Source 1primary paper1995Molecular and Cellular Biology
Toolkit/CRY2-lacZ gene fusion
CRY2-lacZ gene fusion
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
The CRY2-lacZ gene fusion is a Saccharomyces cerevisiae reporter construct used to measure how CRY2 sequence features regulate reporter expression. It was applied to identify cis-acting elements involved in repression of CRY2, linking CRY2-derived nucleotide information to lacZ output.
Usefulness & Problems
Why this is useful
This construct is useful for dissecting gene-specific repression determinants within CRY2 in yeast. The cited study used CRY2-lacZ fusions to assay expression changes associated with cis-acting elements implicated in feedback inhibition of CRY2.
Problem solved
It helps solve the problem of locating CRY2-encoded regulatory elements responsible for selective repression of CRY2 expression. The broader biological context is that CRY2, but not CRY1, is repressed by ribosomal protein 59 expressed from either paralog, and CRY2 mRNA increases 5- to 10-fold when CRY1 is deleted or inactivated.
Problem links
Need conditional protein clearance
DerivedThe CRY2-lacZ gene fusion is a Saccharomyces cerevisiae reporter construct used to measure how CRY2 sequence features regulate reporter expression. It was applied to identify cis-acting elements involved in repression of CRY2, linking CRY2-derived nucleotide information to lacZ output.
Need conditional recombination or state switching
DerivedThe CRY2-lacZ gene fusion is a Saccharomyces cerevisiae reporter construct used to measure how CRY2 sequence features regulate reporter expression. It was applied to identify cis-acting elements involved in repression of CRY2, linking CRY2-derived nucleotide information to lacZ output.
Need tighter control over gene expression timing or amplitude
DerivedThe CRY2-lacZ gene fusion is a Saccharomyces cerevisiae reporter construct used to measure how CRY2 sequence features regulate reporter expression. It was applied to identify cis-acting elements involved in repression of CRY2, linking CRY2-derived nucleotide information to lacZ output.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Mechanisms
cis-acting repressionDegradationfeedback inhibitiongene expression reporting via lacz fusionTechniques
Functional AssayTarget processes
degradationrecombinationtranscriptionImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: reporter
The available evidence indicates a lacZ fusion built from CRY2 sequences and assayed in Saccharomyces cerevisiae. Specific promoter design, fusion boundaries, vector system, selection markers, and readout conditions are not described in the supplied evidence.
The supplied evidence supports use as a reporter for CRY2 repression but does not provide detailed construct architecture, quantitative lacZ performance metrics, or validation across multiple organisms or assay formats. Evidence is limited to a single 1995 study in Saccharomyces cerevisiae.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
In wild-type Saccharomyces cerevisiae cells, CRY1 and CRY2 are expressed at an approximately 10:1 ratio.
The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells.
expression ratio CRY1:CRY2 10:1
In wild-type Saccharomyces cerevisiae cells, CRY1 and CRY2 are expressed at an approximately 10:1 ratio.
The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells.
expression ratio CRY1:CRY2 10:1
In wild-type Saccharomyces cerevisiae cells, CRY1 and CRY2 are expressed at an approximately 10:1 ratio.
The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells.
expression ratio CRY1:CRY2 10:1
In wild-type Saccharomyces cerevisiae cells, CRY1 and CRY2 are expressed at an approximately 10:1 ratio.
The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells.
expression ratio CRY1:CRY2 10:1
In wild-type Saccharomyces cerevisiae cells, CRY1 and CRY2 are expressed at an approximately 10:1 ratio.
The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells.
expression ratio CRY1:CRY2 10:1
In wild-type Saccharomyces cerevisiae cells, CRY1 and CRY2 are expressed at an approximately 10:1 ratio.
The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells.
expression ratio CRY1:CRY2 10:1
In wild-type Saccharomyces cerevisiae cells, CRY1 and CRY2 are expressed at an approximately 10:1 ratio.
The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells.
expression ratio CRY1:CRY2 10:1
Ribosomal protein 59 expressed from either CRY1 or CRY2 represses expression of CRY2 but not CRY1.
Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1.
Ribosomal protein 59 expressed from either CRY1 or CRY2 represses expression of CRY2 but not CRY1.
Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1.
Ribosomal protein 59 expressed from either CRY1 or CRY2 represses expression of CRY2 but not CRY1.
Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1.
Ribosomal protein 59 expressed from either CRY1 or CRY2 represses expression of CRY2 but not CRY1.
Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1.
Ribosomal protein 59 expressed from either CRY1 or CRY2 represses expression of CRY2 but not CRY1.
Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1.
Ribosomal protein 59 expressed from either CRY1 or CRY2 represses expression of CRY2 but not CRY1.
Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1.
Ribosomal protein 59 expressed from either CRY1 or CRY2 represses expression of CRY2 but not CRY1.
Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1.
Deletion or inactivation of CRY1 increases CRY2 mRNA levels by 5- to 10-fold.
Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA.
fold change in CRY2 mRNA 5- to 10-fold increased
Deletion or inactivation of CRY1 increases CRY2 mRNA levels by 5- to 10-fold.
Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA.
fold change in CRY2 mRNA 5- to 10-fold increased
Deletion or inactivation of CRY1 increases CRY2 mRNA levels by 5- to 10-fold.
Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA.
fold change in CRY2 mRNA 5- to 10-fold increased
Deletion or inactivation of CRY1 increases CRY2 mRNA levels by 5- to 10-fold.
Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA.
fold change in CRY2 mRNA 5- to 10-fold increased
Deletion or inactivation of CRY1 increases CRY2 mRNA levels by 5- to 10-fold.
Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA.
fold change in CRY2 mRNA 5- to 10-fold increased
Deletion or inactivation of CRY1 increases CRY2 mRNA levels by 5- to 10-fold.
Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA.
fold change in CRY2 mRNA 5- to 10-fold increased
Deletion or inactivation of CRY1 increases CRY2 mRNA levels by 5- to 10-fold.
Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA.
fold change in CRY2 mRNA 5- to 10-fold increased
The regulatory sequence of CRY2 is phylogenetically conserved, with a very similar sequence present at the 5' end of the RP59 gene in Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved, with a very similar sequence present at the 5' end of the RP59 gene in Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved, with a very similar sequence present at the 5' end of the RP59 gene in Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved, with a very similar sequence present at the 5' end of the RP59 gene in Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved, with a very similar sequence present at the 5' end of the RP59 gene in Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved, with a very similar sequence present at the 5' end of the RP59 gene in Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved, with a very similar sequence present at the 5' end of the RP59 gene in Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis.
Feedback regulation of CRY2 occurs posttranscriptionally.
Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally.
Feedback regulation of CRY2 occurs posttranscriptionally.
Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally.
Feedback regulation of CRY2 occurs posttranscriptionally.
Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally.
Feedback regulation of CRY2 occurs posttranscriptionally.
Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally.
Feedback regulation of CRY2 occurs posttranscriptionally.
Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally.
Feedback regulation of CRY2 occurs posttranscriptionally.
Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally.
Feedback regulation of CRY2 occurs posttranscriptionally.
Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally.
CRY2 pre-mRNA accumulates in mtr mutants and upf1 mutants, suggesting that unspliced CRY2 pre-mRNA is degraded in the cytoplasm in wild-type repressed cells.
Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm.
CRY2 pre-mRNA accumulates in mtr mutants and upf1 mutants, suggesting that unspliced CRY2 pre-mRNA is degraded in the cytoplasm in wild-type repressed cells.
Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm.
CRY2 pre-mRNA accumulates in mtr mutants and upf1 mutants, suggesting that unspliced CRY2 pre-mRNA is degraded in the cytoplasm in wild-type repressed cells.
Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm.
CRY2 pre-mRNA accumulates in mtr mutants and upf1 mutants, suggesting that unspliced CRY2 pre-mRNA is degraded in the cytoplasm in wild-type repressed cells.
Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm.
CRY2 pre-mRNA accumulates in mtr mutants and upf1 mutants, suggesting that unspliced CRY2 pre-mRNA is degraded in the cytoplasm in wild-type repressed cells.
Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm.
CRY2 pre-mRNA accumulates in mtr mutants and upf1 mutants, suggesting that unspliced CRY2 pre-mRNA is degraded in the cytoplasm in wild-type repressed cells.
Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm.
CRY2 pre-mRNA accumulates in mtr mutants and upf1 mutants, suggesting that unspliced CRY2 pre-mRNA is degraded in the cytoplasm in wild-type repressed cells.
Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm.
Sequences necessary and sufficient for regulation of CRY2 lie within the transcribed region, including the 5' exon and the first 62 nucleotides of the intron.
Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron.
intron segment length 62 nucleotides
Sequences necessary and sufficient for regulation of CRY2 lie within the transcribed region, including the 5' exon and the first 62 nucleotides of the intron.
Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron.
intron segment length 62 nucleotides
Sequences necessary and sufficient for regulation of CRY2 lie within the transcribed region, including the 5' exon and the first 62 nucleotides of the intron.
Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron.
intron segment length 62 nucleotides
Sequences necessary and sufficient for regulation of CRY2 lie within the transcribed region, including the 5' exon and the first 62 nucleotides of the intron.
Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron.
intron segment length 62 nucleotides
Sequences necessary and sufficient for regulation of CRY2 lie within the transcribed region, including the 5' exon and the first 62 nucleotides of the intron.
Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron.
intron segment length 62 nucleotides
Sequences necessary and sufficient for regulation of CRY2 lie within the transcribed region, including the 5' exon and the first 62 nucleotides of the intron.
Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron.
intron segment length 62 nucleotides
Sequences necessary and sufficient for regulation of CRY2 lie within the transcribed region, including the 5' exon and the first 62 nucleotides of the intron.
Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron.
intron segment length 62 nucleotides
Both the secondary structure and nucleotide sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Both the secondary structure and nucleotide sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Both the secondary structure and nucleotide sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Both the secondary structure and nucleotide sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Both the secondary structure and nucleotide sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Both the secondary structure and nucleotide sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Both the secondary structure and nucleotide sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Approval Evidence
1 source8 linked approval claimsfirst-pass slug cry2-lacz-gene-fusion
cis-Acting elements involved in repression of CRY2 were identified by assaying the expression of CRY2-lacZ gene fusions
Source:
expression ratiosupports
In wild-type Saccharomyces cerevisiae cells, CRY1 and CRY2 are expressed at an approximately 10:1 ratio.
The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells.
Source:
gene specific repressionsupports
Ribosomal protein 59 expressed from either CRY1 or CRY2 represses expression of CRY2 but not CRY1.
Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1.
Source:
perturbation effect on mrnasupports
Deletion or inactivation of CRY1 increases CRY2 mRNA levels by 5- to 10-fold.
Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA.
Source:
phylogenetic conservationsupports
The regulatory sequence of CRY2 is phylogenetically conserved, with a very similar sequence present at the 5' end of the RP59 gene in Kluyveromyces lactis.
The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis.
Source:
posttranscriptional regulationsupports
Feedback regulation of CRY2 occurs posttranscriptionally.
Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally.
Source:
premrna accumulation in mutantssupports
CRY2 pre-mRNA accumulates in mtr mutants and upf1 mutants, suggesting that unspliced CRY2 pre-mRNA is degraded in the cytoplasm in wild-type repressed cells.
Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm.
Source:
regulatory region mappingsupports
Sequences necessary and sufficient for regulation of CRY2 lie within the transcribed region, including the 5' exon and the first 62 nucleotides of the intron.
Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron.
Source:
sequence structure requirementsupports
Both the secondary structure and nucleotide sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression.
Source:
Comparisons
Source-backed strengths
The construct directly reports the functional consequences of CRY2 sequence determinants in the native yeast gene-regulatory context of the study. It was specifically informative for identifying cis-acting elements involved in CRY2 repression, a process relevant to the approximately 10:1 CRY1:CRY2 expression ratio observed in wild-type Saccharomyces cerevisiae.
Compared with cdiGEBS
CRY2-lacZ gene fusion and cdiGEBS address a similar problem space because they share degradation, recombination, transcription.
Shared frame: same top-level item type; shared target processes: degradation, recombination, transcription; shared mechanisms: degradation
Compared with constitutive SsDHN overexpression in transgenic tobacco
CRY2-lacZ gene fusion and constitutive SsDHN overexpression in transgenic tobacco address a similar problem space because they share degradation, recombination.
Shared frame: same top-level item type; shared target processes: degradation, recombination; shared mechanisms: degradation
Strengths here: looks easier to implement in practice.
Compared with orthogonal degrons
CRY2-lacZ gene fusion and orthogonal degrons address a similar problem space because they share degradation, recombination.
Shared frame: same top-level item type; shared target processes: degradation, recombination; shared mechanisms: degradation
Ranked Citations
- 1.