scholarly journals A short 5'-flanking region mediates glucose repression of amylase gene expression in Drosophila melanogaster.

Genetics ◽  
1993 ◽  
Vol 134 (2) ◽  
pp. 507-515 ◽  
Author(s):  
C Magoulas ◽  
L Bally-Cuif ◽  
A Loverre-Chyurlia ◽  
B Benkel ◽  
D Hickey
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Glucose Repression ◽  
Essential Elements ◽  
Site Directed Mutagenesis ◽  
Deletion Analysis ◽  
Upstream Region ◽  
Directed Mutagenesis ◽  
Amylase Gene ◽  
Recombinant Gene

Abstract Expression of the alpha-amylase gene is highly repressed by dietary glucose in Drosophila melanogaster larvae. Here, we show that glucose repression is controlled by DNA sequences that are located upstream of the transcribed region. Recombinant gene constructions, in which the amylase promoter sequences were fused with the transcribed region of the Adh gene, were expressed in transgenic Drosophila larvae. The expression of ADH from the recombinant gene was shown to be subject to glucose repression. The function of potential regulatory cis-acting elements within the glucose responsive upstream region was examined by deletion analysis and by site-directed mutagenesis, coupled with expression assays in transformed larvae. The upstream deletion analysis showed that essential elements, both for overall activity and for glucose repression of the amylase gene, are located within a 109-bp region upstream of the transcription start site. Site-directed mutagenesis of these upstream sequences showed that the TATA motif, at position -31, and a novel 36-bp element, at position -109, were necessary for full activity of the amylase promoter. None of the introduced mutations resulted in loss of glucose responsiveness. These results indicate that glucose repression, in Drosophila, is mediated by transcriptional mechanisms that involve multiple, functionally redundant DNA elements.

Activation Control of pur Gene Expression inLactococcus lactis: Proposal for a Consensus Activator Binding Sequence Based on Deletion Analysis and Site-Directed Mutagenesis of purC and purD Promoter Regions

1998 ◽  
Vol 180 (15) ◽  
pp. 3900-3906 ◽  
Author(s):  
Mogens Kilstrup ◽  
Stine G. Jessing ◽  
Stephanie B. Wichmand-Jørgensen ◽  
Mette Madsen ◽  
Dan Nilsson
Keyword(s):  
Site Directed Mutagenesis ◽  
Deletion Analysis ◽  
Directed Mutagenesis ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Similar Region ◽  
Low Levels ◽  
High Level ◽  
Transcriptional Start Sites ◽  
Binding Sequence

ABSTRACT A comparison of the purC and purD upstream regions from Lactococcus lactis revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between −79 and −70 nucleotides upstream from the transcriptional start sites. Both promoters have well-defined −10 regions but lack sequences resembling −35 regions for ς70 promoters. Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation. Adjacent to the conserved sequence in purC is a second and similar region required for high-level expression of the gene. A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions. By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at thepurC and purD promoters. Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important. All results support the idea thatpurC and purD transcription is regulated by a transcriptional activator binding to the PurBox sequence.

scholarly journals THE INTERACTION OF GENETIC AND ENVIRONMENTAL FACTORS IN THE CONTROL OF AMYLASE GENE EXPRESSION IN DROSOPHILA MELANOGASTER

Genetics ◽  
1986 ◽  
Vol 114 (3) ◽  
pp. 943-954
Author(s):  
Bernhard F Benkel ◽  
Donal A Hickey
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Environmental Factors ◽  
Molecular Basis ◽  
Amylase Activity ◽  
Glucose Repression ◽  
Amylase Gene ◽  
Dietary Effects ◽  
Eukaryotic Gene ◽  
Genetic And Environmental Factors

ABSTRACT A number of previous studies have established that amylase activity can vary between Drosophila strains which are maintained under identical laboratory conditions. In addition, we have recently shown that all strains examined so far are subject to glucose repression of amylase activity. In this study, we show that the degree of glucose repression can vary between strains. Moreover, the glucose repression effect is much more pronounced in larvae than in adult flies. Our results lead to the conclusion that the strain-specific differences in activity and the dietary effects are not independent phenomena. These results have implications for the interpretation of many studies on amylase activity variation, including those experiments which have been designed to link amylase activity variations with fitness differences in nature. A question that naturally arises concerns the molecular basis for these strain-specific variations in the degree of glucose repression of this eukaryotic gene.

scholarly journals DNA Gap Repair-Mediated Site-Directed Mutagenesis is Different from Mandecki and Recombineering Approaches

2018 ◽  
Author(s):  
George T. Lyozin ◽  
Luca Brunelli
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Products ◽  
E Coli ◽  
Health And Disease ◽  
First Time ◽  
Disease Understanding

AbstractSite-directed mutagenesis allows the generation of mutant DNA sequences for downstream functional analysis of genetic variants involved in human health and disease. Understanding the mechanisms of different mutagenesis methods can help select the best approach for specific needs. We compared three different approaches for in vivo site-directed DNA mutagenesis that utilize a mutant single-stranded DNA oligonucleotide (ssODN) to target a wild type DNA sequence in the host Escherichia coli (E. coli). The first method, Mandecki, uses restriction nucleases to introduce a double stranded break (DSB) into a DNA sequence which needs to be denatured prior to co-transformation. The second method, recombineering (recombination-mediated genetic engineering), requires lambda red gene products and a mutant ssODN with homology arms of at least 20 nucleotides. In a third method described here for the first time, DNA gap repair, a mutant ssODN targets a DNA sequence containing a gap introduced by PCR. Unlike recombineering, both DNA gap repair and Mandecki can utilize homology arms as short as 10 nucleotides. DNA gap repair requires neither red gene products as recombineering nor DNA denaturation or nucleases as Mandecki, and unlike other methods is background-free. We conclude that Mandecki, recombineering, and DNA gap repair have at least partly different mechanisms, and that DNA gap repair provides a new, straightforward approach for effective site-directed mutagenesis.

scholarly journals Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension

BioTechniques ◽  
2020 ◽  
Vol 68 (6) ◽  
pp. 345-348
Author(s):  
Rasmus Hejlesen ◽  
Ernst-Martin Füchtbauer
Keyword(s):  
Dna Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Overlap Extension

We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.

