scholarly journals 5 S rRNA is involved in fidelity of translational reading frame.

Genetics ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 95-105
Author(s):  
J D Dinman ◽  
R B Wickner
Keyword(s):  
Rdna Locus ◽  
Wild Type ◽  
Lacz Reporter ◽  
Lacz Expression ◽  
Lacz Reporter Gene ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Episomal Vector ◽  
Electrophoretic Mobilities ◽  
Insertion Mutations

Abstract Chromosomal mutants (maintenance of frame = mof) in which the efficiency of -1 ribosomal frameshifting is increased can be isolated using constructs in which lacZ expression is dependent upon a -1 shift of reading frame. We isolate a new mof mutation, mof9, in Saccharomyces cerevisiae and show that it is complemented by both single and multi-copy 5 S rDNA clones. Two independent insertion mutations in the rDNA locus (rDNA::LEU2 and rDNA::URA3) also display the Mof- phenotype and are also complemented by single and multi-copy 5 S rDNA clones. Mutant 5 S rRNAs expressed from a plasmid as 20-50% of total 5 S rRNA in a wild-type host also induced the Mof- phenotype. The increase in frameshifting is greatest when the lacZ reporter gene is expressed on a high copy, episomal vector. No differences were found in 5 S rRNA copy number or electrophoretic mobilities in mof9 strains. Both mof9 and rDNA::LEU2 increase the efficiency of +1 frameshifting as well but have no effect on readthrough of UAG or UAA termination codons, indicating that not all translational specificity is affected. These data suggest a role for 5 S rRNA in the maintenance of frame in translation.

scholarly journals The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

1998 ◽  
Vol 18 (3) ◽  
pp. 1506-1516 ◽  
Author(s):  
Ying Cui ◽  
Jonathan D. Dinman ◽  
Terri Goss Kinzy ◽  
Stuart W. Peltz
Keyword(s):  
Site Selection ◽  
Start Site ◽  
Wild Type ◽  
Virus Propagation ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Translational Accuracy ◽  
Its Gene ◽  
Killer Virus

ABSTRACT Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene.mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

scholarly journals Mutagenesis of all Eight avr Genes in Xanthomonas campestris pv. campestris Had No Detected Effect on Pathogenicity, But One avr Gene Affected Race Specificity

2005 ◽  
Vol 18 (12) ◽  
pp. 1306-1317 ◽  
Author(s):  
Adriana Castañeda ◽  
Joseph D. Reddy ◽  
Basma El-Yacoubi ◽  
Dean W. Gabriel
Keyword(s):  
Xanthomonas Campestris ◽  
Subtractive Hybridization ◽  
Wild Type ◽  
Differential Host ◽  
Reading Frame ◽  
Race Specificity ◽  
Avr Gene ◽  
Xanthomonas Campestris Pv Campestris ◽  
Avr Genes ◽  
Insertion Mutations

Suppression subtractive hybridization (SSH) was used to identify genes present in the systemic crucifer black rot pathogen Xanthomonas campestris pv. campestris 528T but missing from the nonsystemic crucifer leaf spot pathogen, X. campestris pv. armoraciae 417. Among the DNA fragments unique to 528T was Xcc2109, one of eight putative avr genes identified in the published 528T genome (NC_003902). Individual and sequential deletion, insertion mutations, or both of all eight 528T avr gene loci were made, but no change in pathogenicity was observed with any combination of avr mutations, including a strain with all eight avr genes deleted. However, insertion or deletion mutants affecting the Xcc2109 locus lost avirulence (i.e., became virulent) on Florida Mustard, an X. campestris pv. campestris race-determining, differential host. The Xcc2109 open reading frame as annotated was cloned and found to be nonfunctional. A longer gene, encompassing Xcc2109 and here designated avrXccFM, was cloned and found to complement the Xcc2109 mutants and to confer avirulence to two additional wild-type X. campestris pv. campestris strains, thereby changing their races. Resistance in Florida Mustard to 528T strains carrying avrXccFM occurred without a typical hypersensitive response (HR) on leaves, although a vascular HR was observed in seedlings.

