Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection.

Integration of retroviral DNA can serve as a paradigm for cellular functions that are affected by the packaging of DNA into chromatin. We have used a novel polymerase chain reaction-based assay to survey DNA and chromatin for the precise distribution of many integration sites. Integration into naked DNA targets is non-uniform, implying a nucleotide sequence bias. In chromatin, integration occurs preferentially at positions where the major groove is on the exposed face of the nucleosomal DNA helix, generating a 10 bp periodic spacing of preferred sites. Chromatin assembly enhances the reactivity of many sites, so that integration occurs most frequently at sites in nucleosomal, rather than nucleosome-free, regions of minichromosomes. In contrast, integration is prevented in a region occupied by a site-specific DNA-binding protein. Comparisons of integration events mediated by viral nucleoprotein complexes or by two different retroviral integrases show that the integration machinery also affects target site selection.     

Defining the consensus sequences of E.coli promoter elements by random selection.

The consensus sequence of E.coli promoter elements was determined by the method of random selection. A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene. The DNA sequences and relative levels of function for -10 and -35 elements were determined. The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters. However, no strong correlation is observed between similarity to the consensus and relative level of function. The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.     

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

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The GCN4 bZIP targets noncognate gene regulatory sequences: quantitative investigation of binding at full and half sites

We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5'-TGACTCA-3') and cAMP-response element (CRE) (5'-TGACGTCA-3') but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and E-box (5'-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full- and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with Kd values of 13 and 12 nM, respectively, to noncognate sites with Kd values of 120 nM to low microM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (Kd = 84 nM) to noncognate half sites (all Kd values > 2 microM). CD shows that comparable levels of alpha-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, alpha-helical proteins with desired DNA recognition properties.     

Determination of adeno-associated virus Rep68 and Rep78 binding sites by random sequence oligonucleotide selection.

To further define the canonical binding site for the P5-promoted Rep proteins of the adeno-associated virus, a modified random oligonucleotide selection procedure was performed, using purified recombinant Rep protein. These results may explain the effects of Rep on cellular gene expression.     

On the structural repertoire of pools of short, random RNA sequences

A detailed knowledge of the mapping between sequence and structure spaces in populations of RNA molecules is essential to better understand their present-day functional properties, to envisage a plausible early evolution of RNA in a prebiotic chemical environment and to improve the design of in vitro evolution experiments, among others. Analysis of natural RNAs, as well as in vitro and computational studies, show that certain RNA structural motifs are much more abundant than others, pointing out a complex relation between sequence and structure. Within this framework, we have investigated computationally the structural properties of a large pool (10⁸ molecules) of single-stranded, 35 nt-long, random RNA sequences. The secondary structures obtained are ranked and classified into structure families. The number of structures in main families is analytically calculated and compared with the numerical results. This permits a quantification of the fraction of structure space covered by a large pool of sequences. We further show that the number of structural motifs and their frequency is highly unbalanced with respect to the nucleotide composition: simple structures such as stem-loops and hairpins arise from sequences depleted in G, while more complex structures require an enrichment of G. In general, we observe a strong correlation between subfamilies—characterized by a fixed number of paired nucleotides—and nucleotide composition. Our results are compared to the structural repertoire obtained in a second pool where isolated base pairs are prohibited.     

Characterization of Theileria species by PCR using specific target sequences

Theileriosis is an infectious disease in tropical countries and in the Mediterranean area. It is caused by Theileria, a haemoprotozoan, transmitted by vectors belonging to the Ixodidae. In Southern Italy and in Sicily the infection is due mainly to T. annulata, but in some cases other species are involved in the disease. The authors describe a method to identify theileriosis in cattle blood samples, using PCR and hybridization techniques. Different primer sets were used to amplify different DNA target sequences, both genus and species specific. Blood samples from cattle were collected in Sicily. The DNA extracted from blood samples was employed first to detect the presence of the 18S ribosomal subunit gene specific for Theileria genus. Successively the positive samples were analysed to identify the species, T. annulata or T. buffeli/orientalis, using as target sequences for amplification respectively a fragment of the TAMS-1 and p33/34 antigens gene. Here the authors describe for the first time the presence of T. buffeli/orientalis infection in Sicilian herds. In fact 66% of positive blood samples were T. buffeli/orientalis infected.     

cis-acting sequences involved in the translational control of GCN4 expression

Four short upstream open reading frames (uORFs) in the mRNA leader are required for the translational control of GCN4 expression in response to amino acid availability. Data are reviewed demonstrating that the fourth (3' proximal) uORF is sufficient to establish the repressed levels of GCN4 expression, while the first uORF functions as a positive regulatory element under starvation conditions to stimulate GCN4 translation. Furthermore, positive and negative trans-acting regulatory factors, the activities of which appear to be modulated according to amino acid availability, exert their effects on GCN4 expression through the uORFs. Direct comparison of the uORFs indicates that there are important nucleotide sequence differences between uORF1 and 4, and that these are located primarily around the termination codons of these elements. Recent findings suggest that the sequences that mediate repression of GCN4 expression are complex, but can be overcome under starvation conditions by ribosomes that have previously translated uORF1.     

A comparison of the different DNA binding specificities of the bZip proteins C/EBP and GCN4.

The bZip proteins GCN4 and C/EBP differ in their DNA binding specificities: GCN4 binds well to the pseudopalindromic AP1 site 5'-A4T3G2A1C0T1C2'A3'T4'-3' and to the palindromic ATF/CREB sequence 5'-A4T3G2A1-C0*G0'T1'C2'A3'T4'-3'; C/EBP preferentially recognizes the palindromic sequence 5'-A4T3T2G1C0*G0'C1'A2'-A3'T4'-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in specific base contacts: asparagine -18, alanine -15, alanine -14, serine -11 and arginine -10 (numbered relative to the start point of the leucine zipper, which we define as +1). In the basic region of C/EBP position -14 is occupied by valine instead of alanine, the other four residues being identical. Here we analyse the role of valine -14 in C/EBP-DNA complex formation. Starting from a C/EBP-GCN4 chimeric bZip peptide which displays C/EBP specificity, we systematically mutated position -14 of its basic region and characterized the DNA binding specificities of the 20 possible different peptides by gel mobility shift assays with various target sites. We present evidence that valine -14 of C/EBP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4, which exclusively contacts thymine 1' of the GCN4 binding sites.