Thermostable mutant variants of Bacillus sp. 406 α-amylase generated by site-directed mutagenesis

2013 ◽  
Vol 8 (4) ◽  
pp. 346-356 ◽  
Author(s):  
Alexandr Kachan ◽  
Anatoliy Evtushenkov
Keyword(s):  
Significant Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Bacillus Sp ◽  
Wild Type ◽  
Amylase Gene ◽  
Wild Type Enzyme ◽  
Triple Mutant ◽  
Heat Stable

AbstractSeveral mutations are known to increase the thermostability of α-amylase of B. licheniformis and other α-amylases. Site-directed mutagenesis was used to introduce similar mutations into the sequence of the α-amylase gene from mesophilic Bacillus sp. 406. The influence of the mutations on thermostability of the enzyme was studied. It was shown that the Gly211Val and Asn192Phe substitutions increased the half-inactivation temperature (Tm) of the enzyme from 51.94±0.45 to 55.51±0.59 and 58.84±0.68°C respectively, in comparison to the wild-type enzyme. The deletion of Arg178-Gly179 (dRG) resulted in an increase of Tm of the α-amylase to 71.7±1.73°C. The stabilising effect of mutations was additive. When combined they increase the Tm of the wild-type amylase by more than 26°C. Thermostability rates of the triple mutant are close to the values which are typical for industrial heat-stable α-amylases, and its ability to degrade starch at 75°C was considerably increased. The present research confirmed that the Gly211Val, Asn192Phe and dRG mutations could play a significant role in thermostabilization of both mesophilic and thermophilic α-amylases.

scholarly journals Differences in the Enterococcus faecalis lsa Locus That Influence Susceptibility to Quinupristin-Dalfopristin and Clindamycin

2005 ◽  
Vol 49 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Kavindra V. Singh ◽  
Barbara E. Murray
Keyword(s):  
Enterococcus Faecalis ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Upstream Region ◽  
Directed Mutagenesis ◽  
Moderate Increase ◽  
Gene Encoding ◽  
Phenotypic Differences ◽  
Abc Protein ◽  
Resistance Expression

ABSTRACT We have previously shown that the Enterococcus faecalis lsa gene, encoding the putative ABC protein Lsa, influences resistance to quinupristin-dalfopristin (Q-D) and clindamycin (CLI). We have now found that, while cloned lsa from E. faecalis strain V583 (lsa V) fully restored resistance to Q-D, CLI, and dalfopristin (DAL) lost by the OG1 lsa disruption mutant TX5332 and also caused increased MICs for Lactococcus lactis LM2301, cloned lsa from OG1 (lsa OG) did not cause any increase in MICs for either species. Sequencing of ca. 2 kb of these two lsa alleles found differences between lsa OG and lsa V in the upstream region as well as in the 5′ and 3′ halves of the lsa gene. To investigate the reason for the phenotypic differences expressed by the two cloned loci, 5′ half plus 3′ half hybrid constructs were created. When introduced into both TX5332 and L. lactis, cloned lsa V5 ′ OG3 ′ conferred increases in MICs of Q-D, CLI, and DAL similar to those of cloned lsa V while cloned lsa OG5 ′ V3 ′ showed a moderate increase in MICs relative to those of lsa OG, indicating that both halves of the locus can influence resistance expression. After site-directed mutagenesis of the cloned lsa alleles at positions −131 and −133 (relative to the putative Lsa start codon ATG), which converted two A's of lsa V to the G and T of lsa OG and vice versa, MIC testing showed that mutagenized lsa OG (lsa OG-M) was strongly influenced by these changes in terms of conferring increased MICs of Q-D, CLI, and DAL relative to lsa OG while the phenotype of mutagenized lsa V (lsa V-M) was less influenced, with moderately decreased MICs, primarily to CLI, relative to lsa V. In conclusion, this study found that changes in different regions of the E. faecalis lsa locus influence the ability of cloned lsa to confer resistance to Q-D, CLI, and DAL.

scholarly journals Autolytic activation and localization in Schneider cells (S2) of calpain B from Drosophila

2004 ◽  
Vol 378 (2) ◽  
pp. 299-305 ◽  
Author(s):  
Attila FARKAS ◽  
Peter TOMPA ◽  
Éva SCHÁD ◽  
Rita SINKA ◽  
Gáspár JÉKELY ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
In Situ Hybridization ◽  
Developmental Stages ◽  
Terminal Segment ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
S2 Cells

Calpain B is one of the two calpain homologues in Drosophila melanogaster that are proteolytically active. We studied its activation by Ca2+ both in vitro and in vivo, in Schneider (S2) cells. Activation involves the autolytic cleavage, at two major sites, of the N-terminal segment, the length of which was earlier underestimated. Site-directed mutagenesis at the autolytic sites did not prevent autolysis, but only shifted its sites. Calpain B mRNA was detectable in all developmental stages of the fly. In situ hybridization and immunostaining showed expression in ovaries, embryo and larvae, with high abundance in larval salivary glands. In S2 cells, calpain B was mainly in the cytoplasm and upon a rise in Ca2+ the enzyme adhered to intracellular membranes.

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