scholarly journals DNA sequence determinants of lambda repressor binding in vivo.

Genetics ◽  
1988 ◽  
Vol 118 (1) ◽  
pp. 21-29
Author(s):  
N Benson ◽  
P Sugiono ◽  
P Youderian
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Operator Function ◽  
Wild Type ◽  
Base Pairs ◽  
Lambda Repressor ◽  
Half Site ◽  
Better Than

Abstract The critical operator determinants for lambda repressor recognition have been defined by analyzing the binding of wild-type repressor to a set of mutant operators in vivo. Base pair substitutions at six positions within the lambda operator half-site impair binding severely, and define these base pairs as critical for operator function. One mutant operator binds repressor better than the consensus operator, and is a superoperator. The model proposed by M. Lewis in 1983 for the binding of lambda repressor to its operator accurately predicts the observed operator requirements for binding in vivo, with several minor exceptions. The order of affinities of the six natural lambda operators has also been determined.

scholarly journals Phage lambda Cro protein and cI repressor use two different patterns of specific protein-DNA interactions to achieve sequence specificity in vivo.

Genetics ◽  
1989 ◽  
Vol 121 (1) ◽  
pp. 5-12
Author(s):  
N Benson ◽  
P Youderian
Keyword(s):  
Specific Protein ◽  
Specific Interactions ◽  
Sequence Specificity ◽  
Base Pairs ◽  
Lambda Repressor ◽  
Protein Dna Interactions ◽  
Cro Protein ◽  
Data Call ◽  
Half Site

Abstract By assaying the binding of wild-type Cro to a set of 40 mutant lambda operators in vivo, we have determined that the 14 outermost base pairs of the 17 base pair, consensus lambda operator are critical for Cro binding. Cro protein recognizes 4 base pairs in a lambda operator half-site in different ways than cI repressor. The sequence determinants of Cro binding at these critical positions in vivo are nearly perfectly consistent with the model proposed by W. F. ANDERSON, D. H. OHLENDORF, Y. TAKEDA and B. W. MATTHEWS and modified by Y. TAKEDA, A. SARAI and V. M. RIVERA for the specific interactions between Cro and its operator, and explain the relative order of affinities of the six natural lambda operators for Cro. Our data call into question the idea that lambda repressor and Cro protein recognize the consensus lambda operator by nearly identical patterns of specific interactions.

scholarly journals Supercoiling DNA locates mismatches

2017 ◽  
Author(s):  
Andrew Dittmore ◽  
Sumitabha Brahmachari ◽  
Yasuhara Takagi ◽  
John F. Marko ◽  
Keir C. Neuman
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Relative Probability ◽  
Base Pairs ◽  
Damage Sensing ◽  
Mismatched Base Pair ◽  
Monovalent Salt

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

scholarly journals Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448 ◽  
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

scholarly journals HOMOLOGOUS RECOMBINATION IN ESCHERICHIA COLI: DEPENDENCE ON SUBSTRATE LENGTH AND HOMOLOGY

Genetics ◽  
1986 ◽  
Vol 112 (3) ◽  
pp. 441-457 ◽  
Author(s):  
Ping Shen ◽  
Henry V Huang
Keyword(s):  
Dna Sequences ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
In Vivo Recombination ◽  
Recombinant Frequency ◽  
Genetic Length ◽  
Efficient Processing ◽  
Low Levels

ABSTRACT We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC  - cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.

Influence of Helix Length on Cleavage Efficiency of Hammerhead Ribozymes

2005 ◽  
Vol 58 (12) ◽  
pp. 851 ◽  
Author(s):  
Philip Hendry ◽  
Maxine J. McCall ◽  
Trevor J. Lockett
Keyword(s):  
Optimal Design ◽  
Base Pair ◽  
Hammerhead Ribozymes ◽  
Base Pairs ◽  
Physiological Conditions ◽  
Rna Transcripts ◽  
Cleavage Efficiency ◽  
Double Helices

The cleavage rates of RNA substrates by trans-acting, hammerhead ribozymes are controlled by interactions between helices I and II. The interactions are affected by the relative lengths of these two double helices and by unpaired nucleotides protruding beyond helix I, either in the substrate or the ribozyme strand. Maximum cleavage rates are observed for ribozyme–substrate complexes with three or more base pairs in helix II and six or less base pairs in helix I. However, for these helix combinations, rates fall sharply with unpaired nucleotides at the end of helix I. Cleavage rates by ribozymes with one or two base pairs in helix II increase as helix I is lengthened, and are unaffected by unpaired nucleotides on the end. Since miniribozymes, with one base pair in helix II, efficiently cleave long RNA transcripts under physiological conditions, they represent the optimal design for the simple hammerheads for application in vivo.

