scholarly journals A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

Genetics ◽  
1987 ◽  
Vol 117 (1) ◽  
pp. 5-12
Author(s):  
Eric Alani ◽  
Nancy Kleckner
Keyword(s):  
Gene Fusion ◽  
Enzyme Assay ◽  
Decarboxylase Activity ◽  
Lacz Gene ◽  
Coding Region ◽  
Ura3 Gene ◽  
Promoter Sequences ◽  
E Coli ◽  
New Type ◽  
Fusion Analysis

ABSTRACT We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5′-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.

scholarly journals Determination of amino acids misincorporation in recombinant protein by mass spectrometry

2013 ◽  
Vol 42 (1) ◽  
pp. 11-19 ◽  
Author(s):  
MZ Alam ◽  
L Regioneiri ◽  
MAS Santos
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Recombinant Protein ◽  
Gene Fusion ◽  
Aminoglycoside Antibiotic ◽  
Protein Functionality ◽  
Lacz Gene ◽  
Host Organism ◽  
E Coli

The synthesis of protein according to genetic code of a gene determines the basis of life and a stable proteome is necessary for cell homeostatis. However, errors occur naturally during translation of protein from its mRNA, which varies from 10-3 to 10-4 per codon. These errors are more frequent in recombinant protein overexpressed in heterologous hosts and affect protein functionality. The increasing amount of nonfunctional protein is often related to mistranslation of a gene under stress. In the present study, Saccharomyces cerevisiae as a host organism to overexpress E. coli lacZ gene fusion with GST to quantify misincorporation of amino acid in GST-? galactosidase recombinant protein. The yeast was treated with various stressors such as ethanol, chromium (CrO3), and aminoglycoside antibiotic - geneticin (G418) to induce protein aggregation. The misincorporation of amino acids was studied in soluble protein fractions by mass-spectrometry to determine how much misincorporation occur. We found that under experimental stress conditions the misincorporation of amino acids ranges from 5.6 ×10-3 to 8 × 10-3, which represents 60-80 fold higher than reported level. DOI: http://dx.doi.org/10.3329/bjas.v42i1.15760 Bang. J. Anim. Sci. 2013. 42 (1): 11-19

scholarly journals The Rhizobium leguminosarum bv. trifolii RosR: Transcriptional Regulator Involved in Exopolysaccharide Production

2007 ◽  
Vol 20 (7) ◽  
pp. 867-881 ◽  
Author(s):  
Monika Janczarek ◽  
Anna Skorupska
Keyword(s):  
Binding Site ◽  
Rhizobium Leguminosarum ◽  
Upstream Region ◽  
Regulatory Sequence ◽  
Lacz Gene ◽  
Sequence Motifs ◽  
Exopolysaccharide Production ◽  
Promoter Sequences ◽  
E Coli ◽  
Multiple Copies

The acidic exopolysaccharide is required for the establishment of symbiosis between the nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii and clover. Here, we describe RosR protein from R. leguminosarum bv. trifolii 24.2, a homolog of transcriptional regulators belonging to the family of Ros/MucR proteins. R. leguminosarum bv. trifolii RosR possesses a characteristic Cys2His2 type zincfinger motif in its C-terminal domain. Recombinant (His)6RosR binds to an RosR-box sequence located upstream of rosR. Deletion analysis of the rosR upstream region resulted in identification of two -35 to -10 promoter sequences, two conserved inverted palindromic pentamers that resemble the cAMP-CRP binding site of Escherichia coli, inverted repeats identified as a RosR binding site, and other regulatory sequence motifs. When assayed in E. coli, a transcriptional fusion of the cAMP-CRP binding site containing the rosR upstream region and lacZ gene was moderately responsive to glucose. The sensitivity of the rosR promoter to glucose was not observed in E. coli ΔcyaA. A rosR frame-shift mutant of R. leguminosarum bv. trifolii formed dry, wrinkled colonies and induced nodules on clover, but did not fix nitrogen. In the rosR mutant, transcription of pssA-lacZ fusion was decreased, indicating positive regulation of the pssA gene by RosR. Multiple copies of rosR in R. leguminosarum bv. trifolii 24.2 increased exopolysaccharide production.

scholarly journals Expression of the Corynebacterium glutamicum panD Gene Encoding l-Aspartate-α-Decarboxylase Leads to Pantothenate Overproduction in Escherichia coli

1999 ◽  
Vol 65 (4) ◽  
pp. 1530-1539 ◽  
Author(s):  
Nicole Dusch ◽  
Alfred Pühler ◽  
Jörn Kalinowski
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Specific Productivity ◽  
Decarboxylase Activity ◽  
Coding Region ◽  
E Coli ◽  
Enhanced Expression

