Q-system (genetics)

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Q-system is a genetic tool that allows to express transgenes in a living organism. [1] Originally the Q-system was developed [2] [3] for use in the vinegar fly Drosophila melanogaster, and was rapidly adapted for use in cultured mammalian cells, [2] zebrafish, [4] worms [5] and mosquitoes. [6] The Q-system utilizes genes from the qa cluster [7] of the bread fungus Neurospora crassa, and consists of four components: the transcriptional activator (QF/QF2/QF2w), the enhancer QUAS, the repressor QS, and the chemical de-repressor quinic acid. Similarly to GAL4/UAS [8] and LexA/LexAop, [9] the Q-system is a binary expression system that allows to express reporters or effectors (e.g. fluorescent proteins, ion channels, toxins and other genes) in a defined subpopulation of cells with the purpose of visualising these cells or altering their function. In addition, GAL4/UAS, LexA/LexAop and the Q-system function independently of each other and can be used simultaneously to achieve a desired pattern of reporter expression, or to express several reporters in different subsets of cells.

Contents

Origin

Repressible Q binary expression system. Q-system shematic.png
Repressible Q binary expression system.

The Q-system is based on two out of the seven genes of the qa gene cluster of the bread fungus Neurospora crassa. [7] The genes of the qa cluster are responsible for the catabolism of quinic acid, which is used by the fungus as a carbon source in conditions of low glucose. [7] The cluster contains a transcriptional activator qa-1F, a transcriptional repressor qa-1S, and five structural genes. The qa-1F binds to a specific DNA sequence, found upstream of the qa genes. The presence of quinic acid disrupts interaction between qa-1F and qa-1S, thus disinhibiting the transcriptional activity of qa-1F. Genes qa-1F, qa-1S and the DNA binding sequence of qa-1F form the basis of the Q-system. The genes were renamed to simplify their use as follows: transcriptional activator qa-1F as QF, repressor qa-1S as QS, and the DNA binding sequence as QUAS. [2] The quinic acid represents the fourth component of the Q-system. The original transactivator QF appeared to be toxic when expressed broadly in Drosophila. To overcome this problem, two new transactivators were developed: QF2 and QF2w. [3]

Use in Drosophila

Basic use

The Q-system functions similarly to, and independently of, the GAL4/UAS [8] and the LexA/LexAop [9] systems. QF, QF2 and QF2w are analogous to GAL4 and LexA, and their expression is usually under the control of cell-type specific promoter, such as nsyb (to target neurons) or tubulin (to target all cells). QUAS is analogous to UAS and LexAop, and is placed upstream of an effector gene, such as GFP. QS is analogous to GAL80, and may be driven by any promoter (e.g. tubulin-QS). Quinic acid is a unique feature of the Q-system, and it must be fed to the flies or maggots in order to alleviate the QS-induced repression. In some ways, quinic acid is analogous to temperature in the case of GAL80ts. In its basic form, two transgenic fly lines, one containing a QF transgene and the other one containing a QUAS transgene, are crossed together. Their progeny that had both a QF transgene and a QUAS transgene will be expressing a reporter gene in a subset of cells (e.g. nsyb-QF2, QUAS-GFP flies express GFP in all neurons). If a fly also expresses QS in some of the cells, the activity of QF will be repressed in these cells, but it may be restored of a fly is fed quinic acid (e.g. a nsyb-QF2, QUAS-GFP, tub-QS fly expresses no GFP when its diet doesn't contain quinic acid, and expresses GFP in its neurons when fed quinic acid). [2] [3] The use of QS repressor and quinic acid allows to fine-tune the temporal control of transgene expression.

Chimeric transactivators

Chimeric transactivators GAL4QF [3] and LexAQF [3] allow to combine the use of all three binary expression systems. GAL4QF binds to UAS, and may be repressed by QS while being unaffected by GAL80. Similarly, LexAQF binds to LexAop, and may be repressed by QS. LexAQF represents a useful extension of the LexA/LexAop system that doesn't have its own repressor.

