Localization of Escherichia coli RNA polymerase-binding sites on bacteriophage S13 replicative form I DNA by protection of restriction enzyme cleavage sites

Protection of restriction endonuclease cleavage sites by Escherichia coli RNA polymerase bound to the replicative form I of bacteriophage S13 DNA has been used to identify a number of regions of RNA polymerase binding. Digestion with HincII, AluI, HinfI, or HaeIII, under conditions optimized for "open" complex formation, revealed 12 regions of RNA polymerase binding. Based on differential salt sensitivities, five of the regions were classified as strong or tight binding sites. These were located before genes A (two sites), B, and D and at the 5' end of gene F. The seven regions which exhibited weaker binding were located at the 5' end of gene C (two sites), in the middle of gene D, just before and at the 3' end of gene F, at the 5' end of gene G, and in the middle of gene H. The sites before genes B and D coincide with sites previously identified as promoters in bacteriophage phi X174. One of the sites before gene A, that at nucleotides 5175-5211, represents a new putative promoter site in bacteriophage S13 and phi X174 located before the previously identified A gene promoter at nucleotides 10-45.     

The human H2A and H2B histone gene complement

Sequences and expression patterns of newly isolated human histone H2A and H2B genes and the respective proteins were compared with previously isolated human H2A and H2B genes and proteins. Altogether, 15 human H2A genes and 17 human H2B genes have been identified. 14 of these are organized as H2A/H2B gene pairs, while one H2A gene and three H2B genes are solitary genes. Two H2A genes and two H2B genes turned outto be pseudogenes. The 13 H2A genes code for at least 6 different amino acid sequences, and the 15 H2B genes encode 11 different H2B isoforms. Each H2A/H2B gene pair is controlled by a divergent promoter spanning 300 to 330 nucleotides between the coding regions of the two genes. The highly conserved divergent H2A/H2B promoters can be classified in two groups based on the patterns of consensus sequence elements. Group I promoters contain a TATA box for each gene, two Oct-1 factor binding sites, and three CCAAT boxes. Group II promoters contain the same elements as group I promoters and an additional CCAAT box, a binding motif for E2F and adjacent a highly conserved octanucleotide (CACAGCTT) that has not been described so far. Five of the 6 gene pairs and 4 solitary genes with group I promoters are localized in the large histone gene cluster at 6p21.3-6p22, and one gene pair is located at 1q21. All group II promoter associated genes are contained within the histone gene subcluster at D6S105, which is located at a distance of about 2 Mb from the major subcluster at 6p21.3-6p22 containing histone genes with group I promoters. Almost all group II H2A genes encode identical amino acid sequences, whereas group I H2A gene products vary at several positions. Using human cell lines, we have analyzed the expression patterns of functional human H2A/H2B gene pairs organized within the two histone gene clusters on the short arm of chromosome 6. The genes show varying expression patterns in different tumor cell lines.     

Localization of Escherichia coli RNA polymerase binding sites on bacteriophage S13 and 0X174 DNAs by electron microscopy.

Complexes between Escherichia coli RNA polymerase and bacteriophage S13 and phage phiX174 replicative form III DNAs have been shown to form at specific locations on the phage genomes. The major locations on S13 have been mapped at 8 to 10 and 92 to 96% of the genome length, starting from the unique Pst I cleavage site. The locations correspond to the beginnings of genes D and B, respectively. Four minor locations map at 18 to 22, 28 to 32, 50 to 56, and 70 to 74% of the genome. The 70 to 74% site corresponds to the beginning of the A gene. The major locations on phiX174 are at 8 to 10, 50 to 54, and 92 to 94% of the genome. The 50 to 54% site is at the start of the H gene and has an equivalent minor site on S13, but it is not a promoter site. Three minor sites on phiX174, at 20 to 24, 26 to 32, and 68 to 74% of the genome, correspond to sites on S13. The data confirm the locations of sites identified by restriction fragment binding experiments (E. Rassart and J. H. Spencer, J. Virol. 27:677--687, 1978) and the assignment of putative promoters at the start of genes A, B and D.     

