Elements of the maize A1 promoter required for transactivation by the anthocyanin B/C1 or phlobaphene P regulatory genes.

The extensive genetic and molecular characterization of the flavonoid pathway's structural and regulatory genes has provided some of the most detailed knowledge of gene interactions in plants. In maize flavonoid biosynthesis, the A1 gene is independently regulated in the anthocyanin and phlobaphene pathways. Anthocyanin production requires the expression of the C1 or PI and R or B regulatory genes, whereas phlobaphene production requires only the P regulatory gene. By deletion analysis of the A1 promoter, we show that the sequences between -123 and -88 are critical for activation by anthocyanin and phlobaphene regulatory genes. Linker-scanner mutations indicated that the -123 to -100 region is more important for transactivation by the P protein. The -98 to -88 region is more important for B/C1 transactivation and shows a strong homology with the region of the Bz1 anthocyanin structural gene promoter shown to be activated by B/C1 and not by P. We identified a 14-bp consensus sequence that is also present in the promoters of three other genes in the anthocyanin pathway, and we propose a model for how the flavonoid regulatory proteins interact with the promoters of the structural genes.     

灰色链霉菌启动子活性片段在大肠杆菌中的亚克隆及其序列分析

通过对重组质粒No.8-1的亚克隆,灰色链霉菌插入到大肠杆菌载体质粒中的序列已缩小到410bp,仍具有启动子功能。序列分析表明,启动子活性片段的G C碱基组成为50.5%。内含链霉菌启动子区域常有的正向重复序列;有1个Alul位点,1个Clal位点,2个Mbol位点,3个Nla Ⅲ位点,1个Pvu Ⅱ位点;具有类似于E.coli启动子的保守序列-10区和-35区,两者间隔18bp;在相应位置上分别有一段序列与E.coli的SD序列和在苄铅青链霉菌的SEP(Streptomyces-E.coil-type promoter)序列中存在的保守序列具有一定的相似性。     

Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor.

The promoter region of the pColV-K30-encoded operon specifying biosynthesis and transport of the siderophore aerobactin was subjected to deletion analysis to determine the smallest DNA sequence affording iron regulation of a iucA'-'lacZ gene fusion. A 78-base-pair (bp) region containing the main (P1) promoter retained the character of inducibility under iron starvation. A 250-bp fragment carrying this sequence was examined for protection against DNase I by the Fur protein, the product of a gene (fur) required for negative control of several iron-regulated functions. The DNase I footprints, in the presence of various divalent heavy-metal ions added as corepressors, revealed two contiguous binding sites with different lengths and affinities for Fur. Increased concentrations of the protein appeared to elicit formation of repressor oligomers which bind to the upstream and downstream regions of the P1 promoter in a metal-dependent fashion, but with a presently undefined stoichiometry. The primary site for Fur binding spans 31 bp and contains two overlapping symmetry dyads which share the sequence 5'-TCATT-3'. It also contains extensive homology with a 19-bp consensus sequence for iron-regulated genes as deduced from comparison with the fhuA and fepA putative promoter sequences.     

棉花nodulin-like基因及其启动子的结构特征和遗传表达

用同尾酶反向PCR技术(Ⅱ-PCR)和快速分离目的基因cDNA 5'未知序列方法(RICUP)首次从陆地棉品种Y18(Gossypium arboreum L.Y18)中分离到nodulin-like基因的全长cDNA、DNA和启动子序列.结果表明,该基因全长为2 353 bp,具有5个内含子和6个外显子.cDNA全长1480 bp,含有一个1125 bp的ORF结构,编码375个氨基酸,在GenBank nr数据库中没有与之同源的基因序列,在EST数据库中仅有一条733 bp的亚洲棉纤维EST序列(GenBank登录号:BF271235)与之有92%的同源性.Nodulin-like基因的启动子全长1 969 bp,启动子区域具有Initiator、TATA box、CAAT-like box和富含AT序列等启动子特征序列.Southern blot结果表明:该基因在棉花基因组中有一个拷贝.Northern blot结果表明:该基因在棉花的蕾、花、纤维和铃壳等生殖器官中呈优势表达.本研究有望为改良棉花生殖器官的农艺性状提供靶基因,并为解决目前转基因抗虫棉"前期抗虫性强、后期抗虫性弱"这一生产实际问题提供有效的表达调控元件.     

Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system.

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.     

Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum

The nucleotide sequence of a 2081-bp fragment of Clostridium acetobutylicum DNA containing the adh1 gene was determined. The butanol dehydrogenase gene is referred to as the adh1 gene since it was shown to have activity using butanol and ethanol as substrates. The adh1 gene consisted of 1164 bp and encoded an alcohol dehydrogenase (ADH) enzyme of 388 aa residues with an Mr of 43,274. The adh1 gene was separated from an upstream open reading frame by an intergenic region of 354 bp. No promoter consensus sequences were identified in the intergenic upstream region and the adh1 gene did not appear to be expressed off its own promoter in Escherichia coli. Three separate types of ADH have been recognized. The ADH1 from C. acetobutylicum exhibited 39% homology with the Fe-containing ADH2 from Zymomonas mobilis and 37% homology with the ADH4 from Saccharomyces cerevisiae, but showed little or no homology with the other characterised types of ADH.     

Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes.

