scholarly journals Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster

1989 ◽  
Vol 86 (17) ◽  
pp. 6696-6698 ◽  
Author(s):  
F A Laski ◽  
S Ganguly ◽  
P A Sharp ◽  
U L RajBhandary ◽  
G M Rubin
Keyword(s):  
Drosophila Melanogaster ◽  
Trna Gene ◽  
P Element ◽  
Female Sterility ◽  
Suppressor Trna ◽  
Amber Codon ◽  
Amber Suppressor ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Male And Female Sterility ◽  
And Function

Drosophila melanogaster strains with a stably incorporated amber suppressor tRNA gene have been generated. A tRNATyr gene was site specifically mutated to produce an anticodon sequence that recognizes the amber codon and then introduced into Drosophila by using P-element-mediated transformation. Transformants from four integration events were recovered. Two integrations resulted in both male and female sterility, whereas the other two resulted in male sterility but female fertility. Strains derived from the two female-fertile integration events were shown to have a low level of amber-suppressing activity by their ability to suppress an amber mutation in a chloramphenicol acetyltransferase gene.

scholarly journals Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

1998 ◽  
Vol 18 (8) ◽  
pp. 4418-4425 ◽  
Author(s):  
Ho-Jin Park ◽  
Uttam L. RajBhandary
Keyword(s):  
Mammalian Cells ◽  
Trna Synthetase ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
E Coli ◽  
Tetracycline Transactivator ◽  
Hela Cell Lines ◽  
Amber Codon ◽  
Amber Suppressor ◽  
Chloramphenicol Acetyltransferase Gene

ABSTRACT As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

Transformation of Drosophila melanogaster with a suppressor tRNA gene from Schizosaccharomyces pombe

Genome ◽  
1988 ◽  
Vol 30 (2) ◽  
pp. 211-217 ◽  
Author(s):  
Charles M. Molnar ◽  
Tove Reece ◽  
James A. Williams ◽  
John B. Bell
Keyword(s):  
Drosophila Melanogaster ◽  
Salivary Gland ◽  
In Situ Hybridization ◽  
Schizosaccharomyces Pombe ◽  
Southern Hybridization ◽  
Trna Gene ◽  
P Element ◽  
Suppressor Trna ◽  
Good Agreement

P-element mediated transformation was utilized to introduce a suppressor tRNA gene [Formula: see text] from Schizosaccharomyces pombe into Drosophila melanogaster. Thirteen independently transformed lines were characterized as to the number and cytological locations of the transposons. It was ascertained that the suppressor tRNA gene of interest was introduced into each transformed strain. The helper P element used (pπ25.1) allows further transposition to occur, and it was determined that from one to seven copies of the heterologous [Formula: see text] gene per strain were present among the respective transformed strains. The number of transposons per transformed line was established by in situ hybridization to salivary gland chromosomes as well as by Southern hybridization analyses and there was good agreement in the totals determined by these two techniques.Key words: Drosophila, Schizosaccharomyces, tRNA suppressor, transformation, transposon.

scholarly journals Cytoplasmic dynein (ddlc1) mutations cause morphogenetic defects and apoptotic cell death in Drosophila melanogaster.

1996 ◽  
Vol 16 (5) ◽  
pp. 1966-1977 ◽  
Author(s):  
T Dick ◽  
K Ray ◽  
H K Salz ◽  
W Chia
Keyword(s):  
Drosophila Melanogaster ◽  
Light Chain ◽  
Blot Analysis ◽  
Cytoplasmic Dynein ◽  
P Element ◽  
Female Sterility ◽  
Dynein Light Chain ◽  
Loss Of Function ◽  
Light Chain Gene

