Molecular weight estimates in situ and after Triton X-100 solubilization

Target size analysis by radiation inactivation is widely used for molecular weight determination of membrane enzymes and receptors in situ without the need for prior solubilization or purification. However, since most molecular weight data available in the literature on membrane proteins involve the use of detergents for solubilization, the target sizes of membrane proteins in situ and after solubilization by detergent treatment have been compared. Using data from the literature and personal results, three different types of behavior of membrane proteins in presence of detergents were found: (i) uncoupling of subunits (electric eel acetylcholinesterase, placental steroid sulfatase, and human nonspecific β-glucosidase); (ii) coupling of protein molecules (mouse liver neuraminidase, and rat liver insulin receptor regulatory component); and (iii) no major change in quaternary structure (rat liver insulin receptor, kidney γ-glutamyltransferase, asialoglycoprotein receptor, insulin degrading enzyme, and human leucocyte neuraminidase). For all these proteins, there is a statistically significant increase in target size of about 24% over the value obtained in situ without detergent. A relatively large body of literature data involving a variety of membrane proteins, membrane types, and irradiation conditions (electron accelerators or ⁶⁰Co sources, and proteins irradiated in lyophilized form or frozen solution) was examined, and it was concluded that target sizes of membrane proteins, irradiated in the presence of Triton X-100, should be diminished by a factor of about 24% to obtain the molecular weight value.     

A functional analysis of the genes Enhancer of split and HLH-m5 during early neurogenesis in Drosophila melanogaster

To assess the functional domains of the proteins encoded by E(spl) and HLH-m5, two genes of the Enhancer of split complex [E(SPL)-C] of Drosophila melanogaster, a number of variants have been made by in vitro mutagenesis, transformed into the germ line of the wild-type, and genetically combined with a chromosomal deletion lacking four of the genes of the E(SPL)-C. All constructs used attenuated the neurogenic phenotype associated with this deletion. However, constructs encoding proteins with truncated carboxy-termini exibited in all cases a higher activity than constructs encoding the full length version of the protein. Neutralization of the basic domain severely reduced, but did not completely abolish the rescuing activity of E(spl), while proteins in which a proline residue within the basic domain had been changed to either threonine or asparagine were slightly less efficient in their rescuing activity than the corresponding wild-type versions. We discuss the possible significance of these results for the function of the protein domains.     

Cloning and expression of a novel component of the CAP superfamily enhanced in the inflammatory response to LPS of the ascidian Ciona intestinalis

The CAP superfamily is a group of proteins that have been linked to several biological functions such as reproduction, cancer, and immune defense. A differential screening between lipopolysaccharide (LPS)-challenged and naive Ciona intestinalis has been performed to identify LPS-induced genes. This strategy has allowed the isolation of a full-length 1471-bp cDNA encoding for a 413-amino-acid protein (CiCAP). In silico analysis has shown that this polypeptide displays a modular structure with similarities to vertebrate CAP-superfamily proteins and to a collagen-binding adhesin of Streptococcus mutans. Domain organization analysis and alignment of CiCAP to other vertebrate CAP proteins have revealed a novel structure suggesting that this protein originated from a common ancestor gene that gave rise to many subfamilies of mosaic proteins with novel functions. Quantitative mRNA expression performed by real-time polymerase chain reaction analysis has demonstrated that this gene is rapidly activated in the pharynx of C. intestinalis a few hours after LPS injection. Moreover, in situ hybridization has shown that CiCAP mRNA is highly expressed by hemocytes with large granules contained inside the pharynx vessels. Thus, CiCAP represents a protein with novel structural domains involved in ascidian immune responses.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay

The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.     

The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state

MR60 is an intracellular membrane protein which has been shown to act as a mannoside specific lectin and to be identical to ERGIC-53, a protein characteristic of the endoplasmic reticulum-Golgi apparatus- intermediate compartment, acting as a shuttle. According to its primary sequence, this MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate recognition domain, a stem, a transmembrane segment and a cytosolic domain. The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers and hexamers). In this paper, we study the relationship between the oligomerization state and the sugar binding capacity by using recombinant proteins. The expression of the recombinant proteins was evidenced by immunocytochemistry and by immunoprecipitation followed by SDS-PAGE analysis. The full size recombinant protein binds mannosides and is oligomeric, up to the hexameric form. Two truncated proteins lacking the transmembrane and the cytosolic domains were prepared and characterized. A long one, containing the cysteine 466 close to the C-terminal end of the recombinant protein but lacking the cysteine 475, close to the C- terminal end of the native protein, does bind mannosides and forms dimers but no higher oligomeric forms. A shorter one, lacking both the cysteines 466 and 475, does not bind mannosides and does not form dimers or higher polymers. The two cysteines in the carbohydrate recognition domain (C190 and C230) are not involved in the stabilization of oligomers. In conclusion, this study shows that the luminal moiety of MR60/ERGIC-53 contains a device allowing both its oligomeric pattern and its sugar binding capability.      

