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Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4-8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-(gamma-maleimidobutryloxy) succinimide ester to a TFO (5'-NH2-[CH2](6 or 12)-MPMPMPMPMPPPPPPT-3', with M being 5-methyl-2'-deoxycytidine and P being 5-[1-propynyl]-2'-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO ('addressed' site: 5'-TTTTTTTCTCTCTCTCN(approximately 10)CAGCTG-3'), leaving 'unaddressed' PvuII sites intact. The preference for cleavage of an 'addressed' compared to an 'unaddressed' site is1000-fold, if the cleavage reaction is initiated by addition of Mg2+ ions after preincubation of scPvuII-TFO and substrate in the absence of Mg2+ ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO.

The specificity of DNA-mediated protein assembly was studied in two in vitro systems, based on (i) the DNA-binding domain of bacteriophage 434 repressor cI (amino acid residues 1–69), or (ii) the DNA-binding domain of the yeast transcription factor GCN4, (amino acids 1–34) and their respective oligonucleotide cognates. In vivo, both of these peptides are part of larger protein molecules that also contain dimerization domains, and the resulting dimers recognize cognate palindromic DNA sequences that contain two half-sites of 4 bp each. The dimerization domains were not included in the peptides tested, so in solution—in the presence or absence of non-cognate DNA oligonucleotides—these molecules did not show appreciable dimerization, as determined by pyrene excimer fluorescence spectroscopy and oxidative cross-linking monitored by mass spectrometry. Oligonucleotides with only one 4 bp cognate half-site were able to initiate measurable dimerization, and two half-sites were able to select specific dimers even from a heterogeneous pool of molecules of closely related specificity (such as DNA-binding domains of the 434 repressor and their engineered mutants that mimic the binding helix of the related P22 phage repressor). The fluorescent technique allowed us to separately monitor the unspecific, ionic interaction of the peptides with DNA which produced a roughly similar signal in the case of both cognate and non-cognate oligonucleotides. But in the former case, a concomitant excimer fluorescence signal showed the formation of correctly positioned dimers. The results suggest that DNA acts as a highly specific template for the recruitment of weakly interacting protein molecules that can thus build up highly specific complexes.

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3'-hydroxyl and 5'-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.

Single-chain repressor RR(Tres) is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain R(TRES) by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of R(TRES) at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic,in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RR(Tres) The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1 x 10(-12) mol/L-1 x 10(11) mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1 x 10(-9) mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5' position. We constructed a new homodimeric single-chain repressor R(TRES)R(TRES) and its DNA-binding specificity was tested by using a series of new operators designed according to the recognition properties previously determined for the R(TREs) domain. These operators containing the consensus sequenceGTAAGAAARNTTACN orGGAAGAAARNTTCCN (R is A or G) were recognized by R(TRES)R(TRES) specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA interactions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

The isolated N-terminal 1-69 domain of the 434-phage repressor, R69, and its covalently linked (head-to-tail and tail-to-tail) dimers have been studied by differential scanning microcalorimetry (DSC) and CD. At neutral solvent conditions the R69 domain maintains its native structure, both in isolated form and within the dimers. The stability of the domain depends highly upon pH within the acidic range, thus at pH 2 and low ionic strength R69 is already partially unfolded at room temperature. The thermodynamic parameters of unfolding calculated from the DSC data are typical for small globular proteins. At neutral pH and moderate ionic strength, the domains of the dimers behave as two independent units with unfolding parameters similar to those of the isolated domain, which means that linking two R69 domains, either by a long peptide linker or by a designed C-terminal disulfide bridge, does not induce any cooperation between them.

Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA ‐ 6 base-pairs ‐ NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator. Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10‐50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target.

Flow cytometric and electron microscopic immunocytochemical studies have been performed in HT-29 human colon tumour cells in vitro, to determine and localise p86 Ku protein, which is a regulatory subunit of DNA-dependent kinase and a specific binding site for somatostatin. We have demonstrated that HT-29 cells contain p86 Ku and that the distribution between the cytoplasm and the nucleus is even. After administration of the somatostatin analogues Sandostatin and TT-232 to HT-29 cells, the p86 Ku content of the cytoplasmic compartment decreased in the first 4 h. An increase in the content of this protein in the nuclear compartment was observed at hour 1 followed by a decrease at hour 4 after treatment. Quantitative differences between the two analogues have been observed in this respect. The practical significance of these findings is discussed.

