L. De Wit

Rapid detection of tuberculous and non-tuberculous mycobacteria by polymerase chain reaction amplification of a 162 bp DNA fragment from antigen 85.

A polymerase chain reaction (PCR) assay was developed for detection of mycobacteria using amplification of a 162 bp region of the genes coding for the mycobacterial antigen 85 complex. Strains belonging to theMycobacterium tuberculosis complex were further differentiated from non-tuberculous mycobacteria by hybridization of the PCR derived Southern blot with an internal oligonucleotide probe and washing under stringent conditions. The method allowed rapid and sensitive detection of mycobacterial DNA in uncultured clinical samples. PCR results obtained forMycobacterium tuberculosis in 206 specimens from 180 untreated patients gave a sensitivity of 93.9% and a specificity of 94.3% compared with the culture. PCR detected DNA fromMycobacterium tuberculosis in seven samples from patients with clinically evident tuberculosis in whom culture was negative. The results suggest that this PCR assay could be used for early and specific diagnosis of tuberculosis.

Stimulation of Fibroblast Interferon Production by a 22K Protein from Human Leukocytes

We have studied the appearance of human interferon-beta (HuIFN-beta) as well as its mRNA in cells treated with a protein, 22K factor, isolated from the culture supernatant of mitogen-stimulated human peripheral blood leukocytes. By itself 22K was found to be unable to induce production of significant amounts of HuIFN-beta protein. However, when aided by treatment with cycloheximide or cycloheximide and actinomycin D (superinduction), 22K caused increases in production ranging from 3- to 20-fold, depending on the cells (diploid or MG-63 osteosarcoma) and the induction schedule. Cells treated with 22K alone produced small amounts of HuIFN-beta mRNA, which was only detectable with a highly sensitive method. In combination with cycloheximide, 22K induced levels of mRNA detectable with less sensitive methods as well. These experiments provide further support for the concept that the antiviral activity of 22K is mediated by its ability to stimulate transcription of the HuIFN-beta gene in cells.

Translation of mouse interferon mRNA in Xenopus laevis oocytes and in rabbit reticulocyte lysates

Abstract Mouse interferon mRNA, extracted from NDV (Newcastle disease virus)-induced L-929 cells has been translated with high efficiency in Xenopus laevis oocytes and rabbit reticulocyte lysates. The translational efficiency of a crude RNA extract was 10 640 interferon units/mg RNA/hour for the Xenopus oocytes and 4 012 interferon units/mg RNA/hour for the reticulocyte lysates. The translation product fulfilled the usual criteria for mouse interferon, viz. species specificity and neutralization by specific anti-mouse interferon antiserum. Upon injection of crude interferon mRNA into Xenopus oocytes, interferon activity appeared both in the oocyte homogenates and the oocyte incubation medium. When analyzed by velocity sedimentation in formamidesucrose, the mouse interferon mRNA showed a rather sharp peak halfway between the 4 S and 18 S RNA markers, as could be expected from a mRNA which codes for a 20,000 dalton protein.

Expression and Preliminary Deletion Analysis of the 42 kDa 2–5A Synthetase Induced By Human Interferon

Among the multiple proteins induced by interferon, the 2–5A synthetase has been shown to be involved in some of its antiviral actions. In the presence of dsRNA, this enzyme catalyzes the synthesis of 2′–5′ linked oligomers of adenosine,ppp(A′ 2p)nA Viral growth is inhibited through mRNA degradation mediated by a latent endoribonuclease which is activated by these 2–5A oliqomers. This effect is transient. The 2–5A oligomers are rapidily degraded by a 2′-phosphodiesterase (1). In addition to this antiviral role, the 2–5A system seems to be involved in the regulation of cell growth and differentiation (2,3). 2–5A activities associated with proteins of different sizes were detected in the cytoplasm and nucleoplasm (4, 5). Two functional mRNAs (1.6 and 1.8 kb in human cells (6, 7) and 1.8 and 3.6kb in mouse cells (5) were shown to encode 2–5A synthetase activity. The relationship between the different mRNAs, which seem to derive from the same gene by tissue specific differential splicing (6, 7) and the different proteins, as well as their cellular localization is still not clear.

Kinetics and distribution of interferon mRNA in interferon-primed and unprimed mouse L-929 cells.

Abstract Mouse L-929 cells that have been primed with 100 U/ml of either crude or electrophoretically pure mouse interferon for two hours before induction with Newcastle disease virus, begin producing interferon about 2–3 hours earlier and produce about 10 times more interferon than do unprimed cells. The kinetics of IF mRNA appearance and decay are very similar for primed and unprimed cells, indicating that the differences in interferon production by primed and unprimed cells cannot simply be explained by differences in the rate of transcription, maturation or degradation of IF mRNA. However, significant differences are noted in the distribution of IF mRNA: priming leads to a marked reduction in the amount of IF mRNA in free cytoplasm and, as a result in total cytoplasmic IF mRNA RNA, whereas polysomal IF mRNA content remains essentially unaltered. Only primed cells showed a detectable level of double stranded (ds)-RNA dependent oligo(2′–5′)adenylate (2–5A) synthetase activity at the time of RNA extraction. It is possible that the 2–5A system may play a role in the altered distribution of IF mRNA in interferon-primed cells.

In Vitro cotranslational processing of human pre-interferon β1 enhances its biological activity

Abstract The addition of dog pancreas microsomes to a micrococcal nuclease-treated rabbit reticulocyte system during cell-free translation of Hu IFN- β 1 mRNA resulted in a 5- to 33-fold increase in the yield of antiviral activity per microgram of mRNA. Size analysis of the immunoprecipitated translation products showed that this increase in biological activity was concomitant with the conversion of an 18,000-dalton species (preIFN) into two products of 16,500 and 20,000 daltons, respectively. In the polyacrylamide gel system used, the peak of the latter product coincided with that of authentic Hu IFN- β 1 . This method of translation has been applied to the rapid scanning of IFN- β 1 mRNA activity after sucrose gradient purification and to the quantitative analysis of IFN- β 1 mRNA.

Cell-free coupling of Newcastle disease virus RNA transcription, translation and Co-translational processing.

A cell-free coupled system for transcription, translation and glycoprotein processing of the Newcastle disease virus genome is described. The system consists of a rabbit reticulocyte lysate preincubated with micrococcal nuclease and of detergent-disrupted purified Newcastle disease virions. [35S]methionine incorporation was linear for 2 h. Polypeptides NP and M, the presumably unglycosylated analogues of glycoproteins HN and possibly F, were identified as translation products. When in vitro synthesis was carried out in the presence of dog pancreas microsomes the HN analogue (pre-HN) was converted to an 80K (approx.) protein which comigrated on polyacrylamide gels with HN synthesized in vivo and which, except for a small fragment, was protected from proteolytic degradation. In immunoprecipitation studies, antiserum against HN purified from virions reacted with both the processed and the unprocessed form of HN synthesized in vitro.