Intersectional expression

Intersectional expression patterns possible using GAL4 and QF binary expression systems. G4QF-intersectionals.png
Intersectional expression patterns possible using GAL4 and QF binary expression systems.

A variety of expression patterns may be achieved by combination of the three binary expression systems and the FLP/FRT or other recombinases. [10] Expression patterns may be constructed as AND, OR, NOR etc. logic gates [1] [2] to e.g. narrow down expression patterns of available GAL4 lines. The resulting expression pattern somewhat depends on the developmental timing of activation of the transcription factors (discussed in [1] ).

Use in other organisms

Q-system appeared to be working successfully in a variety of organisms. It has been used to drive expression of luciferase, as a proof of principle, in cultured mammalian cells. [2] In zebrafish [4] the Q-system has been successfully used with several tissue-specific promoters, and was shown to work independently of the GAL4/UAS system when expressed in the same cell. In C. elegans [5] the Q-system has been shown to work in muscles and in neuronal tissue. In 2016, the Q-system was used to target, for the first time, the olfactory neurons of malaria mosquitoes Anopheles gambiae. [6] In 2019, the Q-system in Anopheles mosquitoes was used to examine the functional responses of olfactory neurons to odors. [11] In 2019, the Q-system was introduced into the Aedes aegypti mosquito to capture tissue specific expression patterns. [12] These successes make the Q-system the system of choice when developing genetic tools for other organisms. Currently the main shortcoming of the Q-system is the low number of available transgenic lines, but it will be overcome as the scientific community creates and shares these resources, such as by the use of the GAL4>QF2 HACK system to convert existing GAL4 transgenic insertions to QF2. [13] DNA binding domain of QF2 fused with VP16 transcriptional activator domain was successfully applied in Penicillium to gain control over the penicillin producing secondary metabolite gene cluster in a scalable manner. [14]

Related Research Articles

Inbred strains are individuals of a particular species which are nearly identical to each other in genotype due to long inbreeding. A strain is inbred when it has undergone at least 20 generations of brother x sister or offspring x parent mating, at which point at least 98.6% of the loci in an individual of the strain will be homozygous, and each individual can be treated effectively as clones. Some inbred strains have been bred for over 150 generations, leaving individuals in the population to be isogenic in nature. Inbred strains of animals are frequently used in laboratories for experiments where for the reproducibility of conclusions all the test animals should be as similar as possible. However, for some experiments, genetic diversity in the test population may be desired. Thus outbred strains of most laboratory animals are also available, where an outbred strain is a strain of an organism that is effectively wildtype in nature, where there is as little inbreeding as possible.

<span class="mw-page-title-main">Regulation of gene expression</span> Modifying mechanisms used by cells to increase or decrease the production of specific gene products

Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products. Sophisticated programs of gene expression are widely observed in biology, for example to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources. Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Often, one gene regulator controls another, and so on, in a gene regulatory network.

<span class="mw-page-title-main">Mosaic (genetics)</span> Condition in multi-cellular organisms

Mosaicism or genetic mosaicism is a condition in which a multicellular organism possesses more than one genetic line as the result of genetic mutation. This means that various genetic lines resulted from a single fertilized egg. Mosaicism is one of several possible causes of chimerism, wherein a single organism is composed of cells with more than one distinct genotype.

<span class="mw-page-title-main">Two-hybrid screening</span> Molecular biology technique

Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.

E2F is a group of genes that encodes a family of transcription factors (TF) in higher eukaryotes. Three of them are activators: E2F1, 2 and E2F3a. Six others act as suppressors: E2F3b, E2F4-8. All of them are involved in the cell cycle regulation and synthesis of DNA in mammalian cells. E2Fs as TFs bind to the TTTCCCGC consensus binding site in the target promoter sequence.

