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NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I), d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) INTRODUCTION [1,2]The role of DNA ligases is well established in DNA replication, in DNA repair and in genetic recombination in prokaryotic and in eukaryotic cells [3]. These enzymes catalyse the restoration of an interruption of a single strand in double-helical DNA. For the joining activity the enzyme requires juxtaposed 3'-hydroxyl and 5'-phosphoryl ends aligned in a duplex structure [4]. The enzymes obtained from unaffected and from T4-infected E. coli have been investigated most thoroughly [4]. However, until now little information is available concerning structural details of the nicked duplex structure.The present work describes an NMR study, augmented with biochemical experiments, of a synthetic nicked duplex structure. The compound consists of a 12 base-paired duplex that features an interruption in the centre of one strand of the double helix. The conformational properties of the nicked duplex are compared with those of the intact doublehelical fragment. Furthermore, the influence of removal of the phosphate at the interruption is demonstrated. Thermodynamic analysis of duplex formation of the nicked as well as of the intact duplex structure is used to study the amount of cooperativity of the two hexamer strands in the melting behaviour of the nicked duplex. Finally, the T4 polynucleotide ligase activity upon this synthetic nicked duplex fragment is reported.

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In a previous study it was shown that RNase P from E. coli cleaves the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA in vitro (Guerrier-Takada et al. (1988) Cell, 53, 267-272). Cleavage takes place at the 3' side of the loop that crosses the deep groove of the pseudoknot structure present in the aminoacyl acceptor domain. In the present study fragments of TYMV RNA with mutations in the pseudoknot, generated by transcription in vitro, were tested for susceptibility to cleavage by RNase P. Changes in the specificity with respect to the site of cleavage and decreases in the rate of cleavage were observed with most of these substrates. The behaviour of various mutants in the reaction catalyzed by RNase P is in agreement with the present model of the TYMV RNA pseudoknot (Dumas et al. (1987), J. Biomol. Struct. Dyn. 263, 652-657). Base substitutions in the loop that crosses the shallow groove of the pseudoknot structure resulted, however, in an unexpected decrease in the rate of cleavage, probably due to conformational changes in the substrates. Studies on other tRNA-like structures revealed an important role in the reaction with RNase P for both the nucleotide at the 3' side of the loop that spans the deep groove and the nucleotide at position 4, which correspond to positions--1 and 73, respectively, in tRNA precursors.

With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T(+)), also had acquired non-selected calf thymus carrier DNA sequences (C(+)), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas(+)), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated ('transforming') foreign DNA sequences 97% of the NT4-like seedlings turned out to be C(+)T(+). Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C(+)T(+):C(-)T(+): C(+)T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes.

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During the infection cycle of Autographa californica multicapsid nuclear polyhedrosis virus, the TATAbinding protein (TBP) of the insect host cell likely participates in early viral transcription, which is mediated by the host RNA polymerase II. However, the role of TBP in late and very late viral transcription, which is accomplished by an alpha-amanitin-resistant RNA polymerase, is unclear. We observed a dramatic increase of TBP protein during the late phases of infection. TBP mRNA levels, however, were not coordinately increased. Indirect-immunofluorescence studies revealed a nuclear redistribution of TBP during infection. After labeling of viral replication centers with bromodeoxyuridine (BrdU), costaining of TBP and BrdU showed that TBP localized to viral DNA replication centers. These results suggest a putative role of TBP during late viral transcription, which may occur in close proximity to viral DNA replication.The TATA-binding protein (TBP) is a universal transcription factor that is required for initiation by all three eukaryotic RNA polymerases. TBP was identified as a subunit of TFIID, a multisubunit complex composed of TBP and TBP-associated factors (for a review, see reference 7). As part of the preinitiation complex at both TATA box-containing and TATA-less promoters, TBP resembles a target for transcriptional activators and repressors (for a review, see reference 19). One unique feature of TBP is its high level of conservation among all eukaryotes and archaea. In most cases, TBP is encoded by a single gene. The carboxy-terminal core domains of all characterized TBPs are more than 75% identical to the human TBP, while the amino-terminal domain is poorly conserved (7). In insects, TBP-encoding cDNAs have been cloned from Drosophila melanogaster cells (8) and from Spodoptera frugiperda cells, which resemble a permissive cell line of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) (20). An amino acid sequence comparison reveals that the C terminus of S. frugiperda TBP is 93% identical to that of Drosophila TBP and about 75% identical to the C termini of all other known TBPs (20).The gene expression cascade of AcMNPV is temporally regulated and characterized by the sequential involvement of two different RNA polymerases. Host RNA polymerase II recognizes early viral, TATA box-containing promoters; thus, TBP is thought to participate in early viral transcription. Late genes and the hyperexpressed very late genes encoding p10 and polyhedrin are transcribed by an alpha-amanitin-resistant RNA polymerase that has been reported to be a complex of four virus-encoded proteins (3, 4). The purified RNA polymerase recognizes late and very late viral promoters, although the burst of very late transcription was not observed by in vitro experiments, suggesting that factors contributing to the hyperexpression of the very late promoters are still unknown (4). Only when in vitro transcription assays were performed with protein extracts of purified cell nuclei was the burst in ver...