The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.

Single-chain (sc) DNA-binding proteins containing covalently dimerized N-terminal domains of the bacteriophage 434 repressor cI have been constructed. The DNA-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural linker sequence that connects the N and C-terminal domains of the intact repressor. Compared to the isolated N-terminal DNA-binding domain, the sc molecule showed at least 100-fold higher binding affinity in vitro and a slightly stronger repression in vivo. The recognition of the symmetric OR1 operator sequence by this sc homodimer was indistinguishable from that of the naturally dimerized repressor in terms of binding affinity, DNase I protection pattern and in vivo repressor function. Using the new, sc framework, mutant proteins with altered DNA-binding specificity have also been constructed. Substitution of the DNA-contacting amino acid residues of the recognition helix in one of the domains with the corresponding residues of the Salmonella phage P22 repressor c2 resulted in a sc heterodimer of altered specificity. This new heterodimeric molecule recognized an asymmetric, artificial 434-P22 chimeric operator with high affinity. Similar substitutions in both 434 domains have led to a new sc homodimer which showed high affinity binding to a natural, symmetric P22 operator. These findings, supported by both in vitro and in vivo experiments, show that the sc architecture allows for the introduction of independent changes in the binding domains and suggest that this new protein framework could be used to generate new specificities in protein-DNA interaction. # 1997 Academic Press Limited

Circular dichroism and electrophoretic mobility shift studies were performed to confirm that dimerized N-terminal domains of bacterial repressors containing helix-turn-helix motifs are capable of high-affinity and specific DNA recognition as opposed to the monomeric N-terminal domains. Specific, high-affinity DNA binding proteins were designed and produced in which two copies of the N-terminal 1-62 domain of the bacteriophage 434 repressor are connected either in a dyad-symmetric fashion, with a synthetic linker attached to the C-termini, or as direct sequence repeats. Both molecules bound to their presumptive cognate nearly as tightly as does the natural (full-length and non-covalently dimerized) 434 repressor, showing that covalent dimerization can be used to greatly enhance the binding activity of individual protein segments. Circular dichroism spectroscopy showed a pronounced increase in the alpha-helix content when these new proteins interacted with their cognate DNA and a similar, although 30% lower, increase was also seen upon their interaction with non-cognate DNA. These results imply that a gradual conformational change may occur when helix-turn-helix motifs bind to DNA, and that a scanning mechanism is just as plausible for this motif class as that which is proposed for the more flexible basic-leucine zipper and basic-helix-loop-helix motifs.

Semisynthetic human Fab libraries were constructed, displayed on the surface of filamentous phage and selected for binding to three hapten conjugates. A number of Fabs were isolated and characterized with respect to affinity and specificity. Fabs exhibited affinities of between 80 and 29 nM, as determined by surface plasmon resonance, for the conjugate on which they were selected. Conservation of Asp101 in the third heavy-chain complementarity determining region (HCDR3) appears to be important in the construction of synthetically diverse repertoires.

The specific ligand binding ability of recombinant human serum albumin produced in yeast using the synthetic gene was studied by affinity Chromatographic method. It was found that synthetic protein possesses those stereoselective binding and binding interactions for several chiral benzodiazepine and coumarin compounds which are characteristic of the natural human serum albumin, suggesting identical tertiary structures.

Interleukin-1 (IL-1) mediates numerous responses on the mouse anterior pituitary cell line AtT-20. We have studied the ligand binding properties of the IL-1 receptors (IL-1R) of the AtT-20 cells and found that they possess 1.5 × 10 3 receptors/cell with a K d of 0.15 nM for [ 125 I]IL-1α. Using oligonucleotide primers, which define a 236 bp region of the cDNA coding for the cytosolic part of the mouse T-cell IL-1R, and cDNA prepared from AtT-20 cells, we obtained by polymerase chain reaction (PCR) a DNA fragment which was shown to posses an identical sequence to that of the corresponding region of the mouse T-cell IL-1R. Thus the AtT-20 pituitary cell line IL-1 receptor appears to be similar to those found on mouse T-cells and fibroblasts.

