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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.

Local binding propensity and curvature of DNA can be characterized using a vector description of DNA bendability, based on a set of parameters derived from deoxyribonuclease I (DNase I) cleavage experiments. Two characteristics — arithmetic and vector averages of bendability — were successfully used to predict experimentally known bendable, rigid and curved segments in DNA. A characteristic distribution of bendability is conserved in evolutionarily related kinetoplast sequences. An analysis of the M. genitalium and H. influenza genomes as well as fragments of human and yeast genomes shows, on the other hand, that highly curved segments — similar to artificially designed curved oligonucleotides — are extremely rare in natural DNA.

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 rapid and simple synthesis of pppGpN type /N equals C, U or A/ diribonucleotide derivatives is described by coupling guanosine 2', 3'-cyclic phosphate 5'-triphosphate with the appropriate ribonucleoside in the presence of ribonuclease T-1.

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.