Novel screening procedure for recombinant plasmids.

Lysed bacterial colonies containing potential recombinant plasmids were mixed with molten agar and sealed into slots of an agarose gel. After electrophoresis overnight, the gel was stained with ethidium bromide, which clearly reveals recombinant plasmids. Xenopus laevis ribosomal DNA and histone DNA of Psammechinus miliaris were ligated into pCRI plasmids and screened by this method.

Expression of the human interferon-beta gene cloned in phage M13 mp7.

The single-stranded DNA phage, M13 mp7 was used in the construction of an expression vector containing the coding sequence for mature interferon-beta (IFN-beta). Two clones expressed a fused polypeptide showing the biological and physicochemical properties of IFN-beta, despite the fact that the N-terminal amino acid sequence had been changed; 10(6) I.U./l of culture were produced with a molecular weight of 20 000.

A comparison of vertebrate interferon gene families detected by hybridization with human interferon DNA.

Cloned human interferon complementary DNAs were used as hybridization probes to detect interferon alpha and beta gene families in restriction endonuclease digests of total genomic DNA isolated from a wide range of vertebrates and invertebrates. A complex interferon-alpha multigene family was detected in all mammals examined, whereas there was little or no cross-hybridization of human interferon-alpha complementary DNA to non-mammalian vertebrates or invertebrates. In contrast, human interferon-beta complementary DNA detected one or two interferon-beta genes in all mammals tested, with the exception of the cow and the blackbuck, both of which possessed a complex interferon-beta multigene family which has presumably arisen by a recent series of gene duplications. Interferon-beta sequences could also be detected in non-mammalian vertebrates ranging from birds to bony fish. Detailed restriction endonuclease mapping of DNA sequences neighbouring the interferon-beta gene in a variety of primates indicated a strong evolutionary conservation of flanking sequences, particularly on the 3' side of the gene.

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Increased expression of a cloned gene by local mutagenesis of its promoter and ribosome binding site.

A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E. coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased beta-galactosidase synthesis, were selected for DNA sequencing. One clone has a G leads to A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C leads to T and T leads to C) between the Shine-Dalgarno sequence and initiation codon which raise expression of beta-galactosidase two-fold. A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present. These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.

Mapping of the Xenopus laevis 5.8S rDNA by restriction and DNA sequencing.

The location of the 5.88 rDNA within the internal transcribed spacer has been found by restriction and sequence analysis. These analyses indicate the deletion of a dinucleotide from the known rRNA sequence. Regions to the 5' and 3' of the gene contain both uncommon sequences and palindromic structures which might provide potential control points. A secondary structure model is suggested for the 5.8S rRNA incorporating the flanking sequences.

More ribosomal spacer sequences from Xenopus laevis.

The base sequence analysis of a Xenopus laevis ribosomal DNA repeat (7) has been extended to cover almost the entire non-transcribed and external transcribed spacer. A compilation of these sequences is presented. All the repetitive and non-repetitive sequence elements of the spacer are identified and their evolution discussed. Comparison of the X.laevis and S.cerevisiae (25,26) ribosomal DNAs shows about 80% sequence conservation in the 18S gene but no sequence conservation, from the available data, in the external transcribed spacer. The sequence coding for the 3' terminus of the X.laevis 40S ribosomal precursor RNA is presented and its structural features analyzed.

High-level expression of an interferon alpha 2 gene cloned in phage M13mp7 and subsequent purification with a monoclonal antibody.

A cloned interferon alpha 2 (IFN-alpha 2) gene was partially digestd with Pvu II to give a fragment that was inserted into the HincII site of the lacZ gene of bacteriophage M13mp7. Two recombinant phages containing the IFN-alpha 2 sequences in the correct orientation for expression from the lac promoter were characterized in detail. DNA sequence analysis showed that the inserted IFN-alpha 2 gene was in phase with the initiation codon of the lacZ gene. The polypeptide product has an additional 19 amino amino acids at the amino terminus of the mature IFN-alpha 2. The first 11 amino acids originate from the amino terminus of beta-galactosidase, and the remaining 8 amino acids are part of the signal sequence of pre-IFN-alpha 2. Infection of Escherichia coli with these phage followed by induction of the lac promoter with isopropyl thiogalactoside gives high yields (up to 10(9) units/liter with an average of 1.5 X 10(8) units/liter) of the modified IFN-alpha 2. This was purified to homogeneity in a single step by immunochromatography using the monoclonal antibody NK2. The nonreduced product had an apparent molecular weight of 20,500 and was shown by immunoradiometric assay to have the same specific activity as IFN made in Namalwa cells. It exhibited the characteristic cross-species antiviral activity of IFN-alpha 2.

Sequence organization of the spacer DNA in a ribosomal gene unit of Xenopus laevis.

A detailed restriction map was constructed for a cloned Xenopus laevis rDNA fragment containing the nontranscribed spacer (NTS) and external transcribed spacer (ETS) together with a portion of both the 18S and 28S rRNA genes. The NTS was found to contain at least three distinct repetitious areas. Region 1 has a repeating unit of approximately 100 bp. The primary structure of this unit has been determined by DNA sequencing. Region 2 is very similar in organization to region 3, and both have an alternating 81/60 bp arrangement as revealed by restriction with Alu I and DNA sequencing. It can be shown that the 81 and 60 bp canons are virtually identical to one another excepting a deletion/insertion of a 21 bp segment. Region 3 differs from region 2 in having sites for Sma I with its 81 bp units. Between these repeated DNA sequences there are two identical, nonrepetitive DNA sequences, each of which is centered around a Bam Hl site. Most of the ETS has been sequenced. It was found to be nonrepetitive and extremely rich in Cs. Close to the 5' end of the 18S coding sequence there is a DNA stretch very rich in purines. About 2.25 kb upstream from the Eco Rl restriction site bisecting the 18S structural gene there is a unique sequence which may be homologous to the 5' end of the 40S precursor RNA. Present evidence suggests that the boundaries between NTS and ETS occur farther downstream than was suggested by electron microscopic data. Sequencing has revealed that the spacer DNA of X. laevis contains different kinds of simple DNA sequences, but no evidence has been found that spacer DNA once arose by saltation of a 15 bp segment. The most surprising finding was that the spacer sequences around the Bam restriction sites (the Bam islands) show high homology with a sequence near the NTS/ETS interface. From the restriction and sequencing analyses it can be deduced that in recent evolutionary times the DNA sequences near the 5' end of the ribosomal transcription unit were reduplicated twice and displaced into spacer by saltation of an intervening short DNA sequence (the 60/81 bp canons). Possible implications of these evolutionary events for spacer functions are consisdered. The sequencing has also provided a molecular basis for a whole range of conclusions arrived at previously by indirect approaches, and these are discussed.

The effect of hypertonic salt on interferon and interferon mRNA synthesis in human MG63 cells.

After infection with Sendai virus or Newcastle disease virus (NDV) strain F, human osteosarcoma MG63 cells produced large amounts of interferon-beta. Both interferon production and overall protein synthesis were strongly inhibited by hypertonic salt. Interferon mRNA synthesis, however, was little affected by hypertonic salt up to twice normal salt concentrations, although cellular RNA synthesis was inhibited under these conditions. The results are compared to those obtained with polyriboinosinic acid: polyribocytidylic acid copolymer [poly(rI) . poly(rC)] inductions of MG63 cells.

The subunit structure of the eukaryotic chromosome.

New strucutral data have been obtained from neutron scattering studies of chromatin. The concentration-dependent meridional peak at 10-11 nm comes from the interparticle spacing of a subunit structure. Peaks at 5.5 and 3.7 nm have a different contrast behaviour to those at 11.0 and 3.7 nm showing that histones and DNA have a different spatial arrangement in the subunit. A globular model in which apolar segments of histones from the core surrounded by DNA complexed with the basic segments of histones agrees with the data.

Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA.

In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.

Differences in the expression of the human interferon-gamma gene in fresh lymphocytes and cultured lymphoblasts.

Fresh human peripheral blood mononuclear lymphocytes and lymphoblasts that had been grown for a period in T-cell growth-factor containing medium were stimulated with staphylococcal enterotoxin A plus mezerein to produce interferon-gamma (IFN-gamma). Growing lymphoblasts produced peak levels of IFN-gamma much earlier after induction than fresh lymphocytes. Quantitation of the steady-state levels of IFN-gamma mRNA showed these to differ markedly between the two cell types over a period of time post-induction. In fresh lymphocytes the steady-state levels of IFN-gamma mRNA increased to a peak level over a period of 4 days while in growing lymphoblasts the peak level occurred after 8 hours. These differences in IFN-gamma mRNA production were shown to be not the result of gross alteration of RNA metabolism following blast transformation.

The arrangement of 18-S and 28-S ribosomal ribonucleic acids within the 40-S precursor molecule of Xenopus laevis.

The arrangement of 18-S rRNA and 28-S rRNA within their 40-S common precursor molecule (pre-rRNA) of Xenopus laevis was investigated by electron microscopic analysis of secondary structure of nascent pre-rRNA chains of oocytes, and by 5'-end analysis of 18-S rRNA and 28-S rRNA hybridized to the EcoRI fragment of rDNA cloned as plasmid pCD42. Secondary structure mapping of phenol-extracted RNA from nucleolar cores revealed complete pre-rRNA chains or molecules at various stages of processing and pre-rRNA molecules apparently lacking one end. In this latter group, which was regarded as representing nascent chains, more than 90% of the molecules had no 28-S rRNA REGION. This shows that the 28-S rRNA sequence is transcribed after the 18-S rRNA region and hence must be located nearer to the 3' end of the pre-rRNA molecule. For 5' end-group determination [3H]uridine-labelled 18-S rRNA and 28-S rRNA were hybridized, as fragments of about 200 nucleotides, to the plasmid pCD42 containing coding sequences for four-fifths of the 18-S rRNA sequence, the external transcribed spacer, the non-transcribed spacer and a tenth of the 28-S rRNA sequence. The RNA was recovered from the hybrids and analyzed for uridine 3',5'-bisphosphate (pUp) after alkaline hydrolysis. The pUp content of the hybridized 18-S rRNA fragments was 20-fold higher than in those of 28-S rRNA, THUS DEMONSTRATING THAT THE 5' END OF THE 18-S rRNA is located next to the external spacer region. From these results it is concluded that the 18-S rRNA is located close to the 5' end of the 40-S pre-rRNA molecule.

Physical studies of chromatin. The recombination of histones with DNA.

Experiments have been carried out to define clearly which histone combinations can induce a higher order structure when combined with DNA. The criterion for a higher order structure being the series of low-angle X-ray diffraction maxima nominally at 5.5 nm, 3.7 nm, 2.7 nm and 2.2 nm. Such a pattern, with resolution similar to that of H1-depleted chromatin, is readily attainable by recombining histones H2A + H2B + H3 + H4 with DNA using a salt-gradient dialysis method. However, the use of urea in the recombination procedure is shown to be detrimental to the production of a higher order structure. Low-angle ring patterns are not obtained by recomgining DNA with single pure histones or any combination of histone pairs exept H3 + H4. The diffraction maxima from the latter are, however, weaker than those from chromatin and there are pronounced semi-equatorial arcs. The presence of a third histone, either H2A or H2B in the H3 + H4 recombination mixture tends to distort the recognised low-angle pattern. It is concluded that the histone pair H3 + H4 is essential for the formation of a regular higher order structure in chromatin, although for a complete structural development the presence of H2A + H2B is also required.