Efficient introduction of aryl bromide functionality into proteins in vivo.

Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20-25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.

Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli.

Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(Phe)amber/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.

The in vitro kinetics of mitochondrial and cytosolic creatine kinase determined by saturation transfer 31P-NMR.

Michaelis- and dissociation constants of sarcomeric mitochondrial creatine kinase (Mi(b)-CK) in solution were determined by enzyme assay and compared to those of cytosolic MM-CK under identical conditions at pH 7.4 and 25 degrees C. Saturation transfer 31P-NMR was used to determine the steady state fluxes mediated by Mi-CK and MM-CK in solution. The NMR detected fluxes of both Mi-CK and MM-CK exhibited, as expected, a linear dependence on Vmax (Vmax range 0-9 mM.s-1). Interestingly, the oligomeric state of Mi-CK, with the Mi-CK octamer/dimer ratio ranging from 2 to 9, did not have a significant effect on the flux/Vmax ratio. Furthermore, the flux/Vmax ratio of Mi-CK was twice as high as that of MM-CK under similar conditions (flux/Vmax for Mi-CK was 0.31 and for MM-CK was 0.15). This difference was primarily due to a 4-fold higher apparent affinity for MgADP of Mi-CK compared to MM-CK (K(m)(MgADP) = 22 +/- 9 microM and 80 +/- 17 microM, resp.). The NMR observed fluxes were in agreement with the fluxes as calculated from the rate equation, using the appropriate metabolite concentrations and the kinetic constants from the spectrophotometric assays. Thus we conclude, that Mi-CK and MM-CK, when in solution, catalyse an exchange-reaction, the flux of which is fully observable by saturation transfer 31P-NMR.

Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments.

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease-sensitive site in M-CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi-CK) into two protein fragments encompassing amino acids [1-167] and [168-380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two-step procedure. In contrast, co-expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi-CK by far- and near-UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C-terminal fragment refolded into a native-like conformation in vitro, whereas the N-terminal fragment was largely unfolded. Prefolded [168-380] interacted in vitro with [1-167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K1 = 17 microM) and then isomerize slowly to an active enzyme (k2 = 0.12 min-1; k-2 = 0.03 min-1). Our data suggest that the C-terminal fragment of Mi-CK represents an autonomous folding unit, and that the folding of the C-terminal part might precede the conformational stabilization of the N-terminal moiety in vivo.

Autophosphorylation of creatine kinase: characterization and identification of a specifically phosphorylated peptide.

We report that several different chicken and rabbit creatine kinase (CK)1 isoenzymes showed an incorporation of 32P when incubated with [gamma-32P]ATP in an autophosphorylation assay. This modification was was shown to be of covalent nature and resulted from an intramolecular phosphorylation reaction that was not dependent on the CK enzymatic activity. By limited proteolysis and sequence analysis of the resulting peptides, the autophosphorylation sites of chicken brain-type CK could be localized within the primary sequence of the enzyme to a 4.5 kDa peptide, spanning a region that is very likely an essential part of the active site of creatine kinase. Homologous peptides were found to be autophosphorylated in chicken muscle-type CK and a mitochondrial CK isoform. Phosphopeptide as well as mutant enzyme analysis provided evidence that threonine-282(2), threonine-289 and serine-285 are involved in the autophosphorylation of CK. Thr-282 and Ser-285 are located close to the reactive cysteine-283. Thr-289 is located within a conserved glycine-rich region highly homologous to the glycine-rich loop of protein kinases, which is known to be important for ATP binding. Thus, it seems likely that the described region constitutes an essential part of the active site of CK.

Multiple-state equilibrium unfolding of guanidino kinases.

The denaturant-induced equilibrium unfolding of octameric mitochondrial creatine kinase, dimeric cytosolic muscle-type creatine kinase, and monomeric arginine kinase was investigated. Stable unfolding intermediates for all three enzymes were manifested by a strongly biphasic red shift of intrinsic protein fluorescence upon increasing denaturant concentrations. In the intermediate state, all proteins were monomeric and enzymatically inactive, but still retained a globular shape. Native tertiary structure interactions were largely disrupted, while at least 50% of the secondary structures were conserved, as suggested by near- and far-UV circular dichroism, respectively. A significantly increased surface hydrophobicity of the intermediate conformation, compared to both the native and the fully unfolded states, was observed by the binding of the hydrophobic fluorescent dye ANS. The observed properties agree formally with the definition of the molten globule state, but can be alternatively explained by a sequential unfolding of individual domains, involving a transient exposure of domain interfaces. Very similar unfolding profiles for all three proteins suggest that the formation of stable unfolding intermediates is not a consequence of the specific oligomeric structures of the CKs but rather due to a common, probably two-domain architecture of the guanidino kinase protomers.

Role of a small RNA pol II subunit in TATA to transcription start site spacing.

The yeast shi mutation affects the spacing between the TATA promoter element and transcription initiation sites; for the H2B and ADH1 genes, a series of start sites located approximately 50-80 bp downstream of TATA is used in addition to the wild-type initiation sites located at around 100 bp from TATA (1). Here, the yeast SHI wild-type gene has been isolated by complementation and shown to be identical to RPB9, the gene encoding a small subunit of RNA polymerase II. A point mutation in the shi gene, changing a cysteine residue in a putative zinc ribbon motif into a phenylalanine residue, was demonstrated to permit the observed usage of upstream initiation sites. Deletion of the non-essential SHI gene also results in usage of upstream initiation sites and causes conditional growth defects.

The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation.

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK. Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.

The structure of mitochondrial creatine kinase and its membrane binding properties.

The biochemical and biophysical characterization of the mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle is reviewed with emphasis on the structure of the octameric oligomer by electron microscopy and on its membrane binding properties. Information about shape, molecular symmetry and dimensions of the Mi-CK octamer, as obtained by different sample preparation techniques in combination with image processing methods, are compared. The organization of the four dimeric subunits into the Mi-CK complex as apparent as apparent in the end-on projections is discussed and the consistently observed high binding affinity of the four-fold symmetric end-on faces towards many support films and towards each other is outlined. A study on the oligomeric state of the enzyme in solution and in intact mitochondria, using chemical crosslinking reagents, is presented together with the results of a search for a possible linkage of Mi-CK with the adenine nucleotide translocator (ANT). The nature of Mi-CK binding to model membranes, demonstrating that rather the octameric than the dimeric subspecies is involved in lipid interaction and membrane contact formation, is resumed and put into relation to our structural observations. The findings are discussed in light of a possible in vivo function of the Mi-CK octamer bridging the gap between outer and inner mitochondrial membranes at the contact sites.

The N-terminal heptapeptide of mitochondrial creatine kinase is important for octamerization.

Mitochondrial creatine kinase (Mi-CK) isoenzymes, in contrast to cytosolic CKs, form octameric molecules composed of four stable dimers. Octamers and dimers are interconvertible. Removal of the N-terminal pentapeptide of chicken cardiac Mi-CK (Mib-CK) by limited proteolysis drastically destabilized the octamer. The role of the charged amino acids within the N-terminal heptapeptide was studied in detail by progressively substituting the four charged residues by uncharged ones. In these altered proteins, the octamer/dimer ratio at equilibrium conditions was shifted toward the dimer. Also, the in vitro dissociation rate of octamers into dimers was increased in correlation to the number of charged residues eliminated. Point mutant E4Q, with only one positive charged amino acid removed, already displayed a 50-fold higher equilibrium constant and a 13-fold increased dissociation rate compared to wild-type Mib-CK. Mutant 4-7, having all four charged residues in the N-terminal heptapeptide substituted, showed a 100-fold higher equilibrium constant and a 146-fold increased dissociation rate. The corresponding values for double mutant E4Q/K5L were intermediate between the single and quadruple mutants. This strongly suggests that the charged amino acids in the N-terminal heptapeptide of Mib-CK, and therefore ionic interactions mediated by the N-terminal moiety, play an important role in forming and stabilizing the octameric molecule. The role of dimer-octamer interconversion in vivo as a possible regulator of contact site formation and of mitochondrial oxidative phosphorylation is discussed.

Creatine kinase: the reactive cysteine is required for synergism but is nonessential for catalysis.

