Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin

DNA sequences, important for the control of Drosophila heat shock gene expression, are packaged in chromatin in a nuclease hypersensitive configuration. Recently, two protein-binding (exonuclease-resistant) sites which cover the TATA box sequence and an upstream control element were shown to occur in vivo amidst the 5' terminal hypersensitive regions of several heat shock genes. Protein-binding at the TATA box is independent of heat shock, but the binding at the upstream element is heat shock dependent, and it was proposed that a heat shock activator protein, HAP, positively regulates the genes. Here, I report the detection of HAP activity in heat shocked cell extracts by reconstituting specific binding to hsp82 gene chromatin in vitro. Inhibition of the binding by free DNA from the 5' region of heat shock genes implies a coordinate regulation of the gene family through HAP interaction with the upstream heat shock consensus sequence. Furthermore, the special ease of induction of the hsp82 gene over other heat shock genes can be explained in molecular terms by the higher affinity of HAP for the hsp82 binding site, which contains a 28 base sequence with almost perfect dyad symmetry, GAAGCCTCTAGAAG/TTTCTAGAGACTTC.     

High-affinity binding site for a specific nuclear protein in the human IgM gene

Proteins binding to specific regions of DNA with high affinity frequently govern or regulate reactions at the gene level. We have identified a high-affinity binding site in the immunoglobulin mu gene that binds a specific nuclear protein, and have now characterized it fully using nuclear factor 1 (NF-1), a protein purified from the nuclei of HeLa cells and required for the in vitro replication of adenovirus (Ad) DNA. NF-1 protects a 25-base pair (bp) double-stranded segment of DNA which shares a consensus sequence, 5' TGGA/CNNNNNGCCAA 3', with similar binding sites in the Ad-5 terminal repeat and the human c-myc gene. Although this site differs from the enhancer region, a biological function is suggested by the fact that it is DNase I hypersensitive in immunoglobulin-producing lymphoblastoid cells. The binding site for the NF-1 protein in the mu gene, by analogy with the site in the Ad-5 terminal repeat, may represent one component of a cellular origin of replication; alternatively, it may be responsible for the activation of the chromatin in this region.     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.     

The 5' ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I

Many specific sites in Drosophila chromatin are hypersensitive to DNase I. The positions of such sites were mapped along the regions of the genome coding for two heat shock proteins. Such sites lie at the 5' ends of heat shock genes and may function as elements for recognition by molecules which regulate gene activity.     

Alteration of c-myc chromatin structure by avian leukosis virus integration

The most common sites of integration of the leukosis virus (ALV) long terminal repeat (LTR) in bursal lymphomas and derivative cell lines correspond to a region encompassed by two major hypersensitive sites in the 5' flanking region of the pre-integration, unrearranged c-myc gene. After integration of the ALV LTR, the major hypersensitive site within the avian c-myc oncogene region is within the proviral LTR, and the major hypersensitive sites normally found in uninfected cells 5' to the first c-myc coding exon are no longer detectable.     

Escherichia coli single-strand binding protein stabilizes specific denatured sites in superhelical DNA

Escherichia coli single-strand binding protein relaxes supercoiled DNA molecules containing the Drosophila melanogaster histone gene repeat unit, by stabilizing denaturation bubbles that map near the boundaries of the genes, at sites that in native chromatin have been shown to be hypersensitive to nucleases. A similar process may contribute to the propagation of such hypersensitive sites after their induction on the activation of gene expression.     

Characterisation of deletions which affect the expression of fetal globin genes in man.

Deletions in the DNA of individuals with hereditary persistence of fetal haemoglobin (HPFH) and 8 beta-thalassaemia have been mapped as a means of identifying regulatory sequences involved in the switch from fetal to adult globin gene expression. The end points of these deletions have been precisely located with respect to restriction endonuclease cleavage sites within and surrounding the gamma-, delta- and beta-globin genes in normal human DNA and the deletion maps were used to obtain definitive evidence for the physical linkage of the fetal and adult beta-like globin genes in the order 5'Ggamma-Agamma-delta-beta 3'. Correlation of haematological data and the location of deletions in two cases of HPFH and one case of deltabeta-thalassaemia suggest that a region of DNA located near the 5'-end of the delta-globin gene may be involved in the suppression in cis of gamma-globin gene expression in adults. The interpretation of a second case of deltabeta-thalassaemia is complicated by the fact that the deletion removes the Agamma-gene in addition to the region near the 5'-end of the delta-globin gene.     

利用Tn5转座子构建杆状病毒AcMNPV随机突变体的初步研究

以杆状病毒模式种AcMNPV为研究对象,应用基于Tn5转座子的随机转座的方法,构建杆状病毒突变体库将果蝇hsp70启动子后接绿色荧光蛋白基因后插入Tn5转座子,构建了可以在昆虫细胞中表达,易于跟踪的转座载体．利用体外转座系统将转座子随机插入AcMNPV基因组,并用转座反应液转染Sf21细胞,得到了表达绿色荧光蛋白的病毒突变体库进一步纯化了两株病毒B9F和Li6A,进行了转座子插入位点的分析,确定两株病毒中,转座子分别插入了94K基因和p10基因．该方法将为杆状病毒功能基因组研究提供重要的手段。     

Unusual sequences in the murine immunoglobulin mu-delta heavy-chain region

The delta heavy (H) chain of mouse immunoglobulin D (IgD) is unusual both in its structure and in its differential expression relative to immunoglobulin M (IgM; reviewed in ref. 1). The region of DNA between IgM and IgD H-chain constant-region genes is probably implicated in this control. So far only fragments of the area have been sequenced. Now, however, we present the complete sequence as well as the sequence of the introns of the C delta gene. We have found several interesting features (Fig. 1), including an open reading frame (ORF) between Cmu and C delta which encodes 146 amino acids that might represent a previously unsuspected domain-like protein; three blocks of simple repetitive sequences; a 162-base pair (bp) unique-sequence inverted repeat; and a domain-like pseudogene in the large intron of C delta. We have not found, however, any sequence 5' of C delta resembling the switch (S) recombination sequences associated with class switching in other heavy chains. Moreover, we have determined the 3' deletion end point of an IgD-producing myeloma and find no sequences reminiscent of switch sites nearby.     

Evolution of the 87A and 87C heat-shock loci in Drosophila

Several species related to Drosophila melanogaster have two loci containing sequences which code for the major heat-shock protein of molecular weight 70,000 (hsp70). These sequences are arranged as two pairs of inverted repeats. The structure of the 87C locus in D. melanogaster seems to have arisen through DNA insertion. Hsp70-coding sequences at the two loci are not evolving independently. Gene conversion between hsp70 genes has apparently occurred both within and between loci.