ABSTRACT The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panDmutant strain. Sequence analysis revealed that the coding region ofpanD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A definedC. glutamicum panD mutant completely lackedl-aspartate-α-decarboxylase activity and exhibited β-alanine auxotrophy. The C. glutamicum panD(panDC.g. ) as well as the E. coli panD (panDE.c. ) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of l-aspartate-α-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed inE. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of thepanDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of thepanDC.g. gene was sufficient β-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external β-alanine supplementation. The enhanced expression ofpanDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)−1h−1.

scholarly journals A Tn 10-lacZ-kanR-URA3 Gene Fusion Transposon for Insertion Mutagenesis and Fusion Analysis of Yeast and Bacterial Genes

Genetics ◽  
1987 ◽  
Vol 116 (2) ◽  
pp. 191-199 ◽  
Author(s):  
Olivier Huisman ◽  
Wendy Raymond ◽  
Kai-Uwe Froehlich ◽  
Patrick Errada ◽  
Nancy Kleckner ◽  
...  
Keyword(s):  
Region Of Interest ◽  
Single Step ◽  
Insertion Mutagenesis ◽  
Lacz Gene ◽  
Ura3 Gene ◽  
New Variant ◽  
Fusion Analysis ◽  
Bacterial Genes ◽  
Yeast Genes ◽  
Insertion Mutations

ABSTRACT We describe here a new variant of transposon Tn 10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes. We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae. This transposon, nicknamed mini-Tn 10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant. The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene. Other yeast genes can easily be substituted for URA3 in the available constructs. The mini-Tn 10-LUK system has several important advantages. (1) Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA. It is easy to obtain enough insertions to sensitively define the functional limits of a gene. (2) Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E. coli. (3) The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene. (4) Under favorable circumstances, usable lacZ protein fusions are created. (5) Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.

scholarly journals Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

2007 ◽  
Vol 189 (14) ◽  
pp. 5108-5118 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Michael G. Kaufman ◽  
Adam K. Bates ◽  
Edward D. Walker
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Gc Content ◽  
Pronounced Effect ◽  
Promoter Elements ◽  
Spacer Length ◽  
Promoter Sequences ◽  
E Coli ◽  
Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

scholarly journals Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

2011 ◽  
Vol 11 (1) ◽  
Author(s):  
Dimitris Dimitriadis ◽  
V Lila Koumandou ◽  
Philip Trimpalis ◽  
Sophia Kossida
Keyword(s):  
Trypanosoma Brucei ◽  
Gene Fusion ◽  
Fusion Analysis

scholarly journals A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box.

1988 ◽  
Vol 8 (11) ◽  
pp. 4634-4641 ◽  
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Gene Fusion ◽  
Regulatory Gene ◽  
Regulatory Region ◽  
Tata Box ◽  
Homologous Gene ◽  
Lacz Gene ◽  
Proline Utilization ◽  
Constitutive Mutation ◽  
Necessary And Sufficient ◽  
Utilization Pathway

Deletion analysis of the promoter of the PUT2 gene that functions in the proline utilization pathway of Saccharomyces cerevisiae identified a PUT2 upstream activation site (UAS). It is contained within a single 40-base-pair (bp) region located immediately upstream of the TATA box and is both necessary and sufficient for proline induction. When placed upstream of a CYC7-lacZ gene fusion, the 40-bp sequence conferred proline regulation on CYC7-lacZ. A 35-bp deletion within the PUT2 UAS in an otherwise intact PUT2 promoter resulted in noninducible expression of a PUT2-lacZ gene fusion. When a plasmid bearing this UAS-deleted promoter was placed in a strain carrying a constitutive mutation in the positive regulatory gene PUT3, expression of PUT2-lacZ was not constitutive but occurred at levels below those found under noninducing conditions. In heterologous as well as homologous gene fusions, the PUT2 UAS appeared to be responsible for uninduced as well as proline-induced levels of expression. Although located immediately adjacent to the PUT2 UAS, the TATA box did not appear to play a regulatory role, as indicated by the results of experiments in which it was replaced by the CYC7 TATA box. A 26-bp sequence containing this TATA box was critical to the expression of PUT2, since a deletion of this region completely abolished transcriptional activity of the gene under both inducing and noninducing conditions. Our results indicate that the PUT2 promoter has a comparatively simple structure, requiring UAS and TATA sequences as well as the PUT3 gene product (directly or indirectly) for its expression.

scholarly journals A Pathoadaptive Deletion in an Enteroaggregative Escherichia coli Outbreak Strain Enhances Virulence in a Caenorhabditis elegans Model

