RLR1 (THO2), required for expressing lacZ fusions in yeast, is conserved from yeast to humans and is a suppressor of SIN4

Gene ◽  
2000 ◽  
Vol 243 (1-2) ◽  
pp. 195-205 ◽  
Author(s):  
Robert W. West ◽  
Brian Kruger ◽  
Sean Thomas ◽  
Junli Ma ◽  
Elena Milgrom
Keyword(s):  
Lacz Fusions

scholarly journals Different structures of selected and unselected araB–lacZ fusions

1997 ◽  
Vol 23 (6) ◽  
pp. 1133-1145 ◽  
Author(s):  
Genevieve Maenhaut-Michel ◽  
Catherine E. Blake ◽  
David R. F. Leach ◽  
James A. Shapiro
Keyword(s):  
Lacz Fusions

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence

1988 ◽  
Vol 8 (12) ◽  
pp. 5439-5447
Author(s):  
P P Mueller ◽  
B M Jackson ◽  
P F Miller ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Normal Growth ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Coding Sequences ◽  
Lacz Fusions ◽  
Upstream Open Reading Frames ◽  
Reading Frames ◽  
Starvation Conditions

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.

scholarly journals New NodW- or NifA-Regulated Bradyrhizobium japonicum Genes

2003 ◽  
Vol 16 (4) ◽  
pp. 342-351 ◽  
Author(s):  
Isabelle Caldelari Baumberger ◽  
Nicole Fraefel ◽  
Michael Göttfert ◽  
Hauke Hennecke
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Plant Cell Wall ◽  
Outer Membrane Proteins ◽  
Cellulase Activity ◽  
Dependent Manner ◽  
Cell Wall Degrading Enzymes ◽  
Reading Frame ◽  
Expression Studies ◽  
Lacz Fusions ◽  
In The Wild

A cluster of genes coding for putative plant cell-wall degrading enzymes (i.e., genes for two endoglucanases [gunA and gunA2], one pectinmethylesterase [pme], and one polygalacturonase [pgl]) was identified by sequence similarities in the symbiotic region of the Bradyrhizobium japonicum chromosome. In addition, a systematic screen of the region revealed several genes potentially transcribed by the σ54-RNA polymerase and activated by the transcriptional regulator NifA (i.e., genes for proteins with similarity to outer membrane proteins [id117 and id525] and a citrate carrier [id331 or citA] and one open reading frame without similarity to known proteins [id747]). Expression studies using transcriptional lacZ fusions showed that gunA2 and pgl were strongly induced by the isoflavone genistein in a NodW-dependent manner, suggesting a role of the gene products in early events of the nodulation process; by contrast, gunA and pme expression was very weak in the conditions tested. The gunA2 gene product was purified and was shown to have cellulase activity. β-Galactosidase activity expressed from transcriptional lacZ fusions to id117, id525, and id747 in the wild type and in nifA and rpoN mutant backgrounds confirmed that their transcription was dependent on NifA and σ54. Despite the presence of a -24/-12-type promoter and a NifA binding site upstream of citA, no regulation could be demonstrated in this case. Null mutations introduced in gunA, gunA2, pgl, pme, citA, id117, id525, and id747 did not impair the symbiosis with the host plants.

scholarly journals HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation.

Genetics ◽  
1992 ◽  
Vol 132 (4) ◽  
pp. 987-1001 ◽  
Author(s):  
M I Chiu ◽  
T L Mason ◽  
G R Fink
Keyword(s):  
Mitochondrial Function ◽  
Nuclear Gene ◽  
Trna Synthetase ◽  
Mitochondrial Enzyme ◽  
Trna Synthetases ◽  
Cytoplasmic Enzyme ◽  
Amino Terminal ◽  
Lacz Fusions ◽  
Targeting Signal ◽  
Amino Terminal Sequence

Abstract Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.

Nucleotide sequence and functional analysis of the RAD1 gene of Saccharomyces cerevisiae

1987 ◽  
Vol 7 (3) ◽  
pp. 1012-1020
Author(s):  
P Reynolds ◽  
L Prakash ◽  
S Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Excision Repair ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Uv Sensitivity ◽  
Protein Mutation ◽  
Discernible Effect ◽  
Lacz Fusions

The RAD1 gene of Saccharomyces cerevisiae is involved in excision repair of damaged DNA. The nucleotide sequence of the RAD1 gene presented here shows an open reading frame of 3,300 nucleotides. Two ATG codons occur in the open reading frame at positions +1 and +334, respectively. Since a deletion of about 2.7 kilobases of DNA from the 5' region of the RAD1 gene, which also deletes the +1 ATG and 11 additional codons in the RAD1 open reading frame, partially complements UV sensitivity of a rad1 delta mutant, we examined the role of the +1 ATG and +334 ATG codons in translation initiation of RAD1 protein. Mutation of the +1 ATG codon to ATC affected the complementation ability of the RAD1 gene, whereas mutation of the +334 ATG codon to ATC showed no discernible effect on RAD1 function. These results indicate that translation of RAD1 protein is initiated from the +1 ATG codon. Productive in-frame RAD1-lacZ fusions showed that the RAD1 open reading frame is expressed in yeasts. The RAD1-encoded protein contains 1,100 amino acids with a molecular weight of 126,360.

Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae

1982 ◽  
Vol 2 (10) ◽  
pp. 1212-1219
Author(s):  
S J Silverman ◽  
M Rose ◽  
D Botstein ◽  
G R Fink
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Base Pair ◽  
Yeast Genome ◽  
Galactosidase Activity ◽  
Lacz Fusions ◽  
Translation Initiation Codon ◽  
Beta Galactosidase ◽  
His4 Gene

The beginning of the Saccharomyces cerevisiae HIS4 gene has been fused to the structural gene for Escherichia coli beta-galactosidase. This construction, which contains HIS4 DNA from -732 to +30 relative to the translation initiation codon, has been integrated into the yeast genome at two chromosomal locations, HIS4 and URA3. At both locations, this 762-base-pair stretch of DNA is sufficient for initiating expression of beta-galactosidase activity in S. cerevisiae and confers upon this activity the regulatory response normally found for HIS4.

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