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Proteins which help in initiation, repression or activation o f transcription are termed transcription factors (TF). These factors bind to their cognate recognition sites on DNA and then act, either positively or negatively, to influence the rate at which pol II initiates transcription at a promoter. Typically, promoters and enhancers bind numerous, different gene-specific transcription factors. Protein-protein interactions between the DNA-bound gene-specific transcription factors can have synergistic or antagonistic effects on transcription. Transcription factors have substantial diversity. Faisst and Meyer, (1992) have reviewed the transcription factors that have been described to date. These proteins generally have separate DNA-binding and transcription activation domains. The DNA-binding and activation regions o f cellular transcriptional activators can be categorized on the basis o f their amino acid sequences. DNA-binding regions include the homeodomain (Gehring 1987; Scott et al. 1989; Scott and Hyashi 1990), which contains the helix-tum-helix motif (Otting et al., 1988; Schaffner, 1989), the POU homeodomain (Herr et al., 1988), the zinc finger domain (Brown et a l , 1985; Miller e t a l , 1985), and the basic region plus leucine zipper domain (Landschulz et al. , 1988; Vinson et al., 1989). Activation regions include the acidic (Gill and Ptashne, 1987; Cress and Triezenberg, 1991), glutamine-rich and proline-rich activators (Coumey and Tjian, 1988).

Many transcription activators are multidomain proteins composed o f distinct polypeptide segments responsible for DNA binding, transcription activation or some other relevant regulatory function. Cellular activators have been shown to encode two essential functions that are typically located within separable domains o f the protein (Brent and Ptashne, 1985; Struhl, 1987; Ptashne, 1988). First, a DNA-binding region binds directly to a specific DNA sequence and is responsible for directing the protein to the promoters it regulates. Second, an activation region, anchored to the promoter by the DNA-binding domain, somehow increases the level o f transcription-most likely by establishing contact with a transcription factor (Ptashne, 1988; Lillie and Green, 1990). This contact may be direct or indirect. At least some TAFs function as transcriptional coactivators by providing a functional link between sequence-specific regulators (e.g., S p l, CTF, and N T F l) and TBP (Pugh and Tjian, 1990, 1991; Tanese et a l , 1991). A recent interesting finding is that TBP is a component o f basal transcription factors for all three eukaryotic RNA polymerases (Lobo et a l , 1991; Comai et a l , 1992; Cormack and Struhl, 1992; Schultz et a l , 1992; White et a l , 1992; Rigby, 1993).

Recently, Hoey et a l (1993) reported the isolation and characterization o f the first gene encoding a TAF protein. The deduced amino acid sequence o f TAFllO revealed the presence o f several glutamine- and serine/threonine-rich regions reminiscent o f the protein-protein interaction domains o f the regulatory transcription factor Spl that are involved in transcription activation and multimerization. TA FllO specifically interacts with the glutamine-rich activation domains o f S p l. Moreover, purified Spl selectively binds recombinant TA FllO in vitro. These findings suggests that TAFllO may function as a coactivator by serving as a site o f protein-protein contact between activators like Spl and the TFIID complex. Human transcription factor Spl contains two glutamine activation domains that can function as potent activators (Coumey and Tjian, 1988) and in protein-protein interactions as surfaces for the formation o f homomultimers (Pascal and Tjian, 1991).

1.8. MOLONEY M URINE LEUKAEMIA VIRUS.

Mo-MuLV is a C-type retrovirus which was obtained by the passage o f a cell-free extract o f the Sarcoma 37 transplantable tumour in neonatal mice (Moloney, 1960). Its general pathology includes thymic leukaemia, disseminated lymphosarcoma or lymphatic

leukaemia and hepatosplenomegaly. The Moloney murine sarcoma virus (Mo-MSV) is a replication defective derivative o f Mo-MuLV in which part o f the env open reading frame has been replaced with the mos oncogene. Mo-MSV was isolated from a sarcoma caused by injecting a BALB/C mouse with Mo-MuLV (Moloney, 1966). The basic structure o f Mo-MuLV, a typical C-type retrovirus, is shown in Fig. 1.5, and the functions o f each region o f the genome are summarized in Table 1.2.

1.8.1 TRANSCRIPTIONAL CONTROL OF MO-MuLV.

The sequences mediating Mo-MuLV and Mo-MSV transcription, and the factors acting at these sites have been investigated in a variety o f cell types. Moloney murine leukaemia virus causes thymic leukaemias when injected into newborn mice. A major determinant o f the thymic disease specificity o f Moloney virus maps genetically to the conserved viral core motif in the virus enhancer. In the U3 sequences Mo-MSV has two single bp additions and three single bp deletions with respect to Mo-MuLV (Hilberg et al., 1987). The 72 bp direct repeat sequences o f the Mo-MSV LTR act as an enhancer o f transcription.

The Mo-MSV enhancer sequences were shown by De Franco et al. (1985) to facilitate activation by the synthetic glucocorticoid hormone dexamethasone in transient transfection experiments. The Mo-MuLV U3 region also contains a thyroid hormone response element (TRE) facilitating activation o f transcription by c-erbA protein (Sap et a l , 1989).

Méthylation interference footprinting and the gel retardation technique defined transcription factor binding sites in the 72 bp direct repeats o f Mo-MuLV (Speck and Baltimore, 1987). Multiple proteins in W EH I231 (a murine B cell line) nuclear extracts were able to interact with the Mo-MuLV sequences. In order to distinguish between these proteins it was necessary to partially fractionate the extract by column chromatography. Six separate binding activities were revealed. Three o f these bound to sites previously characterized:- (i) The GRE site occurring once in each 72bp repeat, (ii) The enhancer core motif with homology to that o f SV40 (Weiher et a l , 1987) also occurring once in each repeat, (iii) Sites with homology to the consensus NF-1 binding site (Nagata et a l , 1982) occurring twice in each repeat. The other three binding sites had not been previously been recognized and were designated LVa, b and c.

Fig. 1.5.structure of the Moloney murine leukaemia virus genome (Adapted from Weiss et ah, 1982; 1985). Details and functions of each regions like R, U3 etc. are given in table 1.2.

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