Bifurcation analysis of Liesegang ring pattern formation.

Bifurcation analysis is introduced to a prototype Liesegang ring (LR) model to explain pattern formation as an instability of a propagating plane reaction front. A theoretical criterion for the onset of patterning is derived and numerically tested. The uneven spacing law of LR bands is explained as a consequence of the time varying velocity of the moving reaction front. Suggestions for controlling pattern formation are provided.

A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II.

The three-dimensional structure of the complex between a T cell receptor (TCR) beta chain (mouse Vbeta8.2Jbeta2.1Cbeta1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR beta chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the beta-SEC3 complex ("hot spot" residues) are strictly conserved among enterotoxins reactive with mouse Vbeta8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vbeta-binding specificities.

Structure of the Vdelta domain of a human gammadelta T-cell antigen receptor.

Antigen recognition by T lymphocytes is mediated by cell-surface glycoproteins known as T-cell antigen receptors (TCRs). These are composed of alpha and beta, or gamma and delta, polypeptide chains with variable (V) and constant (C) regions. In contrast to alphabeta TCRs, which recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC), gammadelta TCRs appear to recognize proteins directly, without antigen processing, and to recognize MHC molecules independently of the bound peptide. Moreover, small phosphate-containing non-peptide compounds have also been identified as ligands for certain gammadelta T cells. These studies indicate that antigen recognition by gammadelta TCRs may be fundamentally different from that by alphabeta TCRs. The three-dimensional structures of several alphabeta TCRs and TCR fragments, and their complexes with peptide-MHC or superantigens, have been determined. Here we report the crystal structure of the Vdelta domain of a human gammadelta TCR at 1.9 A resolution. A comparison with antibody and alphabeta TCR V domains reveals that the framework structure of Vdelta more closely resembles that of VH than of Valpha, Vbeta or VL (where H and L refer to heavy and light chains), whereas the relative positions and conformations of its complementarity-determining regions (CDRs) share features of both Valpha and VH. These results provide the first direct evidence that gammadelta TCRs are structurally distinct from alphabeta TCRs and, together with the observation that the CDR3 length distribution of TCR delta chains is similar to that of immunoglobulin heavy chains, are consistent with functional studies suggesting that recognition of certain antigens by gammadelta TCRs may resemble antigen recognition by antibodies.

Dual conformations of a T cell receptor V alpha homodimer: implications for variability in V alpha V beta domain association.

The crystal structure of a mutant T cell receptor (TCR) V alpha domain containing a grafted third complementarity-determining region (CDR3) from a different V alpha was determined at 2.3 A resolution by molecular replacement using the wild-type V alpha structure as a search model. Like the wild-type V alpha domain, the mutant crystallized as a homodimer very similar to TCR V alpha V beta and antibody V(L)V(H) heterodimers, with the CDR loops disposed to form part of the antigen-binding site. However, the relative orientation of the two chains in the mutant V alpha homodimer differs from that in the wild-type by a rotation of 14 degrees such that the buried surface area in the dimer interface of the mutant is 140 A2 less than in the wild-type. While the residues forming the interface are essentially the same in the two structures, there are only four pairs of interface hydrogen bonds in the case of the mutant compared with eight for the wild-type. These results suggest that multiple relative orientations of the V alpha and V beta domains of TCRs may be possible, providing a significant contribution to TCR combining site diversity.

Cloning, expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.

Crystal structure of the V alpha domain of a T cell antigen receptor.

The crystal structure of the V alpha domain of a T cell antigen receptor (TCR) was determined at a resolution of 2.2 angstroms. This structure represents an immunoglobulin topology set different from those previously described. A switch in a polypeptide strand from one beta sheet to the other enables a pair of V alpha homodimers to pack together to form a tetramer, such that the homodimers are parallel to each other and all hypervariable loops face in one direction. On the basis of the observed mode of V alpha association, a model of an (alpha beta)2 TCR tetramer can be positioned relative to the major histocompatibility complex class II (alpha beta)2 tetramer with the third hypervariable loop of V alpha over the amino-terminal portion of the antigenic peptide and the corresponding loop of V beta over its carboxyl-terminal residues. TCR dimerization that is mediated by the alpha chain may contribute to the coupling of antigen recognition to signal transduction during T cell activation.

A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator.