2010 ◽  
Vol 78 (9) ◽  
pp. 4068-4076 ◽  
Author(s):  
Jennifer Hwang ◽  
Lisa M. Mattei ◽  
Laura G. VanArendonk ◽  
Philip M. Meneely ◽  
Iruka N. Okeke
Keyword(s):  
Escherichia Coli ◽  
Caenorhabditis Elegans ◽  
Epithelial Cells ◽  
Genetic Material ◽  
Decarboxylase Activity ◽  
Multicopy Plasmid ◽  
Lysine Decarboxylase ◽  
Outbreak Strain ◽  
E Coli ◽  
C Elegans

ABSTRACT Enteroaggregative Escherichia coli (EAEC) strains are important diarrheal pathogens. EAEC strains are defined by their characteristic stacked-brick pattern of adherence to epithelial cells but show heterogeneous virulence and have different combinations of adhesin and toxin genes. Pathoadaptive deletions in the lysine decarboxylase (cad) genes have been noted among hypervirulent E. coli subtypes of Shigella and enterohemorrhagic E. coli. To test the hypothesis that cad deletions might account for heterogeneity in EAEC virulence, we developed a Caenorhabditis elegans pathogenesis model. Well-characterized EAEC strains were shown to colonize and kill C. elegans, and differences in virulence could be measured quantitatively. Of 49 EAEC strains screened for lysine decarboxylase activity, 3 tested negative. Most notable is isolate 101-1, which was recovered in Japan, from the largest documented EAEC outbreak. EAEC strain 101-1 was unable to decarboxylate lysine in vitro due to deletions in cadA and cadC, which, respectively, encode lysine decarboxylase and a transcriptional activator of the cadAB genes. Strain 101-1 was significantly more lethal to C. elegans than control strain OP50. Lethality was attenuated when the lysine decarboxylase defect was complemented from a multicopy plasmid and in single copy. In addition, restoring lysine decarboxylase function produced derivatives of 101-1 deficient in aggregative adherence to cultured human epithelial cells. Lysine decarboxylase inactivation is pathoadapative in an important EAEC outbreak strain, and deletion of cad genes could produce hypervirulent EAEC lineages in the future. These results suggest that loss, as well as gain, of genetic material can account for heterogeneous virulence among EAEC strains.

Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5

Development ◽  
1999 ◽  
Vol 126 (17) ◽  
pp. 3795-3809 ◽  
Author(s):  
D. Acampora ◽  
G.R. Merlo ◽  
L. Paleari ◽  
B. Zerega ◽  
M.P. Postiglione ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Brain Regions ◽  
Lacz Gene ◽  
Vestibular Organ ◽  
E Coli ◽  
Branchial Arches ◽  
Homeobox Protein ◽  
Derivatives Of ◽  
Delayed Ossification ◽  
Newborn Animals

The Dlx5 gene encodes a Distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of the mouse embryo. In later developmental stages, it appears in the branchial arches, the otic and olfactory placodes and their derivatives, in restricted brain regions, in all extending appendages and in all developing bones. We have created a null allele of the mouse Dlx5 gene by replacing exons I and II with the E. coli lacZ gene. Heterozygous mice appear normal. Beta-galactosidase activity in Dlx5+/− embryos and newborn animals reproduces the known pattern of expression of the gene. Homozygous mutants die shortly after birth with a swollen abdomen. They present a complex phenotype characterised by craniofacial abnormalities affecting derivatives of the first four branchial arches, severe malformations of the vestibular organ, a delayed ossification of the roof of the skull and abnormal osteogenesis. No obvious defect was observed in the patterning of limbs and other appendages. The defects observed in Dlx5−/− mutant animals suggest multiple and independent roles of this gene in the patterning of the branchial arches, in the morphogenesis of the vestibular organ and in osteoblast differentiation.

Forebrain and midbrain regions are deleted in Otx2−/− mutants due to a defective anterior neuroectoderm specification during gastrulation

Development ◽  
1995 ◽  
Vol 121 (10) ◽  
pp. 3279-3290 ◽  
Author(s):  
D. Acampora ◽  
S. Mazan ◽  
Y. Lallemand ◽  
V. Avantaggiato ◽  
M. Maury ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Null Allele ◽  
External Layer ◽  
Coding Region ◽  
Coding Sequence ◽  
E Coli ◽  
Homozygous Mutant

We have replaced part of the mouse homeogene Otx2 coding region with the E. coli lacZ coding sequence, thus creating a null allele of Otx2. By 9.5 dpc, homozygous mutant embryos are characterized by the absence of forebrain and midbrain regions. From the early to midstreak stages, endomesodermal cells expressing lacZ fail to be properly localized anteriorly. In the ectodermal layer, lacZ transcription is progressively extinguished, being barely detectable by the late streak stage. These data suggest that Otx2 expression in endomesoderm and ectoderm is required for anterior neuroectoderm specification. In gastrulating heterozygous embryos, a post-transcriptional repression acts on lacZ transcripts in the ectoderm, but not in the external layer, suggesting that different post-transcriptional mechanisms control Otx2 expression in both layers.

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