Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.

Residues 32 to 40, which are conserved among ras proteins from different species, are likely to participate in interactions with the p21 effector system. With the goal of understanding the structural basis of the regulatory functions of c-Ha-ras p21, we produced rabbit antisera against a synthetic peptide corresponding to amino acids 33 to 42 of the protein. The affinity-purified antibodies interacted specifically with p21 and with the antigenic peptide. The epitope recognized by the antibodies appeared to be centered on threonine 35. The antibodies inhibited both in vitro p21-induced production of cyclic AMP in detergent extracts of RAS-defective yeast membranes and GAP-stimulated GTPase activity. However, monoclonal anti-ras antibodies Y13-259 and Y13-238 were not capable of specifically inhibiting interactions of p21 with these two putative effector proteins. The apparent inhibitory effect of Y13-259 on stimulation of p21 by GAP was due to a greatly reduced rate of exchange of nucleotides in the binding pocket of the protein. These findings provide additional support for the essential role of the residue 32 to 40 domain as the true effector site and further evidence of the involvement of GAP as a cellular effector of ras proteins.

Control of phenylalanyl-tRNA synthetase genetic expression. Site-directed mutagenesis of the pheS, T operon regulatory region in vitro.

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.

Attenuation control of the Escherichia coli phenylalanyl-tRNA synthetase operon.

The pheST operon codes for the two subunits of the essential enzyme phenylalanyl-tRNA synthetase. The nucleotide sequence of the regulatory regions of the operon, in vitro transcription data and in vivo experiments indicate that the operon is controlled by attenuation in a way similar to many amino acid biosynthetic operons. In this work the control of the pheST operon was studied in vivo by measuring the effect of deletions in the regulatory regions on downstream expression. The presence of a strong promoter followed by an approximately 90% efficient terminator in front of the structural parts of the operon is demonstrated. An open reading frame coding for a 14 amino acid long leader peptide containing five phenylalanine residues is located between the promoter and the terminator. The presence of the transcription terminator is shown to be essential to the operon's regulation. The localization of the promoter and the terminator agrees with the results of previous in vitro experiments. It is also shown that about 30% of the transcripts covering the pheST operon come from the upstream gene, rplT, which codes for the ribosomal protein L20. Although cotranscription exists between rplT and pheST, these genes are not systematically coregulated since reducing the translation of rplT about tenfold, does not change pheST expression. The pheST operon is also shown to be derepressed by a cellular excess of phenylalanyl-tRNA synthetase. This derepression is shown to be due to the pheST attenuator.

Escherichia coli phenylalanyl-tRNA synthetase operon region. Evidence for an attenuation mechanism. Identification of the gene for the ribosomal protein L20.

The nucleotide sequences of pheS and of the beginning of pheT have been determined. The genes pheS and pheT code, respectively, for the small and large subunits of phenylalanyl-tRNA synthetase, an alpha 2 beta 2 enzyme. Upstream from pheS the sequence shows another open reading frame of 354 nucleotides (rplT), which accounts for a protein of Mr 13,400. The product of this gene, previously named "P12", is identified as the ribosomal protein L20. The promoter for the pheS, T operon was located 368 nucleotides in front of pheS by transcription experiments in vitro. The promoter site is followed by a short open reading frame, which codes for a 14-residue peptide containing five phenylalanine residues. Immediately downstream from the stop codon of this open reading frame, the DNA sequence indicates that the transcript can be folded into three alternative secondary structures, one of which is a site of transcription termination. In vitro, 90% of transcription products initiated at the pheS, T promoter terminate at this site. However, long run-off transcripts proceeding through the terminator and covering the pheS structural gene are observed. No other transcription initiation could be detected between the terminator and the pheS structural gene. All these results are consistent with a mechanism by which phenylalanine-mediated attenuation controls the expression of phenylalanyl-tRNA synthetase. Further evidence is provided for this model by the features of pheS, T regulation in vivo (see the accompanying paper).

Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in vivo.

The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.

Escherichia coli phenylalanyl-tRNA synthetase operon is controlled by attenuation in vivo.

The two subunits of phenylalanyl-tRNA synthetase are made from two adjacent, cotranscribed genes that constitute the pheS,T operon. Three different fusions between pheS,T and lac genes were constructed in order to study the regulation of the pheS,T operon in vivo. We show, using these fusions, that phenylalanyl-tRNA synthetase transcription is derepressed when the level of aminoacylated tRNAPhe is lowered by mutational alteration of the synthetase. The pheS,T operon is also derepressed in strains carrying a trpX mutation. The gene trpX codes for an enzyme that modifies both tRNATrp and tRNAPhe and a mutation in that gene causes derepression of the trp and pheA operons, both of which are controlled by attenuation. The in vivo features of the regulation of pheS,T expression described here in correlation with the DNA sequence and in vitro transcription results described in the accompanying paper by Fayat et al. indicate that phenylalanyl-tRNA synthetase is controlled by attenuation in a way analogous to several amino acid biosynthetic operons.