ADR1-mediated regulation of ADH2 requires an inverted repeat sequence

1986 ◽  
Vol 6 (6) ◽  
pp. 1894-1902
Author(s):  
J Shuster ◽  
J Yu ◽  
D Cox ◽  
R V Chan ◽  
M Smith ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Regulatory Protein ◽  
Active Form ◽  
Growth Conditions ◽  
Repeat Sequence ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Mutant Genes ◽  
Latent Activity

DNA sequence analysis of wild-type and mutant ADH2 loci suggested that two unusual features 5' of the promoter, a 22-base-pair perfect dyad sequence and a (dA)20 tract, were important for regulation of this gene (D. W. Russell, M. Smith, D. Cox, V. M. Williamson, and E. T. Young, Nature [London] 304:652-654, 1983). Oligonucleotide-directed mutagenesis was used to construct ADH2 genes lacking the 22-base-pair dyad or the (dA)20 tract (V.-L. Chan and M. Smith, Nucleic Acids Res. 12:2407-2419, 1984). These mutant genes and other ADH2 deletions constructed by BAL 31 endonuclease digestion were studied after replacing the wild-type chromosomal locus with the altered alleles by the technique of gene transplacement (T. L. Orr-Weaver, J. W. Szostak, and R. S. Rothstein, Proc. Natl. Acad. Sci. USA 78:6354-6358, 1981), using canavanine resistance as the selectable marker. Deletions lacking the dyad failed to derepress normally and did not respond to mutations at the ADR1 locus, which encodes a protein necessary to activate ADH2. Deletions of the (dA)20 tract did not have a detectable phenotype. A small deletion located just 3' to the (dA)20 tract (between positions -164 and -146) had a low amount of ADR1-dependent transcription during repressed growth conditions, indicating that the regulatory protein encoded by ADR1 is present in a potentially active form during repression and that alterations of a DNA sequence in the promoter region can unmask its latent activity.

Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

scholarly journals ADR1-mediated regulation of ADH2 requires an inverted repeat sequence.

1986 ◽  
Vol 6 (6) ◽  
pp. 1894-1902 ◽  
Author(s):  
J Shuster ◽  
J Yu ◽  
D Cox ◽  
R V Chan ◽  
M Smith ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Regulatory Protein ◽  
Active Form ◽  
Growth Conditions ◽  
Repeat Sequence ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Mutant Genes ◽  
Latent Activity

DNA sequence analysis of wild-type and mutant ADH2 loci suggested that two unusual features 5' of the promoter, a 22-base-pair perfect dyad sequence and a (dA)20 tract, were important for regulation of this gene (D. W. Russell, M. Smith, D. Cox, V. M. Williamson, and E. T. Young, Nature [London] 304:652-654, 1983). Oligonucleotide-directed mutagenesis was used to construct ADH2 genes lacking the 22-base-pair dyad or the (dA)20 tract (V.-L. Chan and M. Smith, Nucleic Acids Res. 12:2407-2419, 1984). These mutant genes and other ADH2 deletions constructed by BAL 31 endonuclease digestion were studied after replacing the wild-type chromosomal locus with the altered alleles by the technique of gene transplacement (T. L. Orr-Weaver, J. W. Szostak, and R. S. Rothstein, Proc. Natl. Acad. Sci. USA 78:6354-6358, 1981), using canavanine resistance as the selectable marker. Deletions lacking the dyad failed to derepress normally and did not respond to mutations at the ADR1 locus, which encodes a protein necessary to activate ADH2. Deletions of the (dA)20 tract did not have a detectable phenotype. A small deletion located just 3' to the (dA)20 tract (between positions -164 and -146) had a low amount of ADR1-dependent transcription during repressed growth conditions, indicating that the regulatory protein encoded by ADR1 is present in a potentially active form during repression and that alterations of a DNA sequence in the promoter region can unmask its latent activity.

scholarly journals Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 158-167 ◽  
Author(s):  
E Yeh ◽  
J Carbon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Transcriptional Activity ◽  
Intragenic Recombination ◽  
Wild Type ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linked Gene ◽  
Mutant Cells

We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the functional centromere region. We examined the cellular function of one of these tightly centromere-linked transcripts. (CEN11)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell. Diploids heterozygous for the (CEN11)L disruption sporulated at wild-type levels, and the absence of the (CEN11)L gene product had no effect on the viability or mitotic growth of haploid cells. Diploids homozygous for the (CEN11)L disruption were unable to sporulate when induced by the appropriate nutritional cues. The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage. The temporal ordering of (CEN11)L function with respect to the sporulation mutant spo13 suggests that the (CEN11)L gene product may be required at both the first and second meiotic cell divisions. This new sporulation gene has been termed SPO15.

scholarly journals Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (9) ◽  
pp. 2247-2256 ◽  
Author(s):  
L Mathison ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Base Pair ◽  
Sequence Data ◽  
Induced Mutations ◽  
Coding Region ◽  
Base Pairs ◽  
Sequence Recognition ◽  
Frameshift Suppression ◽  
Target Sites

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation. Long consecutive G-C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.

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