Disruption ofGja8(α8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation

Development ◽  
2002 ◽  
Vol 129 (1) ◽  
pp. 167-174 ◽  
Author(s):  
Pei Rong ◽  
Xin Wang ◽  
Ingrid Niesman ◽  
Ying Wu ◽  
Lucio E. Benedetti ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Cell Communication ◽  
Cell Maturation ◽  
Wild Type ◽  
Lacz Reporter ◽  
Lacz Reporter Gene ◽  
Gap Junction Channels ◽  
Protein Levels ◽  
Lens Growth ◽  
And Control

The development of the vertebrate lens utilizes a sophisticated cell-cell communication network via gap junction channels, which are made up of at least three connexin isoforms, α8 (Cx50), α3 (Cx46) and α1 (Cx43), and which are encoded by three different genes. In a previous study, we reported that, with a disruption of Gja3 (α3 connexin), mice developed nuclear cataracts with a normal sized lens. We show that Gja8tm1 (α8–/–) mice develop microphthalmia with small lenses and nuclear cataracts, while the α8 heterozygous (+/–) mice have relatively normal eyes and lenses. A comparative study of these α3 and α8 knockout mice showed that the protein levels of both α3 and α8 were independently regulated and there was no compensation for either the α3 or α8 protein from the wild-type allele when the other allele was disrupted. More interestingly, western blotting data indicated that the presence of α8 in the lens nucleus is dependent on α3 connexin, but not vice versa. The staining of the knock-in lacZ reporter gene showed the promoter activity of α8 connexin is much higher than that of α3 connexin in embryonic lenses and in adult lens epithelium. More importantly, a delayed denucleation process was observed in the interior fibers of the α8–/– lenses. Therefore, α8 connexin is required for proper fiber cell maturation and control of lens size.

scholarly journals Nuclear Proteins Nut1p and Nut2p Cooperate To Negatively Regulate a Swi4p-Dependent lacZ Reporter Gene inSaccharomyces cerevisiae

1998 ◽  
Vol 18 (8) ◽  
pp. 4707-4718 ◽  
Author(s):  
Ramon K. Tabtiang ◽  
Ira Herskowitz
Keyword(s):  
Binding Sites ◽  
Regulatory Region ◽  
Nuclear Proteins ◽  
Upstream Region ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Lacz Reporter Gene ◽  
Reading Frame ◽  
Sensitive Allele ◽  
Activation Sequence

ABSTRACT The URS2 region of the Saccharomyces cerevisiae HOupstream region contains 10 binding sites for the Swi4p/Swi6p transcription factor and confers Swi4p dependence for transcription. Using a hybrid promoter, UAS GAL (upstream activation sequence of GAL1)-URS2R, in which theGAL1-10 regulatory region is fused to the proximal 360 bp of URS2, we isolated mutants in which Swi4p is no longer required for transcription. Mutations of SIN4, ROX3,SRB8, SRB9, SRB10,SRB11, and two novel genes, NUT1 andNUT2, relieve the requirement of Swi4p for expression of this reporter. We found that NUT1 (open reading frame [ORF] YGL151w) is a nonessential gene, that NUT2 (ORF YPR168w) is essential, and that both Nut1p and Nut2p encode nuclear proteins. Deletion of NUT1 causes a constitutive, Swi4p-independent phenotype only in combination with thenut2-1 allele or an allele of CCR4. In contrast, inactivation of a temperature-sensitive allele ofNUT2, nut2-ts70, alone causes constitutivity.nut1Δ nut2-1 cells and sin4Δ cells exhibit Swi4p-independent expression of an ho-lacZ reporter but not of an intact ho gene. Likewise, a pPHO5-lacZconstruct is constitutively expressed in nut1 nut2 mutants relative to their wild-type counterparts. These results suggest that Nut1p, Nut2p, Sin4p, and Ccr4p define a group of proteins that negatively regulate transcription in a subtle manner which is revealed by artificial reporter genes.

