Loss of alpha5beta1-mediated adhesion of monocytic cells to fibronectin by interferons beta and gamma is associated with changes in actin and paxillin cytoskeleton.

INTRODUCTION: Modulation of the adhesive responses of monocytic cells may reflect their motility within the bone marrow and at sites of inflammation. Monocyte alpha5beta1 integrins mediate fibronectin-dependent adhesion. We previously showed that type II IFN-gamma reduces adhesiveness to fibronectin (Fn) whereas TGF-beta1 enhances cell attachment. Here, we investigate the role of type I IFNs (alpha, beta) on the adhesive capacity of monocytic cells. MATERIALS AND METHODS: The influence of IFNs on the human U937 cell line adhesion to fibronectin-coated surfaces was determined. The expression of integrins and cytoskeleton proteins was analyzed by FACS, Western blotting and/or fluorescence microscopy analyses. RESULTS: IFN-alpha did not affect cell adhesion to fibronectin. In contrast, IFN-beta, like IFN-gamma, abrogated U937 adhesion to fibronectin and antagonized TGF-beta1-mediated cell attachment to Fn. The impaired binding of IFN-beta- and IFN-gamma-treated cells to fibronectin was not due to reduced levels of alpha5beta1 integrins. IFN-beta and IFN-gamma re-organized filamentous actin, and such rearrangement differed from that observed in TGF-beta1-adhesive cells. U937 cells dominantly expressed 44 to 46 kDa paxillin forms and treatment with IFNs enhanced the number of 66 to 70 kDa forms of paxillin. CONCLUSION: Our data show that IFN-beta and IFN-gamma induced loss of monocytic adhesion to fibronectin associated with changes in actin and paxillin cytoskeleton, thereby pointing to a possible effect of these cytokines in monocyte trafficking.

Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5[prime] and 3[prime] Sequences.

The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5[prime] and 3[prime] regions of the Flaveria bidentis Me1 gene fused to the [beta]-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5[prime] region of Me1 determines bundle sheath specificity, whereas the 3[prime] region contains an apparent enhancer-like element that confers high-level expression in leaves. The interaction of 5[prime] and 3[prime] sequences was dependent on factors that are present in the C4 plant but not found in tobacco.

Isolation and biochemical characterization of highly purified Escherichia coli molecular chaperone Cpn60 (GroEL) by affinity chromatography and urea-induced monomerization.

Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino-acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns. Pure substrate-free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers. The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence. GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformers. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences.

GroEL/ES-mediated refolding of human carbonic anhydrase II: role of N-terminal helices as recognition motifs for GroEL.

The presence of GroEL/ES during the refolding of human carbonic anhydrase II (pseudo-wild type) was found to increase the yield of active enzyme from 65 to 100%. This chaperone action on the enzyme could be obtained by adding GroEL alone, and the time-course in that case was only moderately slower than the spontaneous process. Truncated forms of carbonic anhydrase, in which N-terminal helices were removed, also served as protein substrates for GroEL/ES. This demonstrates that N-terminally located helices are not obligatory as recognition motifs.

Internal symmetry of the molecular chaperone cpn60 (GroEL) determined by X-ray crystallography.

The Escherichia coli molecular chaperone cpn60 oligomer, [cpn60](14), also called GroEL, has been crystallized and examined by X-ray crystallography and self-rotation function calculations. The crystals show unit-cell dimensions a = 143.3, b = 154.6 and c = 265 A, with alpha = 82, beta = 95 and gamma = 107 degrees. The space group is P1 and crystals diffract to 7 A. X-ray analysis shows that the oligomer has one sevenfold symmetry axis and seven twofold axes that are all perpendicular to the sevenfold. The symmetry suggests that [cpn60](24) consists of two heptamers, [cpn60](7), stacked on top of each other. The orientations of the symmetry axes of the two independent [cpn60](14) oligomers in the triclinic unit cell have been determined relative to the crystallographic axes. The two oligomers in the unit cell are arranged side-by- side, but the second oligomer is rotated 26 degrees around the sevenfold axis relative to the first oligomer.

The mechanism of translational coupling in Escherichia coli. Higher order structure in the atpHA mRNA acts as a conformational switch regulating the access of de novo initiating ribosomes.

Bacterial genes are commonly transcribed to form polycistronic mRNAs bearing reading frames whose respective translational efficiencies are not independently determined. As in many bacterial operons, expression of the atp genes of Escherichia coli is strongly influenced by translational coupling. The gene pair atpHA is tightly coupled, whereby atpA is translated at least three times more efficiently than atpH. However, there is no fixed stoichiometry of coupling: mutations in atpH lead to increases in the translation ratio (atpA/atpH) of up to approximately 40-fold. We have demonstrated that secondary structure sequestering the atpA translational initiation region (TIR) is important to the coupling mechanism in that it inhibits de novo translational initiation at the atpA start codon. Genetic and structural analyses indicate that this inhibitory structure can be induced to refold into a less inhibitory conformation either by introducing two single-base substitutions or as a result of ribosomes translating atpH. We propose a model in which the secondary structure of the atpA TIR acts analogously to a "gating device" in that it restricts de novo ribosomal initiation until it is "switched" into a more open conformation. This contrasts with the function of a stem-loop structure located immediately downstream of atpA and upstream of the Shine-Dalgarno region of atpG, which was found to inhibit translation, but not to mediate tight coupling. Results obtained using the "specialized" ribosome system of Hui and de Boer ((1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4762-4766) indicate that primarily ribosomes reinitiating after termination on atpH are responsible for inducing refolding of the atpA TIR. The principle of alternative mRNA conformations with different functional properties embodied in the model presented here can only be fulfilled by certain types of structure. It is likely to operate in several steps of prokaryotic gene expression, underlying a range of regulatory events including transcriptional attenuation and translational activation.

