Nucleosome positioning in the <Emphasis Type="Italic">NPTII</Emphasis> neomycin phosphotransferase gene on a yeast plasmid in the repressed and active states

A yeast plasmid was constructed to contain a hybrid GAL-CYC promoter, the NPTII neomycin phosphotransferase gene, and the FRT sequence between them. The CYC part of the GAL-CYC promoter harbored four upstream activating sequences (UASs) and two close TATA boxes. NPTII was efficiently expressed upon induction with galactose, conferring G418 resistance on yeast cells. Nucleosome positioning was studied in repressed and induced NPTII in transformed cells. A stable positioning of three nucleosomes was detected under repressive conditions (growth on glucose). Two nucleosomes were on the CYC part of the promoter, one including both of the TATA boxes. The third nucleosome overlapped the FRT sequence and the start of the NPTII coding region. Each of the three nucleosomes displayed multiple positions, suggesting their sliding along DNA. After induction of NPTII expression with galactose, a sliding of two nucleosomes was detected, exposing the TATA box and a long promoter segment. The 5′-distal nucleosome moved closer to the UASs, bringing them closer to the TATA box, which was assumed to facilitate the assembly of the preinitiation complex. The two nucleosomes slid independently of each other. The second nucleosome moved towards the FRT sequence and repositioned at its nucleosome positioning signal. Galactose-induced expression did not affect the nucleosome positioning in the coding region of NPTII. Unidirectional sliding and repositioning were detected without induction after deacetylase inhibition with trichostatin A. Basal NPTII expression was observed without activation of the GAL-CYC promoter and after a spatial uncoupling of the coding sequence and promoter via gene inversion and was probably driven by the FRT TATA-like element, which is in the region permanently exposed in vivo.     

Function-based selection and characterization of base-pair polymorphisms in a promoter of Escherichia coli RNA polymerase-sigma(70)

We performed two sets of in vitro selections to dissect the role of the -10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-sigma(70) forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus -10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.     

Identification of the tobacco and Arabidopsis homologues of the pollen-expressed LAT59 gene of tomato

We describe the complete genomic sequences for the tobacco and Arabidopsis homologues of tomato LAT59, a previously described member of a family of pectate lyase-like genes. Translation of the tobacco gene, Nt59, predicts a protein with 93.5% overall amino acid similarity to LAT59. Nt59 has two introns whose positions are exactly conserved with the two introns of LAT59. Both LAT59 and Nt59 are specifically expressed in pollen and their promoter and 5-UTR sequences are highly similar. Furthermore, two promoter elements shown to be important for pollen expression of LAT59 are conserved in the Nt59 promoter. The Arabidopsis homologue, At59, was found by examination of four candidates. At59 has 72.6% amino acid similarity to LAT59 and the position of one of its two introns is conserved with one of the LAT59 introns. At59 is also pollen-expressed and although its promoter sequence is quite different from the Nt59 and LAT59 promoters, the two promoter elements are somewhat conserved.     

Analysis of the distribution of the nucleotide sequence and electrostatic potential of the Escherichia coli genome

The oligonucleotide composition of the E. coli genome and its sigma70-specific promoters has been analyzed. The promoter DNA was shown to contain mainly AT-rich hexanucleotides having functionally important physical properties such as the ability to form easily melting sites and induce the bending of the double helix. A comparative analysis of the electrostatic characteristics of hexanucleotides within the whole sequence of the E. coli genome and its promoter regions was made. Hexanucleotides possessing a more electronegative surrounding were found to predominate in the nucleotide sequence of the promoter DNA.     

