CorrectionsBIOCHEMISTRY. For the article ''Interaction of RNA polymerase with forked DNA: Evidence for two kinetically significant intermediates on the pathway to the final complex,'' by Laura Tsujikawa, Oleg V. Tsodikov, and Pieter L. deHaseth, which appeared in number 6, March 19, 2002, of Proc. Natl. Acad. Sci. USA (99, 3493-3498; First Published March 12, 2002; 10.1073͞ pnas.062487299), the authors note the following concerning RNA polymerase (RNAP) concentrations. No correction was made for the fraction of RNAP (0.5) that is active in promoter binding. With this correction, the values of K 1 and K app (but not K f ) would increase by about a factor of 2. The relative values would remain essentially unchanged. Also, the legends to Figs. 2, 3, and 5 contain errors pertaining to the symbols used for data obtained with and without heparin challenge, the duration of the challenge, and the concentration of added heparin. The figures and the corrected legends appear below. Fig. 2. Determination of equilibrium affinities by titration of wt Fork with RNAP. The reactions contained 1 nM wt Fork and variable amounts of RNAP as shown and were analyzed by electrophoretic mobility shift immediately (OE; data shown are averages of three independent experiments) or after a challenge with 100 g͞ml heparin for 10 min (F; data shown are averages of four independent experiments). The curves shown reflect the simultaneous errorweighted fits of the data to Eqs. 3 and 4 -7. The parameters are shown in Table 1 (line 1). www.pnas.org͞cgi͞doi͞10.1073͞pnas.013667699 Fig. 3. Kinetics of complex formation. RNAP (65 nM) and wt forked DNA (1 nM) were incubated for various time intervals and then complex formation was determined immediately (Ϫheparin) or after a 2-min challenge with 100 g͞ml heparin (ϩheparin). The Ϫheparin data (s) were fit (error-weighted) with Eq. 8 with a 2 ϭ 0 (kaϪ ϭ 0.10 Ϯ 0.01 s Ϫ1 ) and the ϩheparin data (OE) with both single (k aϩ ϭ 0.036 Ϯ 0.004 s Ϫ1 ; thin line) and double-exponential (ka 1 ϭ 0.044 Ϯ 0.002 s Ϫ1 ; ka 2 ϭ (5 Ϯ 3) ϫ 10 Ϫ4 s Ϫ1 ; thick line) equations. Fig. 5.Comparison of the kinetics for formation and dissociation of competitor-resistant complexes between RNAP and wt Fork. Association data were obtained as described in the text and the legend for Fig. 3 except the concentration of forked DNA was 10 nM. Dissociation kinetics were obtained by challenging with 100 g͞ml heparin a mixture of RNAP and forked DNA that had been incubated for 30 min. The curves represent double-exponential fits of the data to Eq. 10. (A) wt RNAP. The observed association rate constants (s) are shown in the legend for Fig. 3; for the slow phase of the dissociation of the wt Fork-wt RNAP complex (F), kd 2 ϭ (1.3 Ϯ 0.2) ϫ 10 Ϫ4 s Ϫ1 . (B) YYW RNAP. The slow phase of the association reaction (F) has a ka 2 ϭ (1.1 Ϯ 0.3) ϫ 10 Ϫ3 s Ϫ1 ; the slow phase of the dissociation reaction (s), a kd 2 ϭ (6 Ϯ 1) ϫ 10 Ϫ4 s Ϫ1 . Fig. 6. BCL-6 preferentially binds to the wild-type exon 1 in Ly1 cells. Both Ly1 and the control Ly7 cells wer...

There is growing debate over the utility of multiple locus association analyses in the identification of genomic regions harboring sequence variants that influence common complex traits such as hypertension and diabetes. Much of this debate concerns the manner in which one can use the genotypic information from individuals gathered in simple sampling frameworks, such as the case/control designs, to actually assess the association between alleles in a particular genomic region and a trait. In this paper we describe methods for testing associations between estimated haplotype frequencies derived from multilocus genotype data and disease endpoints assuming a simple case/control sampling design. These proposed methods overcome the lack of phase information usually associated with samples of unrelated individuals and provide a comprehensive way of assessing the relationship between sequence or multiple-site variation and traits and diseases within populations. We applied the proposed methods in a study of the relationship between polymorphisms within the APOE gene region and Alzheimer's disease. Cases and controls for this study were collected from the United States and France. Our results confirm the known association between the APOE locus and Alzheimer's disease, even when the 4 polymorphism is not contained in the tested haplotypes. This suggests that, in certain situations, haplotype information and linkage disequilibrium-induced associations between polymorphic loci that neighbor loci harboring functional sequence variants can be exploited to identify disease-predisposing alleles in large, freely mixing populations via estimated haplotype frequency methods.

We have identified and characterized two mutually exclusive nuclear proteins that interact with a single crucial element of the albumin promoter. One, albumin proximal factor (APF), is found only in liver or differentiated hepatoma cells and is probably identical to the liver-specific factors named HNFl, alTFB, or HPl-binding protein. The other, variant albumin proximal factor (vAPF), is present in dedifferentiated hepatoma cells as well as in somatic cell hybrids that show extinction of the expression of liver-specific proteins, including albumin. Reversion to the hepatic phenotype of either a dedifferentiated variant or an extinguished somatic hybrid clone is accompanied by loss of vAPF and reappearance of APF. These two proteins differ in their thermostability and in their molecular weight, while displaying identical sequence specificities. Both proteins interact with a homologous motif present in promoter regions of several other liver-specific genes. In vitro transcription assays, using a rat liver nuclear extract, indicate that the binding of APF to its target sequence is required for albumin transcription. These results suggest that a modification in the primary structure of a transcription factor is correlated with the differentiated state of the hepatic cell.

No abstract

Hepatic Nuclear Factor 1 (HNF1, also referred to as LFB1, HP1 or APF) is a liver-specific transcription factor required for the expression of many hepatocyte specific genes. We report here the purification of this rat liver nuclear protein and the cloning of its cDNA using a PCR-derived approach. Seven independent clones reveal 3 alternative polyadenylation sites and a unique open reading frame. Both a motif homologous to the homeodomain and a distal dimerization domain are required for specific DNA binding. Sequence comparisons reveal several atypical features at key positions in the segment corresponding to helices III and IV of the Antaennapedia homeodomain as well as a potential 24 amino acid loop in place of the universal turn between helices II and III. Together with its property to dimerize in the presence or absence of DNA, these features place HNF1 as the prototype of a novel subclass of transcription factors distantly related to homeoproteins.

An efficient method for producing the covalent closure of oligonucleotides on complementary templates by the action of BrCN was developed. A rational design of linear precursor oligonucleotides was studied, and the effect of factors such as oligonucleotide concentration and oligomer-template length ratio was evaluated. The efficiency of circularization was shown to correlate well with the secondary structure of the precursor oligomer (as predicted by a simple computer analysis), hairpin-like structures bearing free termini clearly favouring the circularization reaction. A novel idea, consisting of the incorporation of non-nucleotide insertions in the precursor oligomer (namely, 1,2-dideoxy-D-ribofuranose residues), may render this method universal and highly effective. An original set of assays was developed to confirm the circular structure of the covalently closed oligonucleotides.

Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity of DNA-binding proteins can be inhibited by the use of double-stranded (ds) oligodeoxynucleotides that compete for the binding to their specific target sequences in promoters and enhancers. As a model, we used phosphodiester dumbbell oligonucleotides containing a binding site for the liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity of HNF-1 to dumbbell oligonucleotides was the same as that to ds oligonucleotides, as determined by gel retardation assays. HNF-1 dumbbells specifically inhibited in vitro transcription driven by the albumin promoter by more than 90%. HNF-1-dependent activation of a CAT reporter plasmid was specifically inhibited when the HNF-1 dumbbell oligonucleotide was added at nM concentration to transiently transfected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which displayed the same activity as the dumbbell oligonucleotides in the in vitro assays, were no more effective in the ex vivo experiments. These results might reflect the increased stability of the circular dumbbell oligonucleotides towards cellular nuclease degradation, as shown in vitro with nucleolytic enzymes. Dumbbell oligonucleotides containing unmodified phosphodiester bonds may efficiently compete for binding of specific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes.

Retroviruses display a strong selective pressure to maintain the dimeric nature of their genomic RNAs, suggesting that dimerization is essential for viral replication. Recently, we identified the cis-element required for initiation of human immunodeficiency virus type I (HIV-I) RNA dimerization in vitro. The dimerization initiation site (DIS) is a hairpin structure containing a self-complementary sequence in the loop. We proposed that dimerization is initiated by a loop-loop kissing interaction involving the self-complementary sequence present in each monomer. We tested the ability of sense and antisense oligonucleotides targeted against the DIS to interfere with a preformed viral RNA dimer. Selfdimerization and inhibition properties of the tested oligonucleotides are dictated by the nature of the loop. An RNA loop is absolutely required in the case of sense oligonucleotides, whereas the nature and the sequence of the stem is not important. They form reversible looploop interactions and act as competitive inhibitors. Antisense oligonucleotides are less efficient in self-dimerization and are more potent inhibitors than sense oligonucleotides. They are less sensitive to the nature of the loop than the antisense oligonucleotides. Antisense hairpins with either RNA or DNA stems are able to form highly stable and irreversible complexes with viral RNA, resulting from complete extension of base pairing initiated by loop-loop interaction.The rational design of antiviral agents is a potential alternative to conventional drug screening in fighting against viral invasion. The antisense oligonucleotide strategy offers an attractive concept, based on the complementarity to a unique site on the RNA target, leading to the selective interference with a given function by "hybridization arrest" (for review, see Refs. 1-3). In the case of human immunodeficiency virus (HIV-I), 1 antisense oligonucleotides have been essentially targeted against sites on the genomic RNA and subgenomic mRNAs controlling the expression of viral proteins (4 -10) or against functional sites located in the 5Ј-untranslated region (4,5,8,11,12). An alternative strategy is to use sense decoy motifs that mimic important RNA structure elements (13-14). Targets on RNA have to be chosen based on the different criteria: functional relevance, sequence conservation, and accessibility to the oligonucleotide within the RNA structure. The latter point is a major problem since the target might be sequestered by the intrinsic structure of the viral RNA and/or by possible interactions with viral or cellular proteins. However, information about the availability of the target is most often missing. A new target site that obeys these three conditions is the recently characterized site that initiates the dimerization of HIV-I genomic RNA.The fact that there is a strong selective pressure to maintain the dimeric nature of genomic RNA suggests that dimerization represents a good target for sense or antisense oligonucleotides. The diploid nature of the RNA genome was suggested ...