Visualization and functional characterization of the developing murine cardiac conduction system

Development ◽  
2001 ◽  
Vol 128 (10) ◽  
pp. 1785-1792 ◽  
Author(s):  
S. Rentschler ◽  
D.M. Vaidya ◽  
H. Tamaddon ◽  
K. Degenhardt ◽  
D. Sassoon ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Optical Mapping ◽  
System Development ◽  
Conduction System ◽  
Cardiac Conduction ◽  
Cardiac Conduction System ◽  
Conductive Network ◽  
Lacz Reporter ◽  
Lacz Expression ◽  
Lacz Reporter Gene

The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. The molecular mechanisms regulating the specification and patterning of cells that form this conductive network are largely unknown. Studies in avian models have suggested that components of the cardiac conduction system arise from progressive recruitment of cardiomyogenic progenitors, potentially influenced by inductive effects from the neighboring coronary vasculature. However, relatively little is known about the process of conduction system development in mammalian species, especially in the mouse, where even the histological identification of the conductive network remains problematic. We have identified a line of transgenic mice where lacZ reporter gene expression delineates the developing and mature murine cardiac conduction system, extending proximally from the sinoatrial node to the distal Purkinje fibers. Optical mapping of cardiac electrical activity using a voltage-sensitive dye confirms that cells identified by the lacZ reporter gene are indeed components of the specialized conduction system. Analysis of lacZ expression during sequential stages of cardiogenesis provides a detailed view of the maturation of the conductive network and demonstrates that patterning occurs surprisingly early in embryogenesis. Moreover, optical mapping studies of embryonic hearts demonstrate that a murine His-Purkinje system is functioning well before septation has completed. Thus, these studies describe a novel marker of the murine cardiac conduction system that identifies this specialized network of cells throughout cardiac development. Analysis of lacZ expression and optical mapping data highlight important differences between murine and avian conduction system development. Finally, this line of transgenic mice provides a novel tool for exploring the molecular circuitry controlling mammalian conduction system development and should be invaluable in studies of developmental mutants with potential structural or functional conduction system defects.

scholarly journals The suppressor of Hairy-wing protein regulates the tissue-specific expression of the Drosophila gypsy retrotransposon.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 215-228 ◽  
Author(s):  
P A Smith ◽  
V G Corces
Keyword(s):  
Reporter Gene ◽  
Leucine Zipper ◽  
Fat Body ◽  
Lacz Reporter ◽  
Lacz Expression ◽  
Lacz Reporter Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Gypsy Retrotransposon ◽  
Beta Galactosidase

Abstract The gypsy retrotransposon of Drosophila melanogaster causes mutations that show temporal and tissue-specific phenotypes. These mutant phenotypes can be reversed by mutations in su(Hw), a gene that also regulates the transcription of the gypsy element. Gypsy encodes a full-length 7.0-kb RNA that is expressed in the salivary gland precursors and fat body of the embryo, imaginal discs and fat body of larvae, and fat body and ovaries of adult females. The su(Hw)-binding region inserted upstream of the promoter of a lacZ reporter gene can induce beta-galactosidase expression in a subset of the embryonic and larval tissues where gypsy is normally transcribed. This expression is dependent on the presence of a functional su(Hw) product, suggesting that this protein is a positive activator of gypsy transcription. Flies transformed with a construct in which the 5' LTR and leader sequences of gypsy are fused to lacZ show beta-galactosidase expression in all tissues where gypsy is normally expressed, indicating that sequences other than the su(Hw)-binding site are required for proper spatial and temporal expression of gypsy. Mutations in the zinc fingers of su(Hw) affect its ability to bind DNA and to induce transcription of the lacZ reporter gene. Two other structural domains of su(Hw) also play an important role in transcriptional regulation of gypsy. Deletion of the amino-terminal acidic domain results in the loss of lacZ expression in larval fat body and adult ovaries, whereas mutations in the leucine zipper region result in an increase of lacZ expression in larval fat body and a decrease in adult ovaries. These effects might be the result of interactions of su(Hw) with activator and repressor proteins through the acidic and leucine zipper domains to produce the final pattern of tissue-specific expression of gypsy.