The fixA, fixB, fixC, and fixX genes of Rhizobium meliloti 1021 constitute an operon and are required for nitrogen fixation in alfalfa nodules. DNA homologous to the R. meliloti fixABC genes is present in all other Rhizobium and Bradyrhizobium species examined, but fixABC-homologous sequences were found in only one free-living diazotroph, Azotobacter vinelandii. To determine whether the fixABCX genes share sequence homology with any of the 17 Klebsiella pneumoniae nif genes, we determined the entire nucleotide sequence of the fixA, fixB, fixC, and fixX genes and defined four open reading frames that code for polypeptides of molecular weights 31,146, 37,786, 47,288, and 10,937, respectively. Neither DNA nor amino acid sequence homology to the R. meliloti fixA, -B, -C, and -X genes was found in the K. pneumoniae nif operon. The fixX gene contains a cluster of cysteine residues characteristic of ferredoxins and is highly homologous to an Azotobacter ferredoxin which has been shown to donate electrons to nitrogenase. The fixABC operon contains a promoter region that is highly homologous to other nifA-activated promoters. We also found a duplication of the 5' end of the fixABCX operon; a 250-bp region located 520 bp upstream of the fixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.     

Sequence and evolution of rhesus monkey alphoid DNA

Summary Analysis of rhesus monkey alphoid DNA suggests that it arose by tandem duplication of an ancestral monomer unit followed by independent variation within two adjacent monomers (one becoming more divergent than the other) before their amplification as a dimer unit to produce tandem arrays. The rhesus monkey alphoid DNA is a tandemly repeated, 343-bp dimer; the consensus dimer is over 98% homologous to the alphoid dimers reported for baboon and bonnet monkey, 81% homologous to the African green monkey alpha monomer, and less than 70% homologous to the more divergent human alphoid DNAs. The consensus dimer consists of two wings (I and II, 172 and 171 bp, respectively) that are only 70% homologous to each other, but share seven regions of exact homology. These same regions are highly conserved among the consensus sequences of the other cercopithecid alphoid DNAs. The three alpha-protein binding sites reported for African green monkey alpha DNA by F. Strauss and A. Varshavsky (Cell 37: 889–901, 1984) occur in wings I and II, but with one site altered in wing I. Two cloned dimer segments are 98% homologous to the consensus, each containing 8 single-base-pair differences within the 343-bp segment. Surprisingly, 37% of these differences occur in regions that are evolutionarily conserved in the alphoid consensus sequences, including the alpha-protein binding sites. Sequence variation in this highly repetitive DNA family may produce unique nucleosomal architectures for different members of an alphoid array. These unique architectures may modulate the evolution of these repetitive DNAs and may produce unique centromeric characteristics in primate chromosomes.     

Nucleotide sequence of chicken myb proto-oncogene promoter region: detection of an evolutionarily conserved element.

The nucleotide sequence of the chicken myb proto-oncogene putative promoter region was determined and compared with the corresponding sequence of the mouse c-myb gene (1). 118 bp upstream from the initiation codon suggested by Gerondakis and Bishop (2) for the chicken c-myb protein, a 124-bp-long conserved element was found (92% identity in chicken and mouse sequences). Sequences homologous to this element were detected on Southern blots of restricted genomic DNAs from mouse, man, lizard, frog, and carp. No hybridization was observed with Drosophila, yeast, or Escherichia coli DNA. In human DNA, sequences homologous to this element were located at the 5' end of the c-myb gene, i.e. in the same position as in the chicken and mouse genes. Several lines of evidence suggest that the element is not a coding exon of a gene overlapping the c-myb gene. It may be of importance that one of the DNase I-sensitive sites and several c-myb mRNA cap sites localized recently in the mouse c-myb gene (3,4) lie within this region. It is suggested that this evolutionarily conserved element is involved in the regulation of myb proto-oncogene expression in vertebrates.     

Identification of the 12-O-tetradecanoylphorbol-13-acetate-responsive enhancer of the MS gene of the Epstein-Barr virus.

We previously located two 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive enhancers, MSTRE-I and MSTRE-II, in the upstream sequence of the MS gene of Epstein-Barr virus (Liu, Q., and Summers, W.C. (1989) J. Virol. 63, 5062-5068). The core sequence of the MSTRE-I enhancer is now determined to be between -718 and -708 of the upstream sequence of the MS gene. The activity of the enhancer is also sensitive to its immediate surrounding sequence on either side. A single copy of a 30-base pair (bp) fragment containing the MSTRE-I sequence was able to confer TPA responsiveness upon the MS promoter even in the absence of an AP-1 binding site. Multiple tandem copies of this 30-bp fragment, regardless of their relative orientations to each other, could function synergistically to enhance the MS promoter activity. At least two copies of the 30-bp fragment were required to bestow TPA induction upon the thymidine kinase gene promoter of herpes simplex virus type 1. The MSTRE-I sequence could also be bound by a Fos-GCN4 chimeric protein but with an affinity much lower than that between the chimeric protein and the AP-1 binding site. This MSTRE-I region has strong homology to one of the TPA-responsive elements (the ZII domain) in the upstream sequence of the EBV BZLF1 gene. In addition, a putative negative regulatory region or silencer was found immediately downstream of the MSTRE-I enhancer. This potential silencer region contains a 14-bp sequence that is homologous to the silencer consensus sequence of the BZLF1 gene. Therefore, the regulation of the MS gene may share the same pathway with the immediate early gene BZLF1.