Identification and characterization of a chicken alpha-globin enhancer.

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.     

Major cis-regulatory elements for rice bidirectional promoter activity reside in the 5′-untranslated regions

Bidirectional promoters are defined as those that regulate adjacent genes organized in a divergent fashion (head to head orientation) and separated by < 1 kb. In order to dissect bidirectional promoter activity in a model plant, deletion analysis was performed for seven rice promoters using promoter-reporter gene constructs, which identified three promoters to be bidirectional. Regulatory elements located in or close to the 5′-untranslated regions (UTR) of one of the genes (divergent gene pair) were found to be responsible for their bidirectional activity. DNA footprinting analysis identified unique protein binding sites in these promoters. Deletion/alteration of these motifs resulted in significant loss of expression of the reporter genes on either side of the promoter. Changes in the motifs at both the positions resulted in a remarkable decrease in bidirectional activity of the reporter genes flanking the promoter. Based on our results, we propose a novel mechanism for the bidirectionality of rice bidirectional promoters.     

Tandem CRP binding sites in the deo operon of Escherichia coli K-12

The locations of DNA binding by the cyclic AMP receptor protein (CRP) in the deo operon of Escherichia coli have been determined by the DNase I footprinting procedure. Two high affinity sites were found around positions -35 and -90, preceding the second deo promoter. In vitro data on induction of gene fusions that join different parts of the deoP -2 regulatory region to the lac genes suggest that: (1) both CRP binding sites are needed for high expression from the deoP -2 region; and (2) negative regulation by the cytR repressor is accomplished by preventing the cAMP-CRP complex from binding to the second target.     

Differential DNA binding properties of three human homeodomain proteins.

The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes.     

Possible promoter regions within the proteolytic system in Streptococcus thermophilus and their interaction with the CodY homolog

Possible promoter regions preceding 14 genes belonging to the proteolytic system of Streptococcus thermophilus KLDS 3.0503 were predicted by a promoter analysis software nnpp . The 14 genes included an extracellular protease gene prtS , an oligopeptide ABC transport system gene amiA1 , and 12 genes, respectively, encoding peptidases pepA, pepS , pepN, pepC , pepB, pepQ , pepV, pepT , pepM, pepXP , pepP , and pepO . These predicted promoter sequences were cloned and inserted into the upstream of a promoterless Escherichia coli gusA (β-glucuronidase) gene in a promoter probe vector pNZ273. The resulting vectors were, respectively, introduced into S. thermophilus KLDS 3.0503 and all 14 predicted promoter sequences were able to drive gusA expression, which indicated that these sequences were functional promoters. These promoters were able to interact with the S. thermophilus CodY homolog in an in vitro DNA binding assay but they did not contain a conserved CodY-box sequence identified in Lactococcus lactis . These results were useful for further studies on the regulation of protein metabolism in S. thermophilus .     

Identification of protein-binding DNA sequences in an auxin-regulated gene of soybean

The promoter region of a soybean auxin-responsive gene, GmAux28, was analyzed to identify protein-binding DNA sequences that may be involved in regulation of expression. Using DNase I footprinting and gel mobility shift assays, multiple regions of interaction, including eight major protein-binding sites, were observed in the GmAux28 gene. Two sequence motifs, TGACGACA and TCCACGTGTC, related to as-1/Hex and G-box elements, respectively, found in several plant promoters, were identified. Four distinct A/T-rich domains were identified; such A/T-rich domains appear to modulate, but not to specify, the expression of many genes. Two new sequence motifs, delta-1 (D1) and delta-4 (D4) were also identified. D1 and D4 share a very similar core sequence, TAGTxxCTGT and TAGTxCTGT, respectively. In gel mobility shift analyses, D1 and D4 elements exhibit a complex interaction of binding proteins. The GmAux22 promoter also contains D1-related elements which compete with the GmAux28 elements. Sequence comparisons have identified D1/D4-like sequences in several other auxin-responsive genes suggesting the possible importance of D1/D4 and the respective binding proteins in the regulation of expression of these genes.