In situ imaging and isolation of proteins using dsDNA oligonucleotides

As proteomics initiatives mature, the need will arise for the multiple visualization of proteins and supramolecular complexes within their true context, in situ. Single-stranded DNA and RNA aptamers can be used for low resolution imaging of cellular receptors and cytoplasmic proteins by light microscopy (LM). These techniques, however, cannot be applied to the imaging of nuclear antigens as these single-stranded aptamers bind endogenous RNA and DNA with high affinity. To overcome this problem, we have developed a novel method for the in situ detection of proteins using double-stranded DNA oligonucleotides. To demonstrate this system we have utilized the prokaryotic DNA-binding proteins LacI and TetR as peptide tags to image fusion proteins in situ using dsDNA oligonucleotides encoding either the Lac or Tet operator. Using fluorescent and fluorogold dsDNA oligonucleotides, we localized within the nucleus a TetR–PML fusion protein within promyelocytic leukaemia protein (PML) bodies by LM and a LacI–SC35 fusion protein within nuclear speckles by correlative light and electron microscopy (LM/EM). Isolation of LacI–SC35 was also accomplished by using biotinylated dsDNA and streptavidin sepharose. The use of dsDNA oligonucleotides should complement existing aptamer in situ detection techniques by allowing the multiple detection and localization of nuclear proteins in situ and at high resolution.     

Attempts to quantitate immunocytochemistry at the electron microscope level

Summary The ability to localize intracellular macromoleculesin situ by high resolution techniques has been made possible by the development of antibody labelling of thin sections obtained either from tissues embedded in an hydrophilic matrix, or by ultracryotomy or from conventional plastic embedded tissue. When particle-tagged immunological reagents are used to visualize intracellular antigens, quantitative information can be obtained by combining particle counts with morphometric estimations of compartment volume. Various detection systems have been used successfully for quantitation, which include ferritin-conjugated antibodies, biotin-avidin-ferritin complexes and, more recently, gold-protein A conjugates. Examples of the use of these techniques the localization of secretory proteins in pancreatic exocrine cells, opsin and a large membrane protein in photoreceptor cells of frog retina, and contractile proteins in skeletal muscle are given. Quantitative data obtained by morphometric analysis, both in bovine and rat pancreatic exocrine cells, are compared with values assessed by biochemical methods.     

Codon usage and base composition inRickettsia prowazekii

Codon usage and base composition in sequences from the A + T-rich genome ofRickettsia prowazekii, a member of the alpha Proteobacteria, have been investigated. Synonymous codon usage patterns are roughly similar among genes, even though the data set includes genes expected to be expressed at very different levels, indicating that translational selection has been ineffective in this species. However, multivariate statistical analysis differentiates genes according to their G + C contents at the first two codon positions. To study this variation, we have compared the amino acid composition patterns of 21R. prowazekii proteins with that of a homologous set of proteins fromEscherichia coli. The analysis shows that individual genes have been affected by biased mutation rates to very different extents: genes encoding proteins highly conserved among other species being the least affected. Overall, protein coding and intergenic spacer regions have G + C content values of 32.5% and 21.4%, respectively. Extrapolation from these values suggests thatR. prowazekii has around 800 genes and that 60–70% of the genome may be coding. Correspondence to: S.G.E. Andersson     

Utilization of tmRNA sequences for bacterial identification

Background  

Ribosomal RNA molecules are widely used for phylogenetic and in situ identification of bacteria. Nevertheless, their use to distinguish microorganisms within a species is often restricted by the high degree of sequence conservation and limited probe accessibility to the target in fluorescence in situ hybridization (FISH). To overcome these limitations, we examined the use of tmRNA for in situ identification. In E. coli, this stable 363 nucleotides long RNA is encoded by the ssrA gene, which is involved in the degradation of truncated proteins.     

Production of polyclonal antibodies against a wheat thioredoxinh by genetic immunization

The recent techniques of genetic immunization, in which DNA constructs are introduced directly into mammalian tissuesin vivo, were used to produce antisera against thioredoxinh, a protein of wheat. Two rabbits and two mice were inoculated intramuscularly with a vector containing the cDNA encoding the protein of interest under control of a cytomegalovirus promoter. No immune response was observed in rabbits, even when a fourfold quantity of DNA and a different inoculation site were used. By contrast, an inoculated mouse was found to produce antisera against the wheat thioredoxinh as analyzed by western blotting. This technique appears useful, therefore, to obtain polyclonal antibodies against plant proteins that are difficult to purify, if the corresponding cDNAs are available.     

Characterization of MSH/ACTH-like immunoreactivity in sciatic nerves of Xenopus laevis by immunocytochemistry,Western blotting and radioimmunoassay

Summary Previous studies have indicated that rat neurofilament protein may contain an endogenous MSH-like epitope with neuroregenerative properties. The presence of such an epitope has now been studied in nerve tissue from Xenopus laevis. Western blot analyses of sciatic nerve tissue using an assortment of sequence-specific MSH/ACTH antisera revealed the presence of two major immunoreactive protein bands of 52 and 50 kDa, which contained a mid-region MSH-like epitope. Weaker staining occurred in another protein band at 135 kDa. Immunocytochemistry revealed the immunoreactivity to reside in the axis cylinders of the nerve fibers. Other antisera, recognizing other regions of MSH/ACTH produced strong staining of Xenopus intermediate lobes, but failed to stain sciatic nerves. Thus, the proteins detected have no clear relation to either Xenopus neurofilament proteins or proopiomelanocortin.     

High quality protein microarray using in situ protein purification

Background  

In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays.