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The IS1-encoded protein InsA binds specifically to both ends of IS1 and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS1 as a result of binding of the host protein Integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903. [ABSTRACT FROM AUTHOR]

Powerful new methods, like expression profiles using cDNA arrays, have been used to monitor changes in gene expression levels as a result of a variety of metabolic, xenobiotic or pathogenic challenges. This potentially vast quantity of data enables, in principle, the dissection of the complex genetic networks that control the patterns and rhythms of gene expression in the cell. Here we present a general approach to developing dynamic models for analyzing time series of whole genome expression. In this approach, a self-consistent calculation is performed that involves both linear and non-linear response terms for interrelating gene expression levels. This calculation uses singular value decomposition (SVD) not as a statistical tool but as a means of inverting noisy and near-singular matrices. The linear transition matrix that is determined from this calculation can be used to calculate the underlying network reflected in the data. This suggests a direct method of classifying genes according to their place in the resulting network. In addition to providing a means to model such a large multivariate system this approach can be used to reduce the dimensionality of the problem in a rational and consistent way, and suppress the strong noise amplification effects often encountered with expression profile data. Non-linear and higher-order Markov behavior of the network are also determined in this self-consistent method. In data sets from yeast, we calculate the Markov matrix and the gene classes based on the linear-Markov network. These results compare favorably with previously used methods like cluster analysis. Our dynamic method appears to give a broad and general framework for data analysis and modeling of gene expression arrays.

The binding of many proteins to DNA is profoundly affected by DNA bending, twisting, and supercoiling. When protein binding alters DNA conformation, interaction between inherent and induced DNA conformation can affect protein binding affinity and specificity. Integration host factor (IHF), a sequence-specific, DNA-binding protein of Escherichia coli, strongly bends the DNA upon binding. To assess the influence of inherent DNA bending on IHF binding, we took advantage of the high degree of natural static curvature associated with an IHF site on a 163-bp minicircle and measured the binding affinity of IHF for its recognition site contained on this DNA in both circular and linear form. IHF showed a higher affinity for the circular form of the DNA when compared to the linear form. In addition, the presence of IHF during DNA cyclization changed the topology of cyclization products and their ability to bind IHF, consistent with IHF untwisting DNA. These results show that inherent DNA conformation anisotropy is an important determinant of IHF binding affinity and suggests a mechanism for modulation of IHF activity by local DNA conformation., (Copyright 2000 Academic Press.)

While the most important radiobiological questions at present can be stated simply, arriving at the answers requires the development of a broad base of understanding to which collaborative interdisciplinary efforts that use a variety of techniques will make important contributions. In spite of the range of questions that remain unanswered, we probably know more about the effects of radiation than any other environmental agent. A sound and well-planned research program into the mechanisms of radiation carcinogenesis can help in many predictable ways. It will provide data needed for setting protection standards, of course, but it will also play a vital role in solving some of the enigmas of the mechanisms of carcinogenesis. In addition, the information gained by the approach focused on basic science will ultimately provide much better methods for extrapolating from cellular and animal studies to health effects in humans and thus for developing sound, science-based radiation risk estimates.

The IS 1-encoded protein InsA binds specifically to both ends of IS1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25 bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903.

The tight-binding I12-X86 lac repressor binds to non-operator DNA in a sequence-specific fashion. Using the DNA of the E. coli I gene we have investigated these sequence-specific interactions and compared them to the operator binding of wild-type repressor. The specific, non-operator DNA interactions are sensitive to the inducer IPTG. One strong binding site in the I gene DNA was found to be one of two expected on the basis of their homology with the lac operator. The binding of I12-X86 repressor to this site was visualized using the footprinting technique, and found to be consistent with an operator-like binding configuration. The protection pattern extends into an adjacent sequence suggesting that two repressor tetramers are bound in tandem.

We have studied the effect on the transposition activity of IS1 of the position and orientation of the element in the donor replicon. Previous studies have shown that the IS1s in the compound transposon Tn9 have different activities in forming plasmid cointegrates. In this study, designed to investigate the sources of this differential activity, we have cloned Tn9 (together with 132 base-pairs of flanking DNA) into several sites in pBR322 and created various derivatives of these plasmids to be used as donor molecules in plasmid fusion experiments. These pBR322-Tn9 plasmids were allowed to form cointegrates with a conjugative, F-derived plasmid, and a large collection of independently formed cointegrates was isolated. The relative activities of the two IS1 elements of Tn9 were estimated by examining the structures of the cointegrates for the frequency of usage of each of the IS1s in cointegrate formation. The results suggest that IS1 activity is modulated by the transcription activity of adjacent DNA sequences in the donor plasmids. Transcription directed into an IS1 inhibits its activity. Confirmatory evidence for this hypothesis is provided by the observation that deletion of adjacent promoters of transcription relieves the inhibitory effect on IS1 activity.

The basic nature of the sequence features that define a promoter sequence for Escherichia coli RNA polymerase have been established by a variety of biochemical and genetic methods. We have developed rigorous analytical methods for finding unknown patterns that occur imperfectly in a set of several sequences, and have used them to examine a set of bacterial promoters. The algorithm easily discovers the "consensus" sequences for the -10 and -35 regions, which are essentially identical to the results of previous analyses, but requires no prior assumptions about the common patterns. By explicitly specifying the nature of the search for consensus sequences, we give a rigorous definition to this concept that should be widely applicable. We also have provided estimates for the statistical significance of common patterns discovered in sets of sequences. In addition to providing a rigorous basis for defining known consensus regions, we have found additional features in these promoters that may have functional significance. These added features were located on either side of the -35 region. The pattern 5', or upstream, from the -35 region was found using the standard alphabet (A, G, C and T), but the pattern between the -10 and the -35 regions was detectable only in a sub-alphabet. Recent results relating DNA sequence to helix conformation suggest that the former (upstream) pattern may have a functional significance. Possible roles in promoter function are discussed in this light, and an observation of altered promoter function involving the upstream region is reported that appears to support the suggestion of function in at least one case.

The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol.

Transposable-element-mediated fusion of the conjugal plasmid pOX38::Tn9 with pBR322 results in the appearance of cointegrates composed of a single copy of each plasmid, and cointegrates which carry a single copy of pOX38 but multiple tandem copies of pBR322. These plasmids are separated by directly repeated copies of the transposable element. We demonstrate here that such multimers can be generated from monomeric cointegrates, probably by unequal crossing over between the flanking Tn9(IS1) elements. Their appearance is thus not necessarily associated with the original transposition (fusion) event. Our study demonstrates that the process of duplication is strongly dependent on the homologous recombination system of Escherichia coli, since it is undetectable by our methods in recA- strains. It is also strongly dependent on the presence of a functional DNA polymerase I in the cell. The major pathway(s) for this duplication thus appears to rely on both the homologous recombination system and the replication of the duplicated segment.