A new temperature-regulated T7 RNA polymerase-driven transcription system has been developed. This system is based on a hybrid regulatory region: the phage T7 late promoter (PT7) linked to the Escherichia coli lac operator (Olac) [Giordano et al., Gene 84 (1989) 209-219], which was located in an earlier obtained [Mashko et al., Gene 97 (1991) 259-266] temperature-controlled amplifiable plasmid, carrying cat under the control of PT7-Olac and, in addition, lambda major early promoter-operator regions and gene cIts857. Plasmids of the pT7-Olac-cat-tsr series were stably maintained at a low-copy-number when grown at low temperature (28 degrees C). In E. coli BL21(DE3), carrying the Plac-controllable T7 RNA polymerase-encoding gene, efficient repression of cat transcription was observed, that was provided by the LacI repressor and, probably, the thermolabile repressor CIts857. At low and moderate temperatures (28/37 degrees C), this 'cooperative' repression was so tight that cat expression was not observed in the cells carrying PT7-Olac on the plasmids, even after IPTG-inducible T7 RNA polymerase biosynthesis. As a result of the thermo-amplification of the recombinant plasmids and temperature-inactivation of CIts857, expression of the T7 RNA polymerase-encoding gene was derepressed due to the titration of LacI by the increasing copies of Olac which in turn, led to the highly efficient T7 RNA polymerase-driven accumulation of CAT in the cells.

[Design of a species-specific DNA probe based on the pFra plasmid of the plague pathogen]. / Konstruirovanie vidospetsificheskogo DNK-zonda na osnove plazmidy pFra chumnogo mikroba.

The recombinant plasmid pBS1 carrying a 2 kb SalGI fragment of Yersinia pestis pFra plasmid was constructed by insertion of the fragment into a vector plasmid pBR327. SalGI-BspRI 400 bp subfragment was recloned into a pBR322 vector plasmid. Open reading frame was found in the fragment by DNA sequencing technique. The subfragment designated F1-probe permits one to identify specifically the Yersinia pestis strains harbouring pFra plasmid, thus, differing them from closely related Yersiniea and other representatives of Enterobacteriaceae family.

[Obtaining human recombinant (serine-17) beta-interferon by the method of oligonucleotide-directed mutagenesis and its expression in Escherichia coli]. / Poluchenie rekombinantnogo (serin-17) beta-interferona cheloveka metodom oligonukleotidnapravlennogo mutageneza i ego ékspresiia v Escherichia coli.

The gene of human mutant (serine-17) fibroblast interferon was isolated with the use of highly efficient oligonucleotide-directed mutagenesis. On the basis of the constructed expression plasmid pPR-IFN Ser17 a strain producing human mutant beta-interferon (VKPM V-4678) was developed. It was shown that the specific activity of the human mutant (serine-17) fibroblast interferon was 1 order of magnitude higher than that of the recombinant interferon which reaches the specific activity of natural fibroblast interferon.

Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-1 beta synthesis in Escherichia coli.

A new dual-replicon recombinant plasmid, pPR53-tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 [Mashko et al., Gene 88 (1990) 121-126]. In contrast to its progenitor, pPR53-tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion. In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and a low Cop at 28 degrees C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the pL promoter. The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53-tsr; the increased plasmid Cop is estimated at approx. 200 per genome 6 h after thermal induction at 42 degrees C. Thus, pPR53-tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids. Here, we employ such an approach for optimization of production of human interleukin-1 beta (hIL-1 beta) in Escherichia coli at a high level. The thermo-induced level of recombinant hIL-1 beta (re-hIL-1 beta) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used. A method based on acidification of the water-soluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1 beta. The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies. The protein yield ranged between 3-5 mg of re-hIL-1 beta/g of wet cells. The re-hIL-1 beta specific activity was about 2 x 10(8) units/mg, coinciding with that of the authentic hIL-1 beta.

TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells.

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the hAng gene in Escherichia coli; isolation and characterization of human recombinant Ser-(-1) angiogenin.

Recombinant human angiogenin has been synthesized in Escherichia coli with the aid of a human angiogenin gene (hAng) cloned by Neznanov et al (1990) from a human complementary DNA (cDNA) library. The gene has been expressed by use of a new type of expression vector called a 'TGATG vector' (plasmid pPR-TGATG-1; Mashko et al 1990a). The highest level of accumulation of the recombinant angiogenin (6%-8% of the total cell protein) was observed in E. coli strain BL21 carrying a temperature-amplifiable version of the plasmid. The synthesized polypeptide carries an additional serine residue at its N terminus in comparison with natural angiogenin. Furthermore, the initiator methionine residue of the recombinant protein is removed with high efficiency by E. coli terminal aminopeptidase. Simple procedures for purification of the recombinant angiogenin from the insoluble fraction of cell protein, and for refolding the protein allowed the isolation of almost 5 mg recombinant angiogenin g-1 wet bacterial biomass. The recombinant Ser-(-1) angiogenin displayed the same biological properties (specific RNAase activity and the ability to induce blood vessel growth on the sclera of experimental animals) as its natural counterpart isolated from human blood.

[Effectiveness of expression of chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli. III. Relative effectiveness of cloned promoters of Escherichia coli and coliphages]. / Issledovanie éffektivnosti ékspressii gena khloramfenikolatsetiltransferazy pod kontrolem chuzherodnykh reguliatornykh oblastei v kletkakh Escherichia coli. III. Izuchenie otnositel'noi éffektivnosti klonirovannykh promotorov Escherichia coli i kolifagov.

