Reverse genetics in zebrafish

2000 ◽  
Vol 2 (2) ◽  
pp. 37-48 ◽  
Author(s):  
ARNE C. LEKVEN ◽  
KATHRYN ANN HELDE ◽  
CHRISTOPHER J. THORPE ◽  
REBECCA ROOKE ◽  
RANDALL T. MOON
Keyword(s):  
Reverse Genetics ◽  
Molecular Genetic ◽  
Point Mutations ◽  
Model System ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Loss Of Function ◽  
Complementation Groups ◽  
Reverse Genetic Approach ◽  
Subsequent Identification

Lekven, Arne C., Kathryn Ann Helde, Christopher J. Thorpe, Rebecca Rooke, and Randall T. Moon. Reverse genetics in zebrafish. Physiol Genomics 2: 37–48, 2000.—The zebrafish has become a popular model system for the study of vertebrate developmental biology because of its numerous strengths as a molecular genetic and embryological system. To determine the requirement for specific genes during embryogenesis, it is necessary to generate organisms carrying loss-of-function mutations. This can be accomplished in zebrafish through a reverse genetic approach. This review discusses the current techniques for generating mutations in known genes in zebrafish. These techniques include the generation of chromosomal deletions and the subsequent identification of complementation groups within deletions through noncomplementation assays. In addition, this review will discuss methods currently being evaluated that may improve the methods for finding mutations in a known sequence, including screening for randomly induced small deletions within genes and screening for randomly induced point mutations within specific genes.

scholarly journals RNA-binding proteins and circadian rhythms in Arabidopsis thaliana

2001 ◽  
Vol 356 (1415) ◽  
pp. 1755-1759 ◽  
Author(s):  
Dorothee Staiger
Keyword(s):  
Arabidopsis Thaliana ◽  
Feedback Loop ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Transcriptional Level ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Circadian Oscillators ◽  
Clock Proteins ◽  
Reverse Genetic Approach

An Arabidopsis transcript preferentially expressed at the end of the daily light period codes for the RNA–binding protein At GRP7. A reverse genetic approach in Arabidopsis thaliana has revealed its role in the generation of circadian rhythmicity: At GRP7 is part of a negative feedback loop through which it influences the oscillations of its own transcript. Biochemical and genetic experiments indicate a mechanism for this autoregulatory circuit: At grp7 gene transcription is rhythmically activated by the circadian clock during the day. The At GPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post–transcriptional level. In this respect, the At GRP7 feedback loop differs from known circadian oscillators in the fruitfly Drosophila and mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that the At GRP7 feedback loop may act within an output pathway from the Arabidopsis clock.

scholarly journals Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3′ to 5′ exoribonuclease Atrimmer 2 in Arabidopsis

2018 ◽  
Vol 115 (28) ◽  
pp. E6659-E6667 ◽  
Author(s):  
Xiaoyan Wang ◽  
Yuan Wang ◽  
Yongchao Dou ◽  
Lu Chen ◽  
Junli Wang ◽  
...  
Keyword(s):  
Small Rnas ◽  
Reverse Genetic ◽  
Asymmetric Effect ◽  
Loss Of Function ◽  
Initiation Step ◽  
Morphological Defects ◽  
Rna Stabilization ◽  
Type 3 ◽  
Argonaute 1 ◽  
Reverse Genetic Approach

The 3′ end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3′ to 5′ exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis. Loss-of-function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3′ to 5′ trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1. Unexpectedly, we observed a marked increase of 3′ to 5′ trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly.

scholarly journals Structural and phenotypic analysis of Chikungunya virus RNA replication elements

2019 ◽  
Vol 47 (17) ◽  
pp. 9296-9312 ◽  
Author(s):  
Catherine Kendall ◽  
Henna Khalid ◽  
Marietta Müller ◽  
Dominic H Banda ◽  
Alain Kohl ◽  
...  
Keyword(s):  
Rational Design ◽  
Chikungunya Virus ◽  
Rna Replication ◽  
Host Cells ◽  
Genome Replication ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Phenotypic Analysis ◽  
Efficient Replication ◽  
Reverse Genetic Approach

Abstract Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5′ region of the CHIKV genome using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5′UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.

scholarly journals A reverse genetic approach for generating gene replacement mutants in Ustilago maydis

2004 ◽  
Vol 272 (4) ◽  
pp. 488-488 ◽  
Author(s):  
A. Brachmann ◽  
J. König ◽  
C. Julius ◽  
M. Feldbrügge
Keyword(s):  
Ustilago Maydis ◽  
Gene Replacement ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Reverse Genetic Approach

scholarly journals Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta

2020 ◽  
Author(s):  
David S. Booth ◽  
Nicole King
Keyword(s):  
Genome Editing ◽  
Reverse Genetics ◽  
Selectable Marker ◽  
Model System ◽  
Genetic Screen ◽  
Reverse Genetic ◽  
Genetic Screens ◽  
Multicellular Development ◽  
Forward Genetic Screen ◽  
Salpingoeca Rosetta

AbstractIn a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.

Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Michel Monod ◽  
Barbara Léchenne ◽  
Olivier Jousson ◽  
Daniela Grand ◽  
Christophe Zaugg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trichophyton Rubrum ◽  
Hydrolytic Activity ◽  
Cdna Libraries ◽  
Sole Nitrogen Source ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Leucine Aminopeptidases ◽  
Dipeptidyl Peptidases ◽  
Reverse Genetic Approach

The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58–65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.

A reverse genetic approach for generating gene replacement mutants in Ustilago maydis

2004 ◽  
Vol 272 (2) ◽  
pp. 216-226 ◽  
Author(s):  
A. Brachmann ◽  
J. König ◽  
C. Julius ◽  
M. Feldbrügge
Keyword(s):  
Ustilago Maydis ◽  
Gene Replacement ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Reverse Genetic Approach
Sign in / Sign up

Export Citation Format

Share Document