Sequences in the 5' leader of Mason-Pfizer monkey virus which affect viral particle production and genomic RNA packaging: development of MPMV packaging cell lines.

We used a series of deletion mutations in the 5' untranslated region of the prototype D type retrovirus, Mason-Pfizer Monkey Virus (MPMV), to analyse RNA encapsidation. A region was identified upstream of the major splice donor which reduced particle production but had a proportionally greater effect on RNA packaging. A small deletion downstream of the splice donor had little effect on RNA production and caused no significant packaging defect. A large deletion encompassing the end of the primer binding site down to the splice donor had a dramatic effect, disrupting viral protein synthesis. Stable cell lines were produced containing packaging-defective virus. These first-generation packaging cell lines were used to package and transfer an MPMV-based vector.

A novel mammalian expression screen exploiting green fluorescent protein-based transcription detection in single cells.

The accumulation of DNA sequence information from large-scale genomic and random library sequencing projects is leading to the rapid identification of many putative genes, virtual transcripts and ESTs of unknown function. There is therefore an increasing need for high throughput, sensitive and robust methods for identification and characterisation of genes, and/or their products, based on function. We describe a high throughput functional expression screen based on semi-quantitative analysis of enhanced green fluorescent protein expression in single cells by confocal microscopy. The assay was implemented in a micro-scale format, requiring around 10(4) cells/test. The system was validated by co-transfection of a series of cDNAs encoding pro-inflammatory cytokine intracellular signal mediators with a d2EGFP reporter containing a cytokine responsive promoter. The majority of the test plasmids gave a detectable signal above background at a pool size of 250-500. Replicate tests indicate that the assay is reproducible at this pool size. At this level we demonstrate that large (>10(6) transformants) libraries can be feasibly screened.

Negative regulatory domains in a trophoblast interferon promoter.

A bovine trophoblast interferon (IFN-tau) gene promoter sequence (-450 to +26 bp relative to the transcription start site) led to expression of reporter gene (CAT) constructs transfected into L929 (murine fibroblast) or JAR (human choriocarcinoma) cells. Expression depended on the presence of an exogenous (SV40) enhancer. Poly(I)(C) activated endogenous IFN production in L929 and JAR cells but had no consistent effect on CAT expression. Similar results were obtained in L929 cells with inactivated Newcastle disease virus. There was no 'priming' effect of exogenous Type I IFN. Deletion mutants revealed sites exerting negative control on expression between - 338 and - 247 bp, and between - 150 and - 71 bp; these regions contained sequences resembling previously identified negative regulatory domains. In the absence of viral inducibility it is proposed that negative regulation contributes towards the stringent control of expression characteristic of IFN-tau genes.

Regulation of trophoblast interferon gene expression.

The trophoblast of the developing ruminant conceptus secretes large quantities (up to 100 micrograms, or 10(7) IU, per 24 h) of a number of characteristic Type I interferons. Secretion (gene expression) commences at Day 8 or 9 (in the sheep; Day 10 in cattle) and ends at about Day 22 (Day 25). The function of this material is in the inhibition of uterine prostaglandin secretion, and hence in the maintenance of the corpus luteum (the 'maternal recognition of pregnancy'). Such rapid onset and cessation of gene transcription begs questions about the function of trophoblast interferon gene promoters, and a limited number of studies have now been carried out with reporter gene constructs, with inconclusive results. It seems unlikely, however, that viral responsiveness accounts for the phenomenon, as in adult interferon genes, although the trophoblast interferons are highly active antiviral agents.

Trophoblast interferons in early pregnancy of domestic ruminants.

A type I interferon (IFN) secreted by the trophoblast of early sheep and cow embryos is thought to be responsible for the maternal recognition of pregnancy. The expression of trophoblast IFN is tissue specific and temporally controlled. However, the isolated bovine trophoblast IFN promoter did not confer tissue specificity on the expression of a bacterial chloramphenicol acetyl transferase (CAT) reporter gene, and could not be induced by virus, unlike other type I IFNs. Trophoblast IFN acts locally within the uterus to prevent luteolysis and prolong progesterone secretion. Endometrial IFN receptors are present, and trophoblast IFN decreases expression of endometrial oxytocin receptor and increases expression of endometrial beta 2-microglobulin, MHC class I antigens and Mx (a mediator of IFN antiviral activity) only in the pregnant horn of pregnant ewes with a transected uterus. The primary effect of trophoblast IFN during early pregnancy appears to be an inhibition of oxytocin receptor expression, although studies in ovariectomized ewes suggest that luteal oxytocin may be required to facilitate the inhibition of prostaglandin F secretion by trophoblast IFN. An investigation of the isolated oxytocin receptor promoter should confirm its critical role in the maternal recognition of pregnancy.