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Related post: in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide position 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence are two of the six GGGCGG SV40 repeat sequences and an 11 nucleotide segment which shows strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone HgA gene. The Rocaltrol Calcitriol position of this upstream promoter sequence can be moved 72 nucleotides closer to the major late cap site without any effect on the efficiency or accuracy of transcription. In vitro promoter competition analysis demonstrates that the upstream promoter sequence, independent of the 294-304 promoter element, is capable of binding polymerase/transcription factors required for SV40 late gene transcription. While two upstream domains control the extent of transcription, DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie Rocaltrol 0.25 Mg downstream of map position 294. Studies have continued to define the molecular architecture of the domain located at base positions 294-304. Using a procedure of base misincorporation, a series of eleven Kpn l resistant mutants have been obtained. The DNA sequences of each of the mutants and their transcription properties Buy Rocaltrol are currently being examined. (N. P. Salzman, Brady, Das, Nandi) B. Improved Procedures for Directed Mutations in Regulatory Control Regions . In the initial studies to generate site specific mutations, single-stranded regions were formed in the SV40 molecule at selected regions, and cytosines in these regions were deaminated using sodium bisulfite. Mutated viruses with altered properties could be generated by this procedure. However, the technique is of restricted value since only one of four deoxynucleotides can be modified and the technique also requires a restriction enzyme cleavage site at the region to be examined. Using chemical synthesis of deoxyol igonucleotides 15 to 20 bases long, it is possible to introduce mutations in any one of the four bases into DNA at any desired site in a molecule. These deoxyol igonucleotides are annealed to specific regions of the SV40 genome that have been inserted into a single-stranded bacteriophage. Using this procedure, we have generated additional mutations into the region that controls the SV40 late promoter. In this same region, the DNA sequences suggest that a hairpin structure can form and that it may serve a role in defining the 5' start site for transcription. 17-3 We have constructed a series of deoxyol igonucleotides that will either strengthen or disrupt this structure. These fragments are presently being incorporated into the S\/40 molecules. In addition to these procedures for directed mutagenesis, we have also generated a series of related mutations at selected sites using the procedure of base misincorporation. With this latter procedure, a number of mutations have been generated in the third T-antigen binding site. (N. P. Salzman, Brady, Das, Nandi, Rodi , and Strike) C. Purified RNA Polymerase II Recognizes Specific Nucleotide Sequences in the Adenovirus Promoter . The properties of RNA polymerase II have been compared when it is purified or when the enzymatic acitvity is contained in the whole cell extract described by Manley et al_. In the whole cell extract, transcription is initiated at the same 5' start sites that are used in vivo . A major initiation site of purified calf thymus RNA polymerase II has been mapped on the cloned Adenovirus 2 major late promoter (Ad2 MLP). The 5' terminus of the major transcript in the TATA box is located 25 to 30 nucleotides upstream of the Ad2 MLP. The specificity of this initiation site has been determined by "runoff" transcriptional analysis and by RNAse Tl analysis which employs single-stranded M13 phage DNA containing the Ad2 MLP as probe. This transcriptional initiation site is used when transcription was carried out on either a superhelical (FI) or linear (Fill) DNA template. Selective initiation of transcription on FI DNA could only be detected at 90, 120, and 150 mM (NHi^)2S0i^ concentrations. In contrast, selective initiation of transcription on Fill DNA could be detected, by RNAse Tl analysis, at (NHit)2S0it concentrations that ranged from 30 mM up to 120 mM. These findings indicate that the purified enzyme enters into a complex with the DNA and migrates to the TATA or binds directly to the TATA box where it initiates transcription. In the presence of other protein factors, it initiates transcription 25-30 nucleotides downstream from this site. ( N. P. Salzman, Mishoe, Vonsover, and Brady) D. Enhanced in Vitro Expression of SV40 and Adenovirus Promoters The whole cell (Manley) extract is able to carry out transcription in vitro , and initiation of transcription occurs at the same 5' start sites that are used in vivo . However, this system does not reflect the level of Rocaltrol 0.25 promoter utilization that is observed in vivo , and some promoters that function in the cell are not expressed in vitro . We have defined Calcitriol Rocaltrol conditions which regulate transcription of the two SV40 late promoters. By altering the ionic conditions of the reaction, there is preferential expression of the promoter that initiates transcription at base position 325. Under these modified conditions, expression of the adenovirus I\/a2 gene is readily observed in vitro , while under standard conditions, it is not transcribed. This adeno gene, like the SV40 late genes, lacks a TATA box upstream from the 5' start site. By constructing a series of upstream deletion mutations, an upstream domain has been identified that controls this gene. (N. P. Salzman, Natarajan) 17-4 PARVOVIRUSES A. Synthesis of Adeno-assodated Virus DNA Has Been Studied Using In Vitro Systems The overall scheme of AAV DNA synthesis in vivo was first described by Straus etal. in 1976 (Proc. Natl. Acad. Sci. 73, p. 742). Briefly, following coinfection of KB cells with AAV and a helper Ad, AAV DNA synthesis is initiated on single-stranded genomic templates by a self-priming mechanism. Subsequent elongation yields a unit length hairpin Intermediate. A second round of self-primed synthesis displaces the 5' -ended arm of the hairpin and leads to either (1) displacement of a complete plus or minus progeny strand (by virtue of a processing/synthesis step at the closed end of the hairpin) or (11) concatemeric molecules If closed end processing/synthesis does not occur. These latter molecules can be eventually processed to unit length templates which, in turn, may also yield progeny strands by new self-primed rounds of displacement
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