The symmetry of Escherichia coli cpn60 (GroEL) determined by X-ray crystallography.

The internal symmetries of the Escherichia coli molecular chaperone cpn60 oligomer, also called GroEL, have been examined by X-ray crystallography and self-rotation functions calculated at a resolution of 8.9 A. The oligomer ([cpn60]14) has one 7-fold symmetry axis and seven 2-fold axes that are all perpendicular to the 7-fold. The symmetry can be explained if oligomeric cpn60 is arranged as two heptamers stacked on top of each other, where the heptameric arrangement generates the 7-fold symmetry axis and the head-to-head assembly of two heptamers results in the seven 2-fold axes. This is an agreement with interpretations of electron microscopy data. However, the experimental determination of the symmetries reported here are made with an independent technique and at higher resolution. In addition self-rotation function calculations show that the symmetries observed are valid also for the internal parts of GroEL and not only for surface views. The orientations of the symmetry axes of the two independent cpn60 oligomers in the triclinic unit cell have been determined relative to the crystallographic axes. The planes formed by the 2-fold axes in the two oligomers deviate by about 2 degrees from the plane formed by the crystallographic a and c axes, while the 7-fold axes form angles of about 16 degrees with the b-axis. The two oligomers in the unit cell are arranged with their 7-fold axis parallel, but the second oligomer is rotated 26 degrees around the 7-fold axis relative to the first oligomer. Knowledge of the symmetry and orientation of the oligomers in the unit cell will be of great help in further crystallographic work.

Crystallization and preliminary X-ray investigation of the Escherichia coli molecular chaperone cpn60 (GroEL).

The Escherichia coli molecular chaperone, cpn60 (GroEL), has been purified from an overproducing E. coli strain and crystallized. Of the two crystal forms that were obtained, one was found to be suitable for crystallographic and structural studies at low resolution. Preliminary X-ray investigation of the crystals show unit cell dimensions: a = 143.3, b = 154.6 and c = 265 A, with alpha = 82 degrees, beta = 95 degrees and gamma = 107 degrees. The space group is P1 and the crystals diffract to a maximum of 7 A when using CuK alpha X-rays from a rotating anode. Both electron microscopy and non-denaturing electrophoretic analysis of redissolved cpn60 crystals show that cpn60 crystallizes in the native oligomeric form. Comparison between the dimensions of oligomeric cpn60 and the crystallographic unit cell volume suggests that the unit cell contains two oligomeric cpn60 molecules. The VM value for two cpn60 molecules per unit cell is 3.5 A3/Da, corresponding to a water content of 65%. Electrophoretic analysis under denaturing conditions shows that the cpn60 in crystals is heterogeneous, and this probably explains the limited resolution of the diffraction data.

Substoichiometric amounts of the molecular chaperones GroEL and GroES prevent thermal denaturation and aggregation of mammalian mitochondrial malate dehydrogenase in vitro.

The molecular chaperones GroEL and GroES were produced at very high levels in Escherichia coli, purified, and shown to protect pig mitochondrial malate dehydrogenase (MDH) against thermal inactivation in vitro. The apparent rate of MDH inactivation at 37 degrees C was reduced by a factor of at least 5 in a process which required only GroEL, GroES, and ATP. GroEL alone did not protect MDH against thermal inactivation but kept the denatured protein soluble and thereby prevented its aggregation. Reactivation of this soluble and inactive form of MDH could be achieved by addition of GroES even after 120 days of storage at -20 degrees C. Protection could be extended for more than 24 hr at 37 degrees C and was observed at molar ratios of chaperones to MDH as low as 1:4, suggesting that GroEL and GroES perform multiple turnovers in the absence of auxiliary chaperones. The availability of these chaperones in large quantities combined with the apparent promiscuity of GroEL binding shows great potential for stabilization of many proteins for which thermostable variants are not available. We speculate that GroEL and GroES perform similar protective roles in vivo and thereby increase the half-life of proteins which otherwise might aggregate under physiological conditions.

Differential gene expression from the Escherichia coli atp operon mediated by segmental differences in mRNA stability.