We report the molecular and genetic characterization of the cytoplasmic dynein light-chain gene, ddlc1, from Drosophila melanogaster. ddlc1 encodes the first cytoplasmic dynein light chain identified, and its genetic analysis represents the first in vivo characterization of cytoplasmic dynein function in higher eucaryotes. The ddlc1 gene maps to 4E1-2 and encodes an 89-amino-acid polypeptide with a high similarity to the axonemal 8-kDa outer-arm dynein light chain from Chlamydomonas flagella. Developmental Northern (RNA) blot analysis and ovary and embryo RNA in situ hybridizations indicate that the ddlc1 gene is expressed ubiquitously. Anti-DDLC1 antibody analyses show that the DDLC1 protein is localized in the cytoplasm. P-element-induced partial-loss-of-function mutations cause pleiotropic morphogenetic defects in bristle and wing development, as well as in oogenesis, and hence result in female sterility. The morphological abnormalities found in the ovaries are always associated with a loss of cellular shape and structure, as visualized by a disorganization of the actin cytoskeleton. Total-loss-of-function mutations cause lethality. A large proportion of mutant animals degenerate during embryogenesis, and the dying cells show morphological changes characteristic of apoptosis, namely, cell and nuclear condensation and fragmentation, as well as DNA degradation. Cloning of the human homolog of the ddlc1 gene, hdlc1, demonstrates that the dynein light-chain 1 is highly conserved in flies and humans. Northern blot analysis and epitope tagging show that the hdlc1 gene is ubiquitously expressed and that the human dynein light chain 1 is localized in the cytoplasm. hdlc1 maps to 14q24.

Drosophila melanogaster Importin α1 and α3 Can Replace Importin α2 During Spermatogenesis but Not Oogenesis

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 157-170 ◽  
Author(s):  
D Adam Mason ◽  
Robert J Fleming ◽  
David S Goldfarb
Keyword(s):  
Drosophila Melanogaster ◽  
Female Sterility ◽  
Degenerate Pcr ◽  
Male And Female ◽  
Importin Α ◽  
Localization Signal ◽  
Male And Female Sterility ◽  
A Site ◽  
Female Gametogenesis

Abstract Importin α’s mediate the nuclear transport of many classical nuclear localization signal (cNLS)-containing proteins. Multicellular animals contain multiple importin α genes, most of which fall into three conventional phylogenetic clades, here designated α1, α2, and α3. Using degenerate PCR we cloned Drosophila melanogaster importin α1, α2, and α3 genes, demonstrating that the complete conventional importin α gene family arose prior to the split between invertebrates and vertebrates. We have begun to analyze the genetic interactions among conventional importin α genes by studying their capacity to rescue the male and female sterility of importin α2 null flies. The sterility of α2 null males was rescued to similar extents by importin α1, α2, and α3 transgenes, suggesting that all three conventional importin α’s are capable of performing the important role of importin α2 during spermatogenesis. In contrast, sterility of α2 null females was rescued only by importin α2 transgenes, suggesting that it plays a paralog-specific role in oogenesis. Female infertility was also rescued by a mutant importin α2 transgene lacking a site that is normally phosphorylated in ovaries. These rescue experiments suggest that male and female gametogenesis have distinct requirements for importin α2.

scholarly journals P-element-induced control mutations at the r gene of Drosophila melanogaster.

1985 ◽  
Vol 5 (10) ◽  
pp. 2567-2574 ◽  
Author(s):  
S Tsubota ◽  
M Ashburner ◽  
P Schedl
Keyword(s):  
Drosophila Melanogaster ◽  
The Other ◽  
P Element ◽  
Female Sterility ◽  
R Gene ◽  
Temporal Expression ◽  
Base Pairs ◽  
P Elements ◽  
Adult Females ◽  
Regulatory Mutations

The P-M hybrid dysgenesis system was used to produce five putative regulatory mutations at the rudimentary locus, r. All five mutations were the result of insertions at the 5' end of the gene, upstream of the proposed start of transcription. All of the mutants displayed a leaky wing phenotype, and four of the mutants showed an uncoupling of the wing and female-sterility phenotypes, suggesting that they altered the normal spatial and temporal expression of the r gene. Four of the insertions were P elements. The fifth insertion, which was larger than an intact P element, consisted of a small P element connected to non-P-element DNA. Two of the mutants produced very little r transcript in adult females and were clustered 80 to 150 base pairs upstream of the start of transcription. The other three mutants had higher levels of r transcript in adult females and were clustered 440 to 500 base pairs upstream of the start of transcription. All of the data suggest that the insertions are in a 5' noncoding region of the r gene involved in the control of its spatial and temporal expression.

scholarly journals Structure and expression of hybrid dysgenesis-induced alleles of the ovarian tumor (otu) gene in Drosophila melanogaster.

Genetics ◽  
1993 ◽  
Vol 133 (2) ◽  
pp. 253-263
Author(s):  
G L Sass ◽  
J D Mohler ◽  
R C Walsh ◽  
L J Kalfayan ◽  
L L Searles
Keyword(s):  
Drosophila Melanogaster ◽  
Ovarian Tumor ◽  
P Element ◽  
Female Sterility ◽  
Protein Coding ◽  
Transcription Start Sites ◽  
Insertion And Deletion ◽  
Undifferentiated Cells ◽  
Egg Chambers ◽  
Mutant Phenotypes

Abstract Mutations at the ovarian tumor (otu) gene of Drosophila melanogaster cause female sterility and generate a range of ovarian phenotypes. Quiescent (QUI) mutants exhibit reduced germ cell proliferation; in oncogenic (ONC) mutants germ cells undergo uncontrolled proliferation generating excessive numbers of undifferentiated cells; the egg chambers of differentiated (DIF) mutants differentiate to variable degrees but fail to complete oogenesis. We have examined mutations caused by insertion and deletion of P elements at the otu gene. The P element insertion sites are upstream of the major otu transcription start sites. In deletion derivatives, the P element, regulatory regions and/or protein coding sequences have been removed. In both insertion and deletion mutants, the level of otu expression correlates directly with the severity of the phenotype: the absence of otu function produces the most severe QUI phenotype while the ONC mutants express lower levels of otu than those which are DIF. The results of this study demonstrate that the diverse mutant phenotypes of otu are the consequence of different levels of otu function.

scholarly journals Molecular genetics of the Drosophila melanogaster ovo locus, a gene required for sex determination of germline cells.

Genetics ◽  
1992 ◽  
Vol 130 (4) ◽  
pp. 791-803
Author(s):  
M D Garfinkel ◽  
A R Lohe ◽  
A P Mahowald
Keyword(s):  
Drosophila Melanogaster ◽  
Sex Determination ◽  
Genomic Dna ◽  
Transcription Unit ◽  
Complementation Group ◽  
P Element ◽  
Female Sterility ◽  
Germline Cells ◽  
Female Specific ◽  
Female Germline

Abstract The Drosophila melanogaster ovo gene is required for survival and differentiation of female germline cells, apparently playing a role in germline sex determination. We recovered 60 kb of genomic DNA from its genetic location at 4E1,2 on the X chromosome. A transcription unit coding for an apparently female-specific germline-dependent 5-kb poly(A)+ RNA size class is located substantially in a 7-kb region, within which three DNA-detectable lesions for mutations that inactivate the ovo function are located at two sites approximately 4 kb apart. The breakpoint of a deficiency that removes the neighboring lethal complementation group shavenbaby (svb) but leaves the ovo function intact maps approximately 5 kb to the molecular left of the leftmost ovo mutant site. A class of mutations that inactivates both the svb function and the ovo function affects genomic DNA between the two ovo sites. Sequences required for the two genetic functions are partly overlapping. In spite of this overlap, P element-mediated gene transfer of a 10-kb genomic DNA segment containing the 5-kb poly(A)+ RNA transcription unit rescues the female sterility phenotypes of ovo mutations, but not the svb lethality.

Sign in / Sign up

Export Citation Format

Share Document