Ectopic is a word used with a prefix, ecto, meaning “out of place.” Ectopic expression is an abnormal gene expression in a cell type, tissue type, or developmental stage in which the gene is not usually expressed. The term ectopic expression is predominantly used in studies using metazoans, especially in Drosophila melanogaster for research purposes.

An enhancer trap is a method in molecular biology. The enhancer trap construct contains a transposable element and a reporter gene. The first is necessary for (random) insertion in the genome, the latter is necessary for identification of the spatial regulation by the enhancer. On top of this, the construct usually includes a genetic marker, e.g., the white gene producing red-colored eyes in Drosophila, or ampicillin resistance in E. coli.

<span class="mw-page-title-main">GAL4/UAS system</span> Biochemical method

The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It is based on the finding by Hitoshi Kakidani and Mark Ptashne, and Nicholas Webster and Pierre Chambon in 1988 that Gal4 binding to UAS sequences activates gene expression. The method was introduced into flies by Andrea Brand and Norbert Perrimon in 1993 and is considered a powerful technique for studying the expression of genes. The system has two parts: the Gal4 gene, encoding the yeast transcription activator protein Gal4, and the UAS, an enhancer to which GAL4 specifically binds to activate gene transcription.

<span class="mw-page-title-main">Brainbow</span> Neuroimaging technique to differentiate neurons

Brainbow is a process by which individual neurons in the brain can be distinguished from neighboring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein in individual neurons, it is possible to flag each neuron with a distinctive color. This process has been a major contribution to the field of neural connectomics.

<span class="mw-page-title-main">TLE1</span> Protein-coding gene in the species Homo sapiens

Transducin-like enhancer protein 1 is a protein that in humans is encoded by the TLE1 gene.

<span class="mw-page-title-main">HES1</span> Protein-coding gene in the species Homo sapiens

Transcription factor HES1 is a protein that is encoded by the Hes1 gene, and is the mammalian homolog of the hairy gene in Drosophila. HES1 is one of the seven members of the Hes gene family (HES1-7). Hes genes code nuclear proteins that suppress transcription.

<span class="mw-page-title-main">SIM2</span> Protein-coding gene in the species Homo sapiens

Single-minded homolog 2 is a protein that in humans is encoded by the SIM2 gene. It plays a major role in the development of the central nervous system midline as well as the construction of the face and head.

<span class="mw-page-title-main">Bioreporter</span> Genetically engineered microbial cells

Bioreporters are intact, living microbial cells that have been genetically engineered to produce a measurable signal in response to a specific chemical or physical agent in their environment. Bioreporters contain two essential genetic elements, a promoter gene and a reporter gene. The promoter gene is turned on (transcribed) when the target agent is present in the cell’s environment. The promoter gene in a normal bacterial cell is linked to other genes that are then likewise transcribed and then translated into proteins that help the cell in either combating or adapting to the agent to which it has been exposed. In the case of a bioreporter, these genes, or portions thereof, have been removed and replaced with a reporter gene. Consequently, turning on the promoter gene now causes the reporter gene to be turned on. Activation of the reporter gene leads to production of reporter proteins that ultimately generate some type of a detectable signal. Therefore, the presence of a signal indicates that the bioreporter has sensed a particular target agent in its environment.

Mosaic analysis with a repressible cell marker, or MARCM, is a genetics technique for creating individually labeled homozygous cells in an otherwise heterozygous Drosophila melanogaster. It has been a crucial tool in studying the development of the Drosophila nervous system. This technique relies on recombination during mitosis mediated by FLP-FRT recombination. As one copy of a gene, provided by the balancer chromosome, is often enough to rescue a mutant phenotype, MARCM clones can be used to study a mutant phenotype in an otherwise wildtype animal.

An upstream activating sequence or upstream activation sequence (UAS) is a cis-acting regulatory sequence. It is distinct from the promoter and increases the expression of a neighbouring gene. Due to its essential role in activating transcription, the upstream activating sequence is often considered to be analogous to the function of the enhancer in multicellular eukaryotes. Upstream activation sequences are a crucial part of induction, enhancing the expression of the protein of interest through increased transcriptional activity. The upstream activation sequence is found adjacently upstream to a minimal promoter and serves as a binding site for transactivators. If the transcriptional transactivator does not bind to the UAS in the proper orientation then transcription cannot begin. To further understand the function of an upstream activation sequence, it is beneficial to see its role in the cascade of events that lead to transcription activation. The pathway begins when activators bind to their target at the UAS recruiting a mediator. A TATA-binding protein subunit of a transcription factor then binds to the TATA box, recruiting additional transcription factors. The mediator then recruits RNA polymerase II to the pre-initiation complex. Once initiated, RNA polymerase II is released from the complex and transcription begins.

Genetic ablation occurs when a gene is deemed “null” through the homologous genetic recombination of a gene. It is utilized in the selective suppression of a specific cell line or cell type. This genetic engineering technique does not limit growth suppression to just the activity of an individual gene. Specific cell ablation enables the examination of the in vivo activity of cells. An example of this method in action can be seen through the production of a knockout mouse. This is accomplished through the administration of one or more transgenes into a fertilized mouse oocyte’s pronucleus. Afterwards, it is reimplanted into a host mother, who then births a transgenic mouse. The transgenic mouse carries one copy of the transgene3 out of several hundred. From these mice, a homozygous colony can be created through breeding.

Pigment dispersing factor (pdf) is a gene that encodes the protein PDF, which is part of a large family of neuropeptides. Its hormonal product, pigment dispersing hormone (PDH), was named for the diurnal pigment movement effect it has in crustacean retinal cells upon its initial discovery in the central nervous system of arthropods. The movement and aggregation of pigments in retina cells and extra-retinal cells is hypothesized to be under a split hormonal control mechanism. One hormonal set is responsible for concentrating chromatophoral pigment by responding to changes in the organism's exposure time to darkness. Another hormonal set is responsible for dispersion and responds to the light cycle. However, insect pdf genes do not function in such pigment migration since they lack the chromatophore.

<span class="mw-page-title-main">Enhancer-FACS-seq</span>

Enhancer-FACS-seq (eFS), developed by the Bulyk lab at Brigham and Women’s Hospital and Harvard Medical School, is a highly parallel enhancer assay that aims for the identification of active, tissue-specific transcriptional enhancers, in the context of whole Drosophila melanogaster embryos. This technology replaces the use of microscopy to screen for tissue-specific enhancers with fluorescence activated cell sorting (FACS) of dissociated cells from whole embryos, combined with identification by high-throughput Illumina sequencing.

<span class="mw-page-title-main">Roger Brent</span> American biologist

Roger Brent is an American biologist known for his work on gene regulation and systems biology. He studies the quantitative behaviors of cell signaling systems and the origins and consequences of variation in them. He is Full Member in the Division of Basic Sciences at the Fred Hutchinson Cancer Research Center and an Affiliate Professor of Genome Sciences at the University of Washington.

The Gal4 transcription factor is a positive regulator of gene expression of galactose-induced genes. This protein represents a large fungal family of transcription factors, Gal4 family, which includes over 50 members in the yeast Saccharomyces cerevisiae e.g. Oaf1, Pip2, Pdr1, Pdr3, Leu3.

References

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  11. ↑ Afify A, Betz JF, Riabinina O, Lahondère C, Potter CJ (October 2019). "Commonly Used Insect Repellents Hide Human Odors from Anopheles Mosquitoes". Current Biology. 29 (21): 3669–3680.e5. doi:10.1016/j.cub.2019.09.007. PMC   6832857 . PMID   31630950.
  12. ↑ Matthews BJ, Younger MA, Vosshall LB (May 2019). "Aedes aegypti". eLife. 8: e43963. doi: 10.7554/eLife.43963 . PMC   6597239 . PMID   31112133.
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