Proviral position effects: possible probes for genes that suppress transcription.

Rous sarcoma virus, an oncogenic avian retrovirus, readily causes morphological transformation of chick cells, but in infected rat cells transformation is rare because proviral transcription is inefficient. This constraint is not due to a lack of positive transcriptional factors, or an excess of negative ones, but reflects the site of proviral integration in rat cell DNA. In most sites the provirus is almost invariably silent, in others it is correspondingly active, whilst in a third category expression fluctuates in concert with transitions in chromatin structure. Transcriptional fluctuations are mediated both by flanking cell DNA in cis and by trans-acting cell genes, suggesting that proviral position effects are sensors for genes that down-regulate transcription, perhaps by determining chromatin configuration. We have tried to identify such genes by gene transfer, karyology and insertional mutagenesis. The variable success of these three approaches indicates that the transcriptional down-regulator(s) need act only transiently. This is consistent with a function that operates in ontogeny or differentiation to down-regulate genes whose silence is then perpetuated by other means. The loss of such functions may predispose to neoplasia.

Transcription of Rous sarcoma proviruses in rat cells is determined by chromosomal position effects that fluctuate and can operate over long distances.

Most Rous sarcoma proviruses integrated in rat DNA are inactive. The active minority are not necessarily a consequence of long terminal repeat mutations and their transcription can fluctuate in parallel with previously demonstrated transitions in chromatin configuration. Transcriptional alternations cannot be attributed to variations in positive or negative factors that operate independently of the site of provirus integration. Moreover, distinct proviruses in the same cell can be differentially expressed and flanking cellular elements can act over several kilobases to inhibit provirus transcription. We postulate that position-dependent fluctuations in proviral repression can be mediated by trans-acting factors, and DNA transfections indicate that initiation and maintenance of this phenomenon are separable entities. Provirus activity presumably reflects otherwise inapparent cell controls that affect chromatin structure over long distances.

Purification of the gam gene-product of bacteriophage Mu and determination of the nucleotide sequence of the gam gene.

The gam gene of bacteriophage Mu encodes a protein which protects linear double stranded DNA from exonuclease degradation in vitro and in vivo. We purified the Mu gam gene product to apparent homogeneity from cells in which it is over-produced from a plasmid clone. The purified protein is a dimer of identical subunits of 18.9 kd. It can aggregate DNA into large, rapidly sedimenting complexes and is a potent exonuclease inhibitor when bound to DNA. The N-terminal amino acid sequence of the purified protein was determined by automated degradation and the nucleotide sequence of the Mu gam gene is presented to accurately map its position in the Mu genome.

Localization of the gam gene of bacteriophage mu and characterisation of the gene product.

Using cloning techniques in conjunction with an in vitro assay for activity of the gam-coded protein (pgam), the gam gene has been located on a 930-bp fragment immediately to the right of an AccI site situated 5.75 kb from the left-hand end of the phage Mu genome. An analysis of the properties of pgam obtained from an overproducing clone indicates that it is a non-specific DNA-binding protein which interacts with linear duplex plasmid DNA having a variety of different termini and confers protection against exonuclease action (Gam function). It also stimulates the frequency with which linear plasmid DNA transforms Escherichia coli to antibiotic resistance (Sot function). The preliminary results reported here suggest that pgam is potentially a useful 'tool' in molecular biology, although the molecular details of pgam activity require further clarification.

Evidence for a conservative pathway of transposition of bacteriophage Mu.

During its lytic cycle bacteriophage Mu uses repeated transposition as a mode of DNA synthesis. These transpositional events are undoubtedly replicative, and presumably semi-conservative. In a Mu lysogen this type of transposition can start immediately after prophage induction. However, in an infective cycle the Mu genome (which is injected into the host cell as a linear molecule flanked by short random sequences of bacterial DNA) must first become integrated into the host chromosome. Little is known about how this occurs apart from the fact that the bacterial sequences at either end of the Mu genome are lost in the process. The integration is thus similar to a transposition event. In an attempt to determine whether this type of Mu transposition (between a linear donor molecule and a circular recipient) is also semi-conservative we have analysed the progeny phage arising from an infective cycle in which the parental DNA was heterozygous for a known genetic marker. The expectation is that if integration of the infecting Mu genome occurs by a single semi-conservative transpositional event then pure phage bursts should be produced as the genetic information on only one strand would be preserved throughout the lytic cycle. The experiments reported here do not support this expectation in that the infected cells yield mixed bursts, suggesting that Mu integration is a conservative, rather than a semi-conservative event.