Structure and Evolution of the Adh Genes of Drosophila Mojavensis

The nucleotide sequence of the Adh region of Drosophila mojavensis has been completed and the region found to contain a pseudogene, Adh-2 and Adh-1 arranged in that order. Comparison of the sequence divergence of these genes to one another and to the Adh region of Drosophila mulleri and other species has allowed the development of a model for the evolution of the duplication of the Adh genes. There have been two major events. An initial duplication of an Adh gene whose dual promoter structure was similar to Drosophila melanogaster, resulted in a species with two Adh genes, one of which may have had only a proximal promoter. A second duplication of this gene generated an Adh region containing three genes. It is proposed that one of these is the ancestral gene having dual promoters, while the other two possess only proximal promoters. Subsequent events have resulted in both a change in the regulation of Adh-2 such that it is expressed as if it had a "distal" type promoter and the mutational inactivation of the most upstream gene resulting in the creation of a pseudogene. The sequence of the D. mojavensis Adh region has also revealed the presence of an element which is composed of juxtaposed inverted imperfectly repeated elements. There is a surprising and not fully explainable strong similarity of the nucleotide sequence of the 5' flanking region of the pseudogene in D. mojavensis and D. mulleri.     

Escherichia coli promoters. I. Consensus as it relates to spacing class, specificity, repeat substructure, and three-dimensional organization

Fifty-two of the best characterized Escherichia coli promoters in the Hawley and McClure [1983) Nucleic Acids Res. 8, 2237-2255) listing were used to determine the distribution of information content in promoters and to describe the basic features underlying the existence of several different promoter spacing classes, which are defined by the number of bases separating the -35 and -10 regions. The contact regions at -35 and -10 do not, on the average, contain sufficient information to specify a promoter. The search for additional specifying bases led to two conclusions: 1) the consensus nucleotide sequence in the noncontact regions of a promoter appears to be distinct for each of the major promoter spacing classes; 2) promoters appear to contain a 15-20 base subset of the 40-50 additional optimal noncontact bases. This improved view of the extended consensus sequence allows the detection of a 10-base degenerate palindrome which may be the basic unit of promoter structure. Contiguous direct repeats of this sequence produce a sequence closely related to the consensus for the 18-base pair spacing class. This underlying structure is also evidenced in the 17- and 16-base pair spacing classes; however, the start points of the fourth and subsequent repetitions of the sequence element are moved one and two bases upstream, respectively, relative to their location in the 18-base pair spacing class. These consensus sequences, when viewed in a helical format, all present the opportunity for two alternative sets of a dyad repeat. The -35 region is common to both sets and is paired with an extended -10 region in one set and with a pseudo-10 region in the other. Possible implications of these arrangements are discussed.     

Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.

The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P-450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys-Cys/His-His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV-1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus-1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the human gene encoding the invariant gamma-chain of class II histocompatibility antigens

The primary structures of a cDNA and the genomic DNA of a gene selectively expressed in chronic lymphocytic leukemia were determined. A computer search of the nucleotide sequence data bank identified this gene as the invariant gamma-chain associated with class II histocompatibility antigens. The invariant gamma-chain genomic sequence spans about 11 kilobases, with eight exons and seven introns. Three of the introns contain members of the Alu repeat family. A putative cap site and promoter sequence were identified at the 5' end of the gene. One or two copies of the gene is present in each haploid genome, and no evidence for amplification or polymorphism was obtained.     

Sequence of the promoter and 5'' coding region of pepN, and the amino-terminus of peptidase N from Escherichia coli K-12

Summary The pepN gene of Escherichia coli K-12 has been cloned onto a multi-copy plasmid and shown to encode a polypeptide which co-migrates with purified peptidase N. Transformed strains have been shown to contain up to a one hundred fold increase in the amount of peptidase N. We isolated the peptidase N protein and determined the sequence of its first 15 amino acids. By restriction mapping, we identified and subcloned the 5 region of the pepN gene and then determined its nucleotide sequence. Comparison of the actual amino acid sequence with that predicted from the extended open reading frame found in the DNA sequence indicated that peptidase N is not synthesized as a pre-protein precursor. The presumed region preceding the open reading frame contained nucleotide sequence having homology to the procaryotic promoter consensus sequences for the -35 and the -10 regions and the ribosome binding site.