A single-stranded DNA binding protein from S. cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences.

A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements. As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein. The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence. Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro. The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.     

The splicing factor SF2/ASF binds to ARS homologs in a human rDNA replication origin

The function of the relatively well-studied DNA replication origins in the yeast Saccharomyces cerevisiae is dependent upon interactions between origin replication complex (ORC) proteins and several defined origin sequence elements, including the 11 bp ARS consensus sequence (ACS). Although the ORC proteins, as well as numerous other protein components required for DNA replication initiation, are largely conserved between yeast and mammals, DNA sequences within mammalian replication origins are highly variable and sequences homologous to the yeast ACS elements are generally not present. We have previously identified several replication initiation sites within the nontranscribed spacer region of the human ribosomal RNA gene, and found that two highly utilized sites each contain a homologue of the yeast ACS embedded within a DNA unwinding element and a matrix attachment region. Here we examine protein binding within these initiation sites, and demonstrate that these ACS homologues specifically bind the alternate splicing factor SF2/ASF as well as GAPDH in vitro, and present evidence that the SF2/ASF interaction also occurs within the nuclei of intact cells. As the moderate upregulation of SF2/ASF has been linked to oncogenesis through the promotion of alternatively spliced forms of several regulatory proteins, our results suggest an additional mechanism by which SF2/ASF may influence the transformed cell phenotype.     

The Drosophila zeste protein binds cooperatively to sites in many gene regulatory regions: implications for transvection and gene regulation.

The Drosophila zeste protein binds in vitro to several sites in the white, Ultrabithorax, decapentaplegic, Antennapedia, and engrailed genes and to at least one site in the zeste gene itself. The distribution of these sites corresponds often with that of regulatory elements in these genes as defined by mutations or, in the case of white, by molecular analysis. A zeste binding site is frequently found in the immediate vicinity of the promoter. zeste binding sites are composed of two or more zeste recognition sequences T/CGAGT/CG. Isolated consensus sequences do not bind or footprint. Cooperative interactions are involved both in binding to a given site and between proteins bound at independent sites. zeste bound to one DNA molecule can in fact bind simultaneously to another DNA molecule. These results suggest a general role for zeste in bringing together distant regulatory elements controlling the activity of a target gene. In this model, transvection effects are a by-product of normal intragenic zeste action.     

牛乳腺核基质附着区多重顺式作用序列表明其功能的多样性

高等真核细胞的染色体DNA通过基质结合区(MAR)不时地与核基质特异性结合而组织成一种空间环状结构。为了研究以DNA套环形式附着于核基质上的DNA序列的特性,从处于泌乳期的乳腺组织中克隆了多个MAR DNA序列。体外结合实验表明,这些序列能够同核基质蛋白共结合成不溶性的复合物,这些复合物可较容易的通过离心去除。其中,两个MAR序列中包含有TL、CA—和GA—阻断以及ATTA基序。这两个序列中含有多个复制／转录因子的结合位点、增强子基序、多个完全的和非完全的反向重复序列以及潜在的DNA弯曲核心序列样结构。同一DNA序列中存在不同元件的组合可能说明在控制一系列细胞的发育过程中,它们可能发挥有正的或负的调控元件的功能。