IS4 transposition in the attenuator region of the Escherichia coli pheS,T operon.

A cis-acting mutation which lowers phenylalanyl-tRNA synthetase operon (pheS,T) transcription about tenfold was previously isolated on a multicopy plasmid [Plumbridge and Springer, J. Bacteriol. 152 (1982) 650-668]. This mutation has now been characterized as an IS4 element inserted in orientation II in the terminator stem of the pheS,T attenuator. The identification of the insertion as IS4 is based on (i) the nature and location of restriction sites internal to the insertion element, and (ii) the DNA sequence of both the left and right Escherichia coli::IS4 junctions. The effect of the IS4 transposition on the expression of pheS,T was studied using pheS,T::lac fusions cloned in lambda phages. IS4 integration into the leader region of the pheS,T operon was shown to abolish the miaA (trpX) allele dependence which characterizes the attenuation mechanism regulating pheS,T expression [Fayat et al., J. Mol. Biol. 171 (1983) 239-261; Springer et al., J. Mol. Biol. 171 (1983) 263-279]. The IS4 insertion site described here is compared to the other known sites and the effect of IS4 transposition on the expression of neighbouring genes is discussed.

Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa.

A Brevibacterium sp. R312 DNA fragment encoding the wide-spectrum amidase (EC 3.5.1.4) has been cloned and sequenced, using limited amino acid (aa) sequence information obtained from the purified enzyme. The deduced aa sequence showed more than 80% strict identity with the Pseudomonas aeruginosa aliphatic amidase, the product of the amiE gene, suggesting a horizontal transfer of the gene during evolution between Gram+ and Gram- bacteria.

Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form.

A synthetic gene coding for human angiogenin was synthesized by solid support phosphoramidite chemistry as eight long oligodeoxynucleotides which were subsequently assembled and cloned in Escherichia coli. The gene was designed to use codons found in highly expressed E. coli proteins. A pBR322-derived expression vector was constructed containing the E. coli trp promoter, the ribosome-binding site of the bacteriophage lambda cII gene, the angiogenin coding sequence, and the transcription terminator region of the E. coli rrnB operon. Under tryptophan deprivation, angiogenin was strongly expressed in E. coli cells at a yield of 5-10% of total protein. The eukaryotic protein was found to be insoluble but could be easily renatured and purified. The purified angiogenin was demonstrated to be active as an angiogenic factor and exhibited a characteristic RNase activity.

Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1 beta.

Expression plasmids carrying the coding sequence of mature human interleukin 1 beta (IL 1 beta) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1 beta from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1 beta fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1 beta maturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1 beta fusion. Immuno-electron microscopy revealed that the sOmpA-IL1 beta fusion was targeted to the inner membrane, whereas the sPhoA-IL1 beta fusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1 beta. Moreover, these E. coli secretion vectors allowed us to produce, in high levels, IL1 beta fragments which otherwise could not be stably accumulated within the cytoplasmic compartment.

Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E. coli.

The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons. The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide. One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator. Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance to pheS, the first structural gene of the operon. The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself. The alternative leader peptide and the terminator peptide are both translated at a negligible rate.

Betulinic acid derivatives: a new class of specific inhibitors of human immunodeficiency virus type 1 entry.

A novel series of omega-aminoalkanoic acid derivatives of betulinic acid were synthesized and evaluated for their activity against human immunodeficiency virus (HIV). The anti-HIV-1 activity of several members of this new series was found to be in the nanomolar range in CEM 4 and MT-4 cell cultures. The optimization of the omega-aminoalkanoic acid side chain is described. The presence of an amide function within the side chain was found important for optimal activity. RPR 103611 (14g), a statine derivative, was found to be inactive against HIV-1 protease, reverse transcriptase, and integrase as well as on gp120/CD4 binding. "Time of addition" experiments suggested interaction with an early step of HIV-1 replication. As syncytium formation, but not virus-cell binding, seems to be affected, betulinic acid derivatives are assumed to interact with the postbinding virus-cell fusion process.

Betulinic acid derivatives: a new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action.

A series of omega-undecanoic amides of lup-20(29)-en-28-oic acid derivatives were synthesized and evaluated for activity in CEM 4 and MT-4 cell cultures against human immunodeficiency virus type 1 (HIV-1) strain IIIB/LAI. The potent HIV inhibitors which emerged, compounds 5a, 16a, and 17b, were all derivatives of betulinic acid (3beta-hydroxylup-20(29)-en-28-oic acid). No activity was found against HIV-2 strain ROD. Compound 5a showed no inhibition of HIV-1 reverse transcriptase activity with poly(C).oligo(dG) as template/primer, nor did it inhibit HIV-1 protease. Additional mechanistic studies revealed that this class of compounds interfere with HIV-1 entry in the cells at a postbinding step.