The atp operon of Escherichia coli directs synthesis rates of protein subunits that are well matched to the requirements of assembly of the membrane-bound H(+)-ATPase (alpha 3 beta 3 gamma 1 delta 1 epsilon 1a1b2c10-15). Segmental differences in mRNA stability are shown to contribute to the differential control of atp gene expression. The first two genes of the operon, atpl and atpB, are rapidly inactivated at the mRNA level. The remaining seven genes are more stable. It has previously been established that the translational efficiencies of the atp genes vary greatly. Thus differential expression from this operon is achieved via post-transcriptional control exerted at two levels. Neither enhancement of translational efficiency nor insertion of repetitive extragenic palindromic (REP) sequences into the atplB intercistronic region stabilized atpl. We discuss the implications of these results in terms of the pathway of mRNA degradation and of the role of mRNA stability in the control of gene expression.

Translational coupling varying in efficiency between different pairs of genes in the central region of the atp operon of Escherichia coli.

A series of atp::lacZ fusions has been constructed for use in a study of translational coupling in the central region of the Escherichia coli atp operon. Five genes, atpE, atpF, atpH, atpA and atpG, were shown to be translationally coupled to various degrees of tightness. A new lac promoter vector, compatible with the atp::lacZ fusion vectors, was used to express individual atp genes in the same hosts as the fusion genes. The H(+)-ATPase subunits thus synthesized exercised no significant trans-regulation on the expression of the atp::lacZ fusions, indicating that the coupling is primarily cis. The mechanism of this coupling was investigated using in vitro mutagenesis. At least in the case of the pair atpHA, coupling seems to involve facilitated binding of fresh ribosomes to the atpA translational initiation regions.

Either of two nod gene loci can complement the nodulation defect of a nod deletion mutant of Rhizobium leguminosarum bv viciae.

A deletion mutant of Rhizobium leguminosarum biovar viciae lacking the host-specific nodulation (nod) gene region (nodFEL nodMNT and nodO) but retaining the other nod genes (nodD nodABCIJ) was unable to nodulate peas or Vicia hirsuta, although it did induce root hair deformation. The mutant appeared to be blocked in its ability to induce infection threads and could be rescued for nodulation of V. hirsuta in mixed inoculation experiments with an exopolysaccharide deficient mutant (which is also Nod-). The nodulation deficiency of the deletion mutant strain could be partially restored by plasmids carrying the nodFE, nodFEL or nodFELMNT genes but not by nodLMN. Surprisingly, the mutant strain could also be complemented with a plasmid that did not carry any of the nodFELMNT genes but which did carry the nodO gene on a 30 kb cloned region of DNA. Using appropriate mutations it was established that nodO is essential for nodulation in the absence of nodFE. Thus, either of two independent nod gene regions can complement the deletion mutant for nodulation of V. hirsuta. Similar observations were made for pea nodulation except that nodL was required in addition to nodO for nodulation in the absence of the nodFE genes. These observations show that nodulation can occur via either of two pathways encoded by non-homologous genes.

Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum.

DNA sequencing of the nodIJ region from Rhizobium leguminosarum biovar trifolii revealed the nodT gene immediately downstream of nodJ. DNA hybridizations using a nodT-specific probe showed that nodT is present in several R. leguminosarum strains. Interestingly, a flavonoid-inducible nodT gene homologue in R. leguminosarum bv. viciae is not in the nodABCIJ operon but is located downstream of nodMN. The sequence of the nodT gene from bv. viciae was determined and a comparison of the predicted amino-acid sequences of the two nodT genes shows them to be conserved; the predicted protein sequences appear to have a potential transit sequence typical of outer-membrane proteins. Mutations affecting nodT in either biovar had no observed effect on nodulation of the legumes tested.

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation.

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.

Characterization of the Rhizobium leguminosarum genes nodLMN involved in efficient host-specific nodulation.

Three nodulation genes, nodL, nodM and nodN, were isolated from Rhizobium leguminosarum and their DNA sequences were determined. The three genes are in the same orientation as the previously described nodFE genes and the predicted molecular weights of their products are 20,105 (nodL), 65,795 (nodM) and 18,031 (nodN). Analysis of gene regulation using operon fusions showed that nodL, nodM and nodN are induced in response to flavanone molecules and that this induction is nodD-dependent. In addition, it was shown that the nodM and nodN genes are in one operon which is preceded by a conserved 'nod-box' sequence, whereas the nodL gene is in the same operon as the nodFE genes. DNA hybridizations using specific gene probes showed that strongly homologous genes are present in Rhizobium trifolii but not Rhizobium meliloti or Bradyrhizobium japonicum. A mutation within nodL strongly reduced nodulation of peas, Lens and Lathyrus but had little effect on nodulation of Vicia species. A slight reduction in nodulation of Vicia hirsuta was observed with strains carrying mutations in nodM or nodN.

Purification of the phoU protein, a negative regulator of the pho regulon of Escherichia coli K-12.

Thermally induced transcription of the phoU gene under control of the major leftward promoter, pL, of phage lambda resulted in production of the PhoU protein to compose approximately 5% of the total cell protein. The PhoU protein was present in the cytoplasm in the form of an aggregate. The amino acid composition and N-terminal amino acid sequence of the purified protein confirmed the reading frame established earlier for the phoU gene.