scholarly journals Creation of a Chloroplast Microsatellite Reporter for Detection of Replication Slippage in Chlamydomonas reinhardtii

2008 ◽  
Vol 7 (4) ◽  
pp. 639-646 ◽  
Author(s):  
Monica GuhaMajumdar ◽  
Ethan Dawson-Baglien ◽  
Barbara B. Sears
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Forensic Analysis ◽  
Direct Repeats ◽  
Reporter Construct ◽  
Chloroplast Microsatellite ◽  
Replication Slippage ◽  
Reading Frame ◽  
Viable Cells ◽  
Short Tandem

ABSTRACT Microsatellites are composed of short tandem direct repeats; deletions or duplications of those repeats through the process of replication slippage result in microsatellite instability relative to other genomic loci. Variation in repeat number occurs so frequently that microsatellites can be used for genotyping and forensic analysis. However, an accurate assessment of the rates of change can be difficult because the presence of many repeats makes it difficult to determine whether changes have occurred through single or multiple events. The current study was undertaken to experimentally assess the rates of replication slippage that occur in vivo in the chloroplast DNA of Chlamydomonas reinhardtii. A reporter construct was created in which a stretch of AAAG repeