Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.     

DNA binding proteins of Schistosoma mansoni recognizing a hexanucleotide motif occurring in genes regulated by steroids

1. Schistosome proteins interacting with a gene (F-10), which is only expressed in adult females, were investigated. 2. These were also tested using a synthetic oligonucleotide of 20 bp bearing a defined sequence derived from the F-10 gene and containing a hexanucleotide motif, TGTCCT, occurring in genes responsive to steroids. 3. Schistosome proteins (male and female) bound to the F-10 DNA, but only the male proteins bound to the synthetic oligonucleotide with high affinity. 4. The other preparations each produced different binding patterns, although this seemed to lack specificity. 5. These results indicated that the F-10 gene binds different proteins along its structure and suggested that proteins present in the male schistosomes may regulate its expression.     

人端粒酶催化亚基hTERT基因启动子的克隆

为了确定人端粒酶催化亚基 h TERT基因的启动子结构特征 ,采用 Panhandle PCR技术 ,从正常人外周血单核细胞基因组 DNA中扩增 h TERT基因 5′端上游旁侧序列 ,结果获得了 h TERT基因翻译起始位点上游 2 0 90 bp的基因组 DNA序列。序列分析表明 h TERT基因的启动子区域缺少典型真核启动子的核心元件 ( TATA box和 CAAT box) ,但含有多个已知转录因子蛋白结合的核心序列 ,如 E box及 Sp1核心序列。提示 h TERT基因的表达可能受特殊的转录因子调控 ,这些转录因子的激活可能与癌变细胞中 h TERT重新组成型表达有关     

Identification of a matrix-associated region 5'' of an immunoglobulin heavy chain variable region gene.

In the accompanying report (C. F. Webb, C. Das, S. Eaton, K. Calame, and P. Tucker, Mol. Cell. Biol. 11:5197-5205, 1991), we characterize B-cell-specific protein-DNA interactions at -500 and -200 bp upstream of the mu immunoglobulin heavy chain promoter whose abundances were increased by interleukin-5 plus antigen. Because of the high A + T/G + C ratio of these sequences and the consistent findings by others that enhancer- and promoterlike regions are often located near matrix-associated regions, we asked whether these sequences might also be involved in binding to the nuclear matrix. Indeed, DNA fragments containing the -500 binding site were bound by nuclear matrix proteins. Furthermore, UV cross-linking studies showed that the DNA binding site for interleukin-5-plus-antigen-inducible proteins could also bind to proteins solubilized from the nuclear matrix. Nuclear matrix-associated sequences have also been demonstrated on either side of the intronic immunoglobulin heavy chain enhancer. Our data suggest a topological model by which interactions among proteins bound to the promoter and distal enhancer sequences might occur.     

The structure of two fast-white myosin heavy chain promoters. A comparative study

Two complete myosin heavy chain genes were isolated from chicken genomic libraries, and shown to code for fast-white isoforms. Isoform specific probes were developed from the 5' nontranslated regions of the two genes and used to identify the developmental stages at which each of the genes are expressed. One of the genes is transcribed in the embryo and the other only in the adult. The 5' flanking regions of the two genes were sequenced along with the first three exons. The 5' untranslated sequences in both genes are not contiguous, one intron is present in the adult gene while the embryonic gene contains two. The promoters of both genes contain the conserved CAAT and TATA box elements observed in other eucaryotic genes. A computer assisted comparison was performed on the two genes at the nucleotide and amino acid levels. No homology could be detected in the 5' flanking regions of the genes except in and around the CAAT and TATA elements, however, structural sequences at the 5' ends were highly conserved as well as the position of the first three introns. The amino acids in and around the ATP binding site are completely conserved between the two isoforms.     

Transcription from the adenovirus major late promoter uses redundant activating elements

The adenovirus major late promoter (MLP) has been analyzed by constructing recombinant viral genomes containing mutations in possible promoter elements. Single base pair changes in the TATA box had no effect on viral replication, and MLP expression, as measured by the accumulation of late mRNAs, was at wild type levels. However, a double mutation in the TATA box reduced viral replication and MLP expression, demonstrating that the TATA box is important, although not essential, for maximal activity in virus. Primer extension analysis showed that the mRNAs were initiated at the correct position. A mutation in the CAAT box was viable, and had only minor effects on MLP expression. However, this mutation when coupled to a single mutation in the TATA box, severely reduced viral replication and expression from the MLP. Similarly, a viable mutation in the UPE, shown previously to abolish binding of USF, coupled to a single mutation in the TATA box was lethal. These results suggest that both USF and the CAAT box binding factor CP1 can interact with TFIID to effect activation, and thus that the mechanism of activation is functionally redundant.     

Identification of sequence-specific DNA-binding factors by label transfer: application to the adenovirus-2 major late promoter.

A method of affinity labelling proteins specifically associated with DNA target sequences is proposed. The method utilizes covalent UV-crosslinking of proteins to highly labelled DNA (e.g. in crude cell or nuclear extracts) followed by degradation of the DNA to short oligonucleotides. Proteins selectively labelled by attached residual oligonucleotides are readily amenable to molecular mass determination. Using this approach, we have characterized a HeLa polypeptide specifically bound to a short segment of the adenovirus-2 major late promoter (Ad2 MLP). A molecular mass value (approximately 51 kD) and precise location of the crosslinking site(s) of the protein within the MLP (-55 with respect to the cap site) were determined.