Tryptophan promoter derivatives on multicopy plasmids: a comparative analysis of expression potentials in Escherichia coli.

A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the -35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at -40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.

High-cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1 beta.

A high-productivity process has been developed for the production of mature human interleukin-1 beta (IL-1 beta) from recombinant Escherichia coli strains. Conditions were found that allow high IL-1 beta expression levels in high cell density cultures. Improved fed-batch fermentation strategies are described which include maintenance of glucose and acetate concentrations below 1 g/l and sparging the fermentor with an O2-enriched air supply. Using the E. coli tryptophan promoter control of transcription, a 2.2 g/l production level of IL-1 beta was achieved in E. coli B at cell densities of 55 g dry weight per litre. Another genetic construction involving the bacteriophage lambda cIts-PR expression cassette allowed a similar IL-1 beta production level (1.9 g/l) in E. coli E103S, albeit at a lower cell density (30 g/l). A simplified procedure allowing the purification of fully active IL-1 beta is also presented.

Zinc(II)-dependent synthesis of diadenosine 5', 5"' -P(1) ,P(4) -tetraphosphate by Escherichia coli and yeast phenylalanyl transfer ribonucleic acid synthetases.

A new activity of Escherichia coli and yeast phenylalanyl-tRNA synthetases, the conversion adenosine 5' -triphosphate into diadenosine 5' ,5"' -P(1) ,P(4) -tetraphosphate, is reported. This activity is followed by (31)P NMR and chromatography on poly(ethylenimine)-cellulose. It is revealed by the addition of ZnCl2 to a reaction mixture containing the enzyme, ATP-Mg(2+), L-phenylalanine, and pyrophosphatase It reflects the reaction enzyme-bound phenylalanyl adenylate with ATP instead of PPi and strongly depends on the hydrolysis of pyrophosphate in the assay medium. The zinc dependence of this reaction parallels that of the inhibition of tRNA(phe) aminoacylation which is described in the accompanying paper [Mayaux, J. F., & Blanquet, S. (1981) Biochemistry (preceding paper in this issue)]. In the presence of an unlimiting pyrophosphatase activity, diadenosine tetraphosphate synthesis by E. coli and yeast phenylalanyl-tRNA synthetases occurs at maximal rates of 0.5 and 2 s-1, respectively (37 degrees C, pH 7.8, 150 mM KC1, 5 mM ATP, 10 mM MgCl2, 2 mM L-phenylalanine, and 80 muM ZnCl2). Under identical experimental conditions, E coli isoleucyl-, methionyl-, and tyrosyl-tRNA synthetases produce small amounts of diadenosine tetraphosphate at rates 2 or 3 orders of magnitude lower than that achieved by phenylalanyl-tRNA synthetase. In the case of E. coli phenylalanyl-tRNA synthetase, it is shown that the diadenosine tetraphosphate synthetase activity is accompanied by a diadenosinetetraphosphatase activity. This activity, actually supported by phenylalanyl-tRNA synthetase, is responsible for the appearance of ADP in the assay medium. It requires also the presence of both ZnCl2 and L-phenylalanine. The formation of ADP from diadenosine tetraphosphate and its reaction with enzyme-bound aminoacyl adenylate account for the appearance in the reaction mixture of diadenosine 5' ,5"' -P(1) ,P(3)-triphosphate, after that of diadenosine tetraphosphate. The significance of these findings in the context of the role of diadenosine tetraphosphate in controlling cellular growth is discussed.

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase.

An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 mumol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30 degrees C under the control of an E. coli promoter and ribosome-binding site.

Molecular cloning of a functional human galanin receptor.

The ubiquitous neuropeptide galanin controls numerous functions such as endocrine secretions, intestinal motility, and behavioral activities. These regulatory effects of galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive guanine nucleotide binding proteins Gi/Go as transducing elements. We report here the isolation of a cDNA coding for a human galanin receptor from a Bowes melanoma cell line cDNA expression library, by using a radioligand binding strategy. The nucleotide sequence of the cloned receptor reveals an open reading frame encoding a 349-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the guanine nucleotide binding protein-coupled neuropeptide receptor family. The cloned receptor expressed in COS cells specifically binds human, porcine, and rat galanin with high affinity (Kd in the nanomolar range) and mediates the galanin inhibition of adenylate cyclase. A 2.8-kb galanin receptor transcript was identified in several human tissues. Cloning of this galanin receptor should enhance our knowledge of its distribution, structure, and function in human physiology and pathophysiology.

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase.

A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrile hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSR1-derived shuttle vector.