scholarly journals Interactions of Drosophila Ultrabithorax Regulatory Regions With Native and Foreign Promoters

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 123-137 ◽  
Author(s):  
Fernando Casares ◽  
Welcome Bender ◽  
John Merriam ◽  
Ernesto Sánchez-Herrero
Keyword(s):  
P Element ◽  
Lacz Gene ◽  
Bithorax Complex ◽  
Lacz Reporter ◽  
Normal Pattern ◽  
Lacz Reporter Gene ◽  
Regulatory Regions ◽  
Long Range Interactions ◽  
In Trans ◽  
Dependent Interaction

The Ultrabithorax (Ubx) gene of the Drosophila bithorax complex is required to specify parasegments 5 and 6. Two P-element “enhancer traps” have been recovered within the locus that contain the bacterial lacZ gene under the control of the P-element promoter. The P insertion that is closer to the Ubx promoter expresses lucZ in a pattern similar to that of the normal Ubx gene, but also in parasegment 4 during embryonic development. Two deletions have been recovered that remove the normal Ubx promoter plus several kilobases on either side, but retain the lacZ reporter gene. The lacZ patterns from the deletion derivatives closely match the normal pattern of Ubx expression in late embryos and imaginal discs. The lacZ genes in the deletion derivatives are also negatively regulated by Ubx and activated in trans by Contrabithorax mutations, again like the normal Ubx gene. Thus, the deleted regions, including several kilobases around the Ubx promoter, are not required for long range interactions with Ubx regulatory regions. The deletion derivatives also stimulate transvection, a pairing-dependent interaction with the Ubx promoter on the homologous chromosome.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

scholarly journals A K+ Uptake Protein, TrkA, Is Required for Serum, Protamine, and Polymyxin B Resistance in Vibrio vulnificus

2004 ◽  
Vol 72 (2) ◽  
pp. 629-636 ◽  
Author(s):  
Yu-Chung Chen ◽  
Yin-Ching Chuang ◽  
Chun-Chin Chang ◽  
Chii-Ling Jeang ◽  
Ming-Chung Chang
Keyword(s):  
Human Serum ◽  
Vibrio Vulnificus ◽  
Polymyxin B ◽  
Wild Type ◽  
Gene Encoding ◽  
Reading Frame ◽  
Isogenic Mutant ◽  
Virulence Attenuation ◽  
Insertional Inactivation ◽  
Transposon Mutants

ABSTRACT Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world. To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of V. vulnificus and screened them for hypersensitivity to human serum. Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as trkA, a gene encoding an amino acid sequence showing high identity to that of TrkA of Vibrio alginolyticus, a protein required for the uptake of potassium. A trkA isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type. At K+ concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain. In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.

Differential elimination of rDNA genes in bobbed mutants of Drosophila melanogaster

1986 ◽  
Vol 6 (4) ◽  
pp. 1023-1031
Author(s):  
R Terracol ◽  
N Prud'homme
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Copy Number ◽  
Rdna Locus ◽  
Gene Copy ◽  
Specific Gene ◽  
Type I ◽  
28S Rrna ◽  
Wild Type ◽  
Y Chromosomes ◽  
Genes Encoding

In Drosophila melanogaster, the multiply repeated genes encoding 18S and 28S rRNA are located on the X and Y chromosomes. A large percentage of these repeats are interrupted in the 28S region by insertions of two types. We compared the restriction patterns from a subcloned wild-type Oregon R strain to those of spontaneous and ethyl methanesulfonate-induced bobbed mutants. Bobbed mutations were found to be deficiencies that modified the organization of the rDNA locus. Genes without insertions were deleted about twice as often as genes with type I insertions. Type II insertion genes were not decreased in number, except in the mutant having the most bobbed phenotype. Reversion to wild type was associated with an increase in gene copy number, affecting exclusively genes without insertions. One hypothesis which explains these results is the partial clustering of genes by type. The initial deletion could then be due either to an unequal crossover or to loss of material without exchange. Some of our findings indicated that deletion may be associated with an amplification phenomenon, the magnitude of which would be dependent on the amount of clustering of specific gene types at the locus.

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