The study on the rate of initiation of model gene cat transcription under the control of E. coli (Plac UV5, Ptrp, Pcat, Ptac), phage lambda (PL, PR), phi X174 (PD) promotors was carried out by means of hybridization of pulse labelled in vivo mRNA with the DNA coding parts. The presence of gene bla(Apr) with its own constitutive promoter in all the recombinants permitted to use the level of appropriate mRNA in the cell as an internal standard. This method allowed to evaluate the true efficiency of the promoters in question. The strength of the promoters studied is shown to be equal within the limit of one order value.

[Creation of artificial hybrid operons with partially overlapping genes to achieve an expression of heterologous genes in Escherichia coli cells]. / Sozdanie "iskusstvennykh gibridnykh operonov s chastichno perekryvaiushchimisia genami" dlia dostizheniia ékspressii geterologichnykh genov v kletkakh Escherichia coli.

A new method of optimization of foreign gene expression in E. coli, based on the construction of hybrid operons with partially overlapping genes is described. The partial overlapping of the translation termination and initiation sites in the formed operon must provide translational coupling of appropriate gene product synthesis. Such an approach has provided the synthesis of human interferon alpha F in E. coli cells under the control of the lacUV5-promotor up to about (3-4).10(7) units per liter of bacterial culture. The reinitiation of the distal gene translation is shown to take place in the intercistronic region. Substitution of the lacUV5 promotor by the more efficient tac one allowed to increase the synthesis level of interferon alpha F to (1-2).10(8) units per liter. The conclusion is made about the equimolarity of distal and proximal to the promotor genes products syntheses when the intercistronic region of E. coli trpE-trpD genes are used for translational coupling.

[Optimization of the gene expression for human alpha F- and beta 1-interferons in Escherichia coli cells]. / Optimizatsiia ékspressii genov al'fa F- i beta 1-interferonov v kletkakh Escherichia coli.

The basic results of the studies on expression of the genes of human alpha F- and beta 1-interferons in E. coli cells are presented. To synthesize the fibroblast interferon, the respective fragment of the human chromosome was cloned, the complete nucleotide sequence of the structural moiety of mature beta-interferon was determined and the genes of "hybrid (interferon-like) proteins" and "hybrid sites of ribosome binding" were constructed with control of the beta-interferon gene by the prokaryotic regulatory areas. Synthesis of beta-interferon was achieved (1.10(7)-5.10(7) IU per 1 l of the bacterial culture) with the use of the tryptophan operon promoter. A new procedure for optimization of allogenic genetic information in E. coli cells: constructing of "hybrid operons with partially overlapping genes" or artificial "overlappons" was developed following the example of the alpha F-interferon gene cloned in the Laboratory headed by E. D. Sverdlov at the Institute of Bioorganic Chemistry of the USSR Academy of Sciences. The use of this procedure enabled production of up to 5.10(7) IU/l of alpha F-interferon under the control of the lacUV5-promoter. On the basis of the newly constructed vector molecules expression of the genes of alpha F- and beta 1-interferons was amplified with the "overlappon" procedure.

[Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulator regions in Escherichia coli cells. II. Molecular cloning of promoters]. / Issledovanie éffektivnosti ékspressii gena khloramfenikolatsetiltransferazy pod kontrolem chuzherodnykh reguliatornykh oblastei v kletkakh Escherichia coli. II. Molekuliarnoe klonirovanie promotorov.

The trpOP, lacUV5, tacOP, PR and PL-promotors were cloned in the previously obtained pML4 vector plasmid. The expression of structural gene cat was studied by the chloramphenicolacetyltransferase determination in cell extracts. The level of protein synthesis by appropriate recombinant plasmids was analysed in vivo and in vitro. It was shown that the efficiency of the gene expression is determined by both the "strength" of the promotors and mRNA translation specificity. The obtained collection of the plasmids might be used for the determination of the promotor strength by the hybridization of pulse-labeled mRNA with DNA and an effective expression of the genes by means of "hybrid protein gene" and "hybrid operon" constructions.

[Expression of the chloramphenicol acetyltransferase gene is under control of various promoters of E. coli and phage lambda]. / Ekspressiia gena khloramfenikolatsetiltransferazy pod kontrolem nekotorykh promotorov E. coli i bakteriofaga lambda.

Plasmids have been constructed with the structural region of the cat gene being under the control of the lactose (lacUV5), tryptophane (trpOP), operons of Escherichia coli, the hybrid trp-lac (tac) promoter and early bacteriophage lambda promoters (PL, PR and PLIT). The expression of chloramphenicolacetyltransferase gene in Escherichia coli cells harbouring such recombinant plasmids and pBR325 as well has been examined by determining the chloramphenicol resistance and studying the enzyme activity of Cm-acetylase. A high level of enzyme synthesis is connected with transcription from PL, PR and tac promoters.