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Three oligonucleotide probe sequences were inferred from the 16S ribosomal ribonucleic acid (rRNA) and the 16S–23S rRNA spacer sequences of Bordetella pertussis ATCC 10380. These probes were used in hybridization tests with deoxyribonucleic acid from Bordetella species and other relevant bacterial taxa. A probe from the spacer region hybridized exclusively to the B. pertussis strains tested and not to strains from other species. Using a combination of three probes, B. pertussis B. parapertussis/B. bronchiseptica and B. avium could be specifically identified and differentiated from other taxa. Differentiation between B. parapertussis and B. bronchiseptica was not possible with the probes used. Using the spacer probe, a colorimetric hybridization assay specific for B. pertussis was developed based on enzymatic amplification of the 16S–23S rRNA spacer and reverse hybridization in microtitre wells. As compared with results using agarose gel electrophoresis, and Southern and dot-spot hybridization with a 32P-labelled probe, this assay proved to be faster and easier to perform and was found to be at least as sensitive and specific.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.

The nucleotide sequence at the beginning of the restriction fragment Hind-D from Simian virus 40 DNA has been derived by partial chemical degradation of (5'-32P)-labeled restriction fragments followed by analysis on polyacrylamide gel according to Maxam and Gilbert [Proc. Natl Acad. Sci. U.S.A. 74, 560-564 (1977)]. The sequence reported here is 140 nucleotides long. It contains an ATG codon which presumably corresponds to the initiator codon of the VP2 protein. This codon is preceded by an untranslated region which shows several interesting features, such as an alternation of (dA+dT)-rich and (dG+dC)-rich blocks.

The sequences of two minicircles from the kinetoplast DNA of the CL strain and one of the Y strain of Trypanosoma cruzi are reported. These 1.4 kb molecules have a peculiar sequence organization, the most distinctive feature being the occurrence of a 120 bp sequence repeated four times, located at 0, 90, 180 and 270 degrees along each circle. We have termed these conserved regions in this species 'minirepeats'. Minirepeats have a 3-fold higher concentration of cytosine residues in comparison with the variable regions and contain the universal 12-mer motif GGGGTTGGTGTA present in all sequenced minicircles and which was shown to be involved in DNA replication. A consensus sequence of T. cruzi minirepeats was determined using the 20 minirepeats present in five known T. cruzi minicircle sequences. This consensus sequence contains regions which have been remarkably well preserved in strains which show great biological diversity. In addition a low level of intraminicircle sequence similarity was also observed within the variable region, but this similarity did not extend between strains. The abundance of conserved minirepeat sequences containing invariant restriction sites in T. cruzi cells may prove valuable for the development of new direct diagnostic methods for Chagas' disease based on DNA probe technology.

The HindII + III restriction fragment J (Hind-J) represents 4.58% of the simian virus 40 genome. The information present in Hind-J is expressed as part of the major, late 16-S messenger RNA, which codes for the structural protein VP1. The nucleotide sequence of the 240-base-pairs-long Hind fragment J has been determined by analysis of each oligonucleotide from both strands resulting from T1 or pancreatic RNase digestion of RNA transcribed from the DNA and from RNase digestion of ribo-substituted DNA. Large oligonucleotide blocks which could be constructed mainly on the basis of complementarity were subsequently ordered by partial chemical degradation of terminally labeled DNA. This direct DNA sequencing approach also completely confirmed the results obtained by both aforementioned RNase degradation methods. In the strand with the same polarity as the late mRNA, triplets corresponding to termination codons are present in two of the three reading frames. The one open reading frame connects in phase with the open reading frame of the neighboring HindII + III fragments K, F and G, which have been published previously and which together with Hind-J span the total VP1 gene. Some features of the primary nucleotide sequence of this VP1 gene and the derived VP1 amino acid sequence are discussed.

Tumour necrosis factor (TNF), released by induced macrophages, causes tumour necrosis in animals and kills preferentially transformed cells in vitro. mRNA induced in the established mouse monocytic PU 5.1.8 cell line by lipopolysaccharide, was converted into double-stranded cDNA and cloned in the pAT153 vector. Recombinant plasmids were screened by plus-minus hybridization and TNF-specific oligonucleotide probes constructed on the basis of partial amino acid sequences of rabbit TNF. A series of TNF specific clones were identified and confirmed by hybrid selection of mouse TNF-specific mRNA. The sequence codes for a 235 amino acids long polypeptide, of which 156 amino acids presumably correspond to the mature product. It can be concluded that mature mouse TNF is a glycosylated dimer. Biologically active TNF was secreted by both Cos-I and CHO-cells transfected with the chimaeric expression vector pSV2d2-mTNF containing the coding region of the mouse TNF cDNA gene.

We report here the nucleotide sequence of the wild-type simian virus 40 (strain 776) restriction fragment Hind-C-P1 DNA and of the homologous region of various mutant DNAs which lack part of this fragment. During this work, we detected between EcoRII fragments N and G an additional, 17-base-pair EcoRII fragment, fragment P, which had previously been overlooked. Also, an additional dTpdG dinucleotide at residues L 339--340 was observed by sequence analysis of the DNA minus (E) strand; the presence of this dinucleotide was masked on sequencing patterns of the plus strand due to the persistence (during gel electrophoresis) of some secondary structures in the strand's 5'-terminal region. These nucleotide additions raise the total length of SV40 DNA to 5243 base pairs. The longest tandemly repeated segment in SV40 DNA now extends over 72 base pairs. SV40 deletion mutants dl 893 and dl 894 and SV40 strains Rh 911 and 1801 all lack an identical 72-base-pair-long DNA segment in the Hind-C region. This deletion corresponds precisely to one of the two aforementioned large tandemly repeated sequences. Mutant dl 895 lacks 66 base pairs, 63 of which are part of the former repetition. All these mutants, except dl 895, very probably were generated by an intramolecular, homologous recombination event. The 40-base-pair deletion in mutant dl 1811 includes the major capping site of SV40 late RNA. dl 1812 lacks only three base pairs, which are part of the overlapping HhaI and HpaII restriction sites at position 0.725--0.726.

Fragment Hind-B-C2, the second largest DNA restriction fragment generated by digestion of fragment Hind-B with the endo R ·EcoRII enzyme, represents 4.4% of the Simian virus 40 (SV40) genome. The complete nucleotide sequence (231 base pairs) of this fragment has been determined by application of the procedure of Maxam and Gilbert (1977). This region is believed to code for the carboxyl-terminal part of the T antigen. On the basis of the termination code words in the strand with the same polarity as the early mRNA, two open reading frames for translation could be defined. Only one of these, however, extends through the adjacent Hind-B-Ci fragment and is therefore believed to dictate the primary structure of the T antigen. It terminates within Hind-B with a TAA signal. The region, determined by the second open frame, is large enough to code for a protein of Mr about 11000. The regions in SV40 DNA from which the non-translated regions of the early and late mRNAs are transcribed overlap over nearly their entire length. Although this spacer region is very (dA + dT)-rich, a secondary structure model can be proposed which is centered around map position 0.17. The RNA copy of the untranslated region also contains the sequence A-A-U-A-A-A, common to all other eukaryotic mRNA molecules sequenced so far; it is present once on the late mRNA and twice on the early messenger RNA. Also a sequence of 6 or 7 successive thymidine residues is present in the early and late DNA strand respectively, 131 and 95 nucleotides beyond the polypeptide termination codons, and these may perhaps play a role in termination of transcription. Of particular interest may be the carboxyl terminus of the T antigen; the terminal tryptic peptide is 10 residues long, six of which are proline.