The microwave spectrum of 4-fluoro-1-butene has been reinvestigated in the 10-26.5 GHz spectral range. The ground and several vibrationally excited states have been assigned for two «new» rotamers denoted Skew-Gauche I and Skew-Gauche II. The dipole moments of the «new» conformers have been determined

α-Values evaluated from solvent deuterium isotope effects and excess acidity correlations have been observed to be very similar in the acid-catalyzed hydrations of both norbornenes (0.77±0.11 and 0.77±0.05, respectively) and nortricyclanes (0.78±0.15 and 0.80±0.03, respectively). By using these, together with reaction constants, the α-values were also estimated for the solvolyses of 2-norbornyl tosylates. They are reasonable (0.88±0.10) if the first-formed carbocations are of similar character in the solvolyses and hydrations, but unreasonable if their characters are different

CASPER, a computer program by which structural analysis of polysaccharides can be performed, has been tested for accuracy. This was done by simulation of the 1 H and 13 C NMR spectra of the Shigella flexneri type Y O-polysaccharide using data from the constituent disaccharides and then comparing these with experimental data. A close fit between experimental and calculated 1 H and 13 C NMR spectra was observed. Conformational and NMR studies on the disaccharides, as methyl glycosides, were also performed. Furthermore, an extraction procedure, aimed at improving the database in CASPER is demonstrated

A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described. Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps. 24 synthetic, 69-85 nucleotides long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks. Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene as large fragments. Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids. The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichia coli-Saccharomyces cerevisiae shuttle vector. This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminal amino acid sequence.

We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases.

Single-chain derivatives of the 434 repressor containing one wild-type and one mutant DNA-binding domain recognize the general operator ACAA-6 base pairs-NNNN, where the ACAA operator subsite is contacted by the wild-type and the NNNN tetramer by the mutant domain. The DNA-binding specificities of several single-chain mutants were studied in detail and the optimal subsites of the mutant domains were determined. The characterized mutant domains were used as building units to obtain homo- and heterodimeric single-chain derivatives. The DNA-binding properties of these domain-shuffled derivatives were tested with a series of designed operators of NNNN-6 base pairs-NNNN type. It was found that the binding specificities of the mutant domains were generally maintained in the new environments and the binding affinities for the optimal DNA ligands were high (with K(d) values in the range of 10(-11)-10(-10) M). Considering that only certain sequence motifs in place of the six base pair spacer can support optimal contacts between the mutant domains and their subsites, the single-chain 434 repressor mutants are highly specific for a limited subset of 14 base pair long DNA targets.

The Pvu II restriction endonuclease has been converted from its natural homodimeric form into a single polypeptide chain by tandemly linking the two subunits through a short peptide linker. The arrangement of the single-chain Pvu II (sc Pvu II) is (2-157)-GlySerGlyGly-(2-157), where (2-157) represents the amino acid residues of the enzyme subunit and GlySerGlyGly is the peptide linker. By introducing the corresponding tandem gene into Escherichia coli , Pvu II endonuclease activity could be detected in functional in vivo assays. The sc enzyme was expressed at high level as a soluble protein. The purified enzyme was shown to have the molecular mass expected for the designed sc protein. Based on the DNA cleavage patterns obtained with different substrates, the cleavage specificity of the sc Pvu II is indistinguishable from that of the wild-type (wt) enzyme. The sc enzyme binds specifically to the cognate DNA site under non-catalytic conditions, in the presence of Ca 2+ , with the expected 1:1 stoichiometry. Under standard catalytic conditions, the sc enzyme cleaves simultaneously the two DNA strands in a concerted manner. Steady-state kinetic parameters of DNA cleavage by the sc and wt Pvu II showed that the sc enzyme is a potent, but somewhat less efficient catalyst; the k cat / K M values are 1.11 × 10 9 and 3.50 × 10 9 min −1 M −1 for the sc and wt enzyme, respectively. The activity decrease is due to the lower turnover number and to the lower substrate affinity. The sc arrangement provides a facile route to obtain asymmetrically modified heterodimeric enzymes.

Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA--6 base-pairs--NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator. Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10-50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target.

The mechanism of oxidative decarboxylation of 2-hydroxy-2-phenylethanoic acid (mandelic acid) by aqueous alkaline sodium hypochlorite (commercial bleach) has been studied. Kinetic studies for a series of 4-substituted mandelic acids (CH 3 O, CH 3 , H, F, Cl, CF 3 , NO 2 substituents) gave a Hammett o value of −0.23. When deuterium was substituted for hydrogen on the α-carbon atom of mandelic acid, a secondary kinetic isotope effect, (k H /k D ), of 1.07 was obtained. On the basis of these results a model for the transition state is proposed

A novel single-chain (sc) protein framework containing covalently dimerized DNA-binding domains (DBD) of the phage 434 repressor was used to construct combinatorial mutant libraries in order to isolate mutant DBDs with altered specificities. The library members contain one wild-type DBD and one mutant domain with randomized amino acids in the DNA-contacting region. Based on previous studies, the mutant sc derivatives are expected to recognize a general ACAA-6 bp-NNNN sequence, where ACAA is contacted by the wild-type and NNNN by the mutant domain. In principle, any sequence can stand for NNNN and serve as a selection target. Here an in vivo library screening method was used to isolate mutant sc repressors that interact with an asymmetric operator containing the TTAA target. Several mutants showed high affinity in vitro binding to operators containing the target and strong (up to 80-fold) preference for the TTAA target over the wild-type TTGT. Specificity studies revealed that certain mutants bound with substantially higher affinities (K(d) approximately 10(-11)M) to operators containing the TTAC sequence, a close homolog of the TTAA target. Thus, we have fortuitously isolated mutant sc repressors that show up to a several hundred-fold preference for TTAC over TTGT.

Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA - 6 base-pairs - NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator. Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10 to 50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target

Single-chain derivatives of the phage 434 repressor, termed single-chain repressors, contain covalently dimerized DNA-binding domains (DBD) which are connected with a peptide linker in a head-to-tail arrangement. The prototype RR69 contains two wild-type DBDs, while RR*69 contains a wild-type and an engineered DBD. In this latter domain, the DNA- contacting amino acids of thealpha3 helix of the 434 repressor are replaced by the corresponding residues of the related P22 repressor. We have used binding site selection, targeted mutagenesis and binding affinity studies to define the optimum DNA recognition sequence for these single-chain proteins. It is shown that RR69 recognizes DNA sequences containing the consensus boxes of the 434 operators in a palindromic arrangement, and that RR*69 optimally binds to non-palindromic sequences containing a 434 operator box and a TTAA box of which the latter is present in most P22 operators. The spacing of these boxes, as in the 434 operators, is 6 bp. The DNA-binding of both single-chain repressors, similar to that of the 434 repressor, is influenced indirectly by the sequence of the non-contacted, spacer region. Thus, high affinity binding is dependent on both direct and indirect recognition. Nonetheless, the single-chain framework can accommodate certain substitutions to obtain altered DNA-binding specificity and RR*69 represents an example for the combination of altered direct and unchanged indirect readout mechanisms.

The interleukin-1 (IL-1) system possesses two distinct receptors (type I and type II) which, together with the accessory protein, mediate a multitude of responses to IL-1 alpha and IL-1 beta, including fever. So far, no receptor subtype-specific ligands have been described. Since both types of IL-1 receptors occur in the thermoregulatory areas it was unclear which IL-1 receptor type mediates fever. We report here that for a series of deletion mutants of human recombinant IL-1 beta (hrIL-1 beta), the affinity of these ligands for the type I IL-1 receptor correlates with their efficacy to evoke the fever response (hrIL-1 betades-SND52-54des-QGE48-50des-I56). Thus, the results suggest that agonist occupancy of the type I IL-1 receptor is essential for IL-1 beta-mediated fever.

The occurrence of the endogenous receptor antagonist for the cytokine interleukin-1 in the rat adrenal gland was analyzed y polymerase chain reaction and by immunohistochemistry using a rabbit polyclonal antiserum. Expression of interleukin-1 receptor antagonist mRNA was demonstrated in both adrenal medulla and cortex, and a marked increase in the transcription was observed after systemic administration of lipopolysaccharides. Interleukin-1 receptor antagonist immunoreactivity was seen in the adrenal medulla, and the immunofluorescence intensity was stronger in the adrenergic, phenylethanolamine N-methyltransferase-positive cells than in the noradrenergic chromaffin cells. The distribution of interleukin-1 receptor antagonist protein is complementary to that of interleukin-1 alpha-like immunoreactivity found in phenylethanolamine N-methyltransferase-negative cells and overlaps with and resembles the distribution of interleukin-1 beta-immunoreactive material. The expression of the interleukin-1 receptor antagonist in the adrenal gland complements previous findings of large constitutive pools of interleukin-1 alpha and interleukin-1 beta in this neuroendocrine organ and also suggests participation of adrenal interleukin-1 receptor antagonist in neuroimmune modulation.

The existence of an endogenous high affinity interleukin 1 receptor antagonist (IL-1ra) suggests that this molecule lacks some structural motif(s) which are present in the closely homologous agonist interleukin 1 beta (IL-1 beta) and which serve as the 'agonist switch' causing signal transduction by the agonist-receptor complex. The primary sequence alignment of IL-1 beta and IL-1ra sequences from different species reveals a six amino acid long motif that is quasi conserved among IL-1 beta sequences, but is missing from the IL-1ra sequences. The three-dimensional structure of human IL-1 beta was used as a template for building structural models of deletion mutants (delta SND 52-54 and delta EESNDK 50-55) using molecular graphics. These models indicated that the middle three residues SND 52-54 from the EESNDK 50-55 loop may be deleted without causing major changes in the tertiary structure of the mutant as compared to that of IL-1 beta. Residues SND 52-54 from the above loop were deleted. When compared with IL-1 beta the IL-1 beta-delta SND analog (delta SND 52-54) binds with the same affinity to type 2 IL-1 receptor but with a more than 10-fold lower affinity to type 1 IL-1 receptor. Despite of this small decrease in affinity at the type 1 receptor the delta SND 52-54 has a 1000-fold lower biological activity than IL-1 beta when tested in a thymocyte activating factor assay.(ABSTRACT TRUNCATED AT 250 WORDS)

The specific ligand binding ability of recombinant human serum albumin produced in yeast using the synthetic gene was studied by affinity chromatographic method. It was found that synthetic protein possesses those stereoselective binding and binding interactions for several chiral benzodiazepine and coumarin compounds which are characteristic of the natural human serum albumin, suggesting identical tertiary structures.

A general method to convert single-stranded, chemically synthesized oligonucleotides into cloned duplexes is described. Oligonucleotides supplied with 3'-terminal extensions that are complementary to 3'-protruding ends obtained by certain restriction enzymes can be cloned either directly or with the help of an adapter molecule into double-stranded vectors. Two methods have also been developed for consecutive cloning applications. According to these methods, the synthetic oligonucleotides (and their enzymatically prepared complementary strands) are joined, one after the other, inside a cloning vector, each joining requiring one cloning step. Synthetic genes are thus built up from oligonucleotides corresponding to only one strand of the DNA. The sequential assembly of the cloned duplex takes place in the 5' to 3' direction. Each oligonucleotide is supplied with a four-nucleotide-long 3'-terminal extension, but this sequence is eliminated when the joining takes place, leaving no limiting sequence between the oligonucleotides. The two consecutive cloning methods, the adapter and the polycloning site methods, are illustrated by the assembly of short artificial genes.

N-terminal amino acid sequence analysis of the enzymatic subunit S1 of pertussis toxin from Bordetella pertussis showed the structure of the first 25 residues. 2 different oligodeoxyribonucleotide probes were synthesized as mixed 32-mers corresponding to the DNA sequence deduced. Southern blot analysis of pertussis DNA digested with restriction endonuclease ClaI showed that both mixed probes hybridized with a DNA fragment of about 10 kb, thus identifying a B. pertussis gene corresponding to the protein structure determined.

Synthetic genes coding for elongated precursors of the vasoactive intestinal polypeptide (VIP) were synthesized and cloned in a highly efficient Escherichia coli expression vector. The synthetic genes code for VIP with its methionine (at position 17) replaced by leucine and elongated at the C-terminus by Gly (vasoactive intestinal polypeptide-Gly, i.e. VIPa) or by Gly-Lys-Arg (vasoactive intestinal polypeptide-Gly-Lys-Arg, i.e. VIPb). The synthetic genes fused to the N-terminal part of the E. coli beta-galactosidase gene were expressed to yield high amounts of fusion proteins reaching upon induction at least 60% of the total cellular protein. The fusion proteins of 314 and 316 amino acids carrying in their C-terminal portion either the 29 or 31 amino acids long VIP precursor polypeptide were shown to be immunoreactive with VIP antisera and were further purified and cleaved by CNBr. The resulting purified peptide precursors (VIPa and VIPb) were recognized by VIP receptors in rat liver plasma membranes and by antibodies to porcine VIP in a radioimmunoassay. Both precursors activated adenylate cyclase in rat liver membranes and stimulated pancreatic secretion in the cat. The affinity and potency of the cloned precursors is close to that of VIP purified from porcine intestine, suggesting that the elongated VIP precursors may act even without being converted into the C-terminal amide form of the peptide. The elongated VIP precursors expressed in E. coli may provide a cheap, large-scale source of experimental material for studies on VIP actions.

The chemical synthesis of ppGpp (guanosine 3′,5′-dipyrophosphate) is described. The synthesis started from guanosine 3′,5′-diphosphate. The acidlabile ethoxyethyl group was used to protect the 2′-OH functional group. The pyrophosphate linkages were built up simultaneously according to the anionexchange method and the protecting group was removed by dilute aqueous acetic acid. In an analogous manner the isomeric non-natural ppGpp (guanosine 2′,5′-dipyrophosphate) and uridine 2′(3′),5′-dipyrophosphate were synthesized as well.

The stability of phosphodiester-amide internucleotide bond is compared in the deoxyribo and ribo series. The destabilizing effect of the 2′-OH group in the ribo series is discussed.

A simple, two-step method is described for the synthesis of the 5'-pyro- and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. The readily accessible 2'(3')-5' ApA, ApG, GpA and GpG were converted in one step to the corresponding 5'-phosphoramidate derivatives which were then transformed to the 5'-pyro- and triphosphates. CD spectra of 3'-5' pn GpG (n = 0,1,2 or 3) derivatives, measured at pH 1, indicated stabilization of the (syn) G+p (anti)G conformation by the 5'-phosphate groups.

Irradiation of 5'-[32P]-phosphate labeled DNA fragments with ultraviolet light in the presence of primary amines followed by piperidine treatment resulted in base-specific cleavage of the DNA chain at T residues, accompanied by a less intensive G reaction. This simple, T greater than G cleavage offers an alternative method for determining T residues in chemical DNA sequencing.

A 47-base-pair (bp) conserved sequence in the 5′-flanking regions of three transcriptional units coding for nodulation functions ( nod ABC, nod EFG, and nod H) has been identified in Rhizobium meliloti strain 41. The conserved region contains subsequences of 7 bp, 5 bp, and 25 bp. The conserved 25-bp sequence was synthesized and used as a hybridization probe; three additional copies of the sequence were identified in R. meliloti 41; all three were localized in the 135-kb nod/nif region of the symbiotic megaplasmid. Nucleotide sequence analysis of the six regions revealed that all contained the 47-bp conserved sequence but, with one exception, adjacent DNA regions did not have long conserved DNA stretches. The position of the 47-bp region was about 200-240 bp upstream of the translational start codons of the three nod genes. This conserved sequence is present in several other Rhizobium species and located adjacent to nod genes. We have demonstrated the involvement of this sequence in the expression of nodulation functions, which suggests that these extended promoter regions may have a role in the coordinated regulation of nodulation genes.

Limited T1 ribonuclease digestion of the family of protamine mRNA's purified from rainbow trout testis yields several large oligoribonucleotide fragments ranging in size from 12--54 nucleotides in length. Several of these fragments purified by two dimensional gel electrophoresis contain several G residues and must represent nuclease-resistant, base-paired regions of the mRNA. Sequence analysis of these oligonucleotides by the method of Simoncsits, A., Brownlee, G.G., Brown, R.S., Rubin, J.R. and Guilley, H. (1977) Nature 269: 833-836, shows that these oligoribonucleotides arise from the 5'- and 3'-non-coding regions of the mRNA. Comparisons of the sequences of the large RNA fragment with DNA sequences obtained after cloning double-stranded protamine cDNA in the plasmids pBr322 and pmB9 show precise correspondence of a 54 nucleotide RNA fragment with positions 49--100 from the 3'-poly(A) tract and extending to within 5 nucleotides of the termination codon. Two other RNA fragments of 21 and 25 nucleotides in length arise from the 5'-non-coding region of the message and possess an AUG-sequence at their 3'-termini which is the initiation codon. The presence of distinct by homologous sequences in several sets of large RNA fragments is consistent with the presence of several closely related protamine mRNA's.

Nucleotide pyrophosphate transferase isolated from Streptomyces griseus is used to transfer pyrophosphate group from gamma-32P-ATP to the 3'-OH of tRNA, generating a strictly terminal label at its 3' end. Using yeast tRNAPhe as model compound, it is demonstrated that the labelled molecule is suitable for rapid gel sequencing by both enzymatic and chemical methods. RNA molecules terminated by pyrimidine nucleoside are poor pyrophosphate acceptors. To label RNAs of this kind, first guanosine 5'-phosphate 3'-(beta-32P)-pyrophosphate (pGpp) is prepared from gamma-32P-ATP and GMP by nucleotide pyrophosphate transferase. pGpp is then ligated to the 3' end of RNA by T4 RNA ligase. The complete nucleotide sequence of 5S RNA from Streptomyces griseus is established by rapid gel sequencing methods performed on 3'-(beta-32P)-pyrophosphate labelled molecule.

A simple way of preparing nucleoside 5'-phosphordiamidate is described. The procedure is based on the ammonolysis of nucleoside 5'-phosphordichloridates by dilute aqueous ammonium hydroxide. The behaviour of nucleoside phosphordiamidates under acidic and alkaline conditions is also reported. Alkaline hydrolysis results in the formation of the parent nucleoside, whereas one amide group can be removed selectively by mild acid hydrolysis. This property of nucleoside phosphordiamidates served as a basis for the elaboration of a simple synthesis of nucleoside phosphoramidates starting from nucleosides.

A simple one-step chemical synthesis of ribonucleoside 2′,3′-cyclic phosphate 5′-phosphates is described. The method is a combination of the phosphorylation of ribonucleosides by pyrophosphoryl chloride with subsequent neutral buffered hydrolysis.

Rat liver nuclear 30 S ribonucleoprotein particles containing pre-mRNA and nuclear sap proteins have been shown to modify in vitro the synthetic dinucleotide ppGpC in the presence of GTP and S-adenosyl-L-methionine (SAM) by the formation of a blocked and methylated (capped) structure 7meG(5′)ppp(5′)-Gm epC. In the absence of SAM the predominant reaction product was GpppGpC. Our results indicate that the 30S ribonucleoprotein particles (informofers) as well as the proteins of the nuclear sap possess both guanylyltransferase, N7-, and 2-o-methyltransferase activities.

METHODS for sequencing nucleic acids have improved significantly in the last few years due to the development of rapid ‘read-off’ gel methods. The first method to be developed was the ‘plus and minus’ copying method for DNA1 and an analogous copying method for RNA now exists2. A different and more direct method for sequencing DNA has been developed3, but no direct method is available for RNA. We have developed such a method and here we describe its use in establishing the sequence of tyrosine tRNA of Bacillus stearothermophilus. This sequence is especially interesting as the tyrosine tRNA and its specific synthetase in B. stearothermophilus offer an attractive experimental system for studying the specific interaction of tRNA and synthetases, which is now possible because of progress in studying the three-dimensional structure of the protein by X-ray crystallography4.

The sequence of the 3′-terminal 43 nucleotides of 16S ribosomal RNA from the cyanobacterium Synechococcus AN PCC 6301 has been determined. This sequence is almost identical with the 3′-terminal sequence of 16S ribosomal RNA from maize chloroplasts. The evolutionary implications of these observations are discussed.