Chemical modification of rabbit muscle creatine kinase (CK) with thiol-specific reagents led to partial or complete inactivation of the enzyme. Using site-directed mutagenesis, we have substituted the corresponding reactive Cys278 in the chicken cardiac mitochondrial creatine kinase (Mib-CK) with either glycine, serine, alanine, asparagine, or aspartate. The resulting mutant Mib-CK enzymes showed qualitatively similar changes in their enzymatic properties. In both directions of the CK reaction, a shift of the pH optimum to lower values was observed. Mutant Mib-CKs were severalfold more sensitive to inhibition by free ADP in the reverse reaction (ATP synthesis) and to free ATP in the forward reaction (phosphocreatine synthesis). With the exception of C278D, all mutant enzymes were specifically activated by chloride and bromide anions. C278D and wild-type Mib-CK were significantly inhibited under the same conditions. At low chloride concentrations, the Vmax of C278D was about 12-fold higher than that of C278N. Thus, Cys278 probably provides a negative charge which is directly or indirectly involved in maximizing CK activity. Under near-optimal conditions in the reverse reaction, mutants C278G and C278S showed about an 11-fold increase in Km(PCr), but only 1.7- and 2.8-fold reductions in Vmax, respectively, compared to wild-type Mib-CK. Thus, the reactive cysteine clearly is not essential for catalysis. For rabbit muscle CK, substrate binding had been shown to be synergistic (i.e., Kd > Km). We confirmed this finding for wild-type Mib-CK by determining the Kd and Km values for both substrates in the forward reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli.

Sarcomeric mitochondrial creatine kinase (Mib-CK) of chicken was expressed in Escherichia coli as a soluble enzyme by using an inducible phage-T7 promoter. Up to one third of the protein in E. coli extracts consisted of soluble recombinant Mib-CK in an enzymically active form. Approx. 20 mg of nearly-homogenous Mib-CK was isolated in a two-step isolation procedure starting with 1 litre of isopropyl beta-D-thiogalactopyranoside-induced E. coli culture, whereas previous attempts to express other CK genes in E. coli have resulted in 20-fold lower yields and inclusion-body formation. Selection of the Mib-CK expression plasmid on media containing kanamycin rather than ampicillin extended the time period of maximal Mib-CK expression. Recombinant Mib-CK displayed an identical N-terminal amino acid sequence, identical Km for phosphocreatine and Vmax. values, the same electrophoretic behaviour and the same immunological cross-reactivity as the native enzyme isolated from chicken heart mitochondria. The recombinant Mib-CK had the same molecular mass as native chicken Mib-CK in m.s. analysis, indicating that post-translational modification of the enzyme in chicken tissue does not occur. As judged by gel-permeation chromatography and electron microscopy, recombinant enzyme formed predominantly octameric oligomers with the same overall structure as the chicken heart enzyme. Furthermore, the enzymes isolated from both sources formed protein crystals of space group P42(1)2, when grown in the absence of ATP, with one Mi-CK octamer per asymmetric unit. The indistinguishable X-ray-diffraction patterns indicate identical structures for the native and recombinant proteins.

Endonucleolytic cleavage of a long 3'-trailer sequence in a nuclear yeast suppressor tRNA.

Transcripts of Saccharomyces cerevisiae nuclear tRNA genes are normally terminated within a few nucleotides of the tRNA coding region, in contrast to mitochondrially encoded tRNAs, which are contained within polycistronic transcripts and thus require 3'-processing by mitochondrial endonucleases. We show that 3'-processing activities capable of removing artificially extended 3'-trailer sequences from some tRNA substrates are also present in the yeast nucleus. Correct 3'-processing in vivo resulted in the formation of functional suppressor tRNA. The 3'-processing activities were also identified in vitro through analysis of transcription-processing products in cell-free yeast S-100 extracts. Comparison of several pre-tRNA substrates showed that the tRNA structure played a major role in determining the processability of a substrate but that the nature of the 3'-trailer sequence also modulated the rate of 3'-processing. Pre-tRNA containing mitochondrial tRNA(Val) sequence was a good substrate for in vitro processing, independent of its 3'-trailer. A 200-nt-long pre-tRNA, encoding the nuclear SUP4 tRNA gene and a mitochondrial 3'-trailer, was processed in yeast S-100 extract in a multistep pathway into mature-sized tRNA(Tyr). Part of the 3'-processing was due to an endonuclease which cleaved near or precisely at the 3'-end of the coding region of the tRNA. A short sequence around this endonucleolytic 3'-cleavage site was crucial for the formation of active suppressor tRNA in vivo. A 9-nt-long sequence motif derived from the mitochondrial 3'-trailer allowed processing, while sequences derived from lacZ or pBR322 DNA were processed neither in vitro nor in vivo.

A complex chitinolytic system in exponentially growing mycelium of Mucor rouxii: properties and function.

Enzymological evidence has been sought for the purported involvement of chitinolysis in vegetative growth of filamentous fungi. A procedure has been developed for the production of fast growing and morphologically homogeneous exponential phase mycelium of the non-septate dimorphic zygomycete Mucor rouxii. A partially purified extract of this material has been subjected to gel-permeation chromatography and the chitinolytic activity of eluate fractions has been assessed using colloidal and nascent chitin and 3,4-dinitrophenyl tetra-N-acetylchitotetraoside [3,4-DNP-(GlcNAc)4] as substrates. Exponentially growing (td = 1.1 h) mycelium consisting of single short-branched hyphae contains at least seven chitinases. The two particulate ones have not been studied in detail. The soluble chitinases hydrolyse (pseudo)chito-oligomers by random cleavage of internal beta-1,4-bonds (and not by processing) and have a minimum chain-length requirement of n = 4. They are clearly distinct from beta-N-acetylglucosaminidase (beta-GlcNAc'ase) with respect to their chromatographic behaviour, substrate chain-length specificity, inhibition by chitobionolactone oxime (Ki = 175 microM), and non-inhibition by the specific beta-GlcNAc'ase inhibitor N-acetylglucosaminono-1,5-lactone oxime. Their pH optima are similar (6.5-7.0), and all can hydrolyse 3,4-DNP-(GlcNAc)4 as well as nascent chitin. With respect to their charge, response to protease treatment, behaviour upon gel-permeation chromatography and ability to use colloidal chitin as a substrate, the soluble chitinases do, however, represent two distinct groups. Type A chitinases are acidic, display partial latency, show an unusual affinity to dextran gel and act weakly on colloidal chitin. Type B chitinases are basic (or neutral) and non-zymogenic, do not behave anomalously upon gel filtration and can degrade performed chitin. An hypothesis is presented for the function of the complex chitinolytic system of the fungal hypha in branching and, possibly, also in apical growth.

Substances in nuclear wheat germ extracts which interfere with polymerase III transcriptional activity in vitro.

Wheat germ nuclear extracts inhibited an active yeast polymerase III (pol III) transcription extract. We isolated two chromatin-associated fractions which harbored biochemically distinguishable inhibitory activities, each contributing about 40-50% to the total inhibitory activity. One fraction, which was released from the chromatin upon treatment with 350 to 900 mM NaCl, was purified to homogeneity and identified as histone H1. It inhibited the yeast extract by excluding the transcription machinery from the template DNA. It can be partially antagonized by additional nontemplate DNA together with templates that have strong pol III promoters. The other fraction, which was released from the chromatin between 0 and 350 mM NaCl, inhibited transcription by affecting transcription complex formation partially through transcription factor-inhibitor interactions. Furthermore, it affected the rate of transcription reinitiation but not the elongation rate. Ways to move towards an active DNA-dependent pol III plant extract are discussed.

SHI, a new yeast gene affecting the spacing between TATA and transcription initiation sites.

In a genetic selection for Saccharomyces cerevisiae genes involved in transcription start site specification, two mutant genes which restore alcohol dehydrogenase activity to a functionally defective S. pombe ADH gene were recovered. Examination of S. pombe ADH initiation sites showed that mutations in the SHI gene shift the location of the transcription initiation window closer to TATA. The shi mutant also affected initiation site selection for two S. cerevisiae genes that were tested. For H2B mRNA, initiation occurred in the shi mutant at a series of initiation sites located 43 to 80 bp 3' of the histone H2B TATA sequence and at the usual initiation sites 102 and 103 bp downstream of the TATA sequence. Weakly used initiation sites ranging from 51 to 80 bp downstream of the TATA sequence were observed for the S. cerevisiae ADH1 gene in shi strains, in addition to the normal ADH1 initiation sites 89 and 99 bp from the TATA sequence. Restoration of function to the defective S. pombe ADH gene occurs only when this gene contains a TATA sequence; a single-base-pair TATA-to-TAGA change is sufficient to prevent this restoration of function. Genetic mapping placed the SHI locus on the left arm of chromosome VII, 22.3 centimorgans from cyh2; it does not correspond to any previously mapped gene.

Specific transcription and reinitiation of class III genes in wheat embryo nuclei and chromatin.

Chromatin isolated from wheat germ embryos has a transcription efficiency for RNA polymerase III (pol III) closely approaching that for isolated wheat germ nuclei. Transcription in nuclei and chromatin is inhibited 5-10-fold by the addition of heparin, suggesting that free pol III molecules bind to chromatin and initiate transcription during thein vitro incubation. Nuclei were shown to have similar transcriptional activity in potassium chloride and potassium acetate. Nuclei and chromatin exhibited different salt optima for transcription. Neither nuclei nor chromatin were strongly stimulated by exogenous protein fractions. The data presented here suggest that in wheat germ nuclei the complete transcriptional apparatus is stably bound to the chromatin. Wheat germ nuclei may serve therefore as an enriched source for a solublein vitro transcription system.

Regulation of the TRP4 gene of Saccharomyces cerevisiae at the transcriptional level and functional analysis of its promotor.

The TRP4 gene of Saccharomyces cerevisiae, which encodes anthranilate phosphoribosyl transferase (E.C. 2.4.2.18), is subject to the general control of amino acid biosynthesis. The regulation takes place at the transcriptional level by increasing the amount of initiation and not by changing the stability of mRNA. We have observed a change in the utilization of TRP4 mRNA start sites, depending on whether cells were grown under repressing or derepressing conditions. The function of promoter elements has been tested by deletion analysis with a plasmid-encoded TRP4 gene. A routinely practicable method was used for copy-number calibration of plasmids based on 2 micron DNA. Promoter structures and spacing problems in the TRP4 promoter region are discussed.

Cloning of the ARO3 gene of Saccharomyces cerevisiae and its regulation.

Regulation of the two isozymes of 3-deoxy-D-arabino-heptulosonate-7phosphate synthase (DAHP synthase; EC 4.1.2.15) encoded by the genes ARO3 and ARO4 of Saccharomyces cerevisiae was studied. Both genes were shown to respond equally well to the general control of amino acid biosynthesis. Strains with mutations in these two genes were obtained by selecting first for a single aro3 mutation and afterwards for a double aro3 aro4 mutation. Gene ARO3, coding for the phenylalanine-dependent isozyme of DAHP synthase was cloned on the 2 micron multicopy vector pJDB207 by complementation of mutation aro3-1 in yeast. The ARO3 gene, carried originally on a 9.6 kb BamHI fragment (plasmid pME541A), was subcloned on a 1.9 kb HindIII-XbaI fragment (plasmid pME543). A transcript of about 1.5 kb was shown to proceed from the HindIII towards the XbaI site. Expression from the 9.6 kb as well as from the 1.9 kb fragment was normal on a multicopy vector, since in both cases DAHP synthase levels of about 50-fold the wild-type level were observed.

The TRP4 gene of Saccharomyces cerevisiae: isolation and structural analysis.

The TRP4 gene of Saccharomyces cerevisiae, encoding the anthranilate phosphoribosyl transferase, was isolated and subcloned by functional complementation in yeast. A 2 kb fragment containing information for a polypeptide of 380 amino acids and the 5'- and 3'-flanking regions was sequenced. The TRP4 transcript was identified and mapped with S1 nuclease. Homologies to two prokaryotic genes encoding the same function, and sequences potentially involved in transcription start and termination and in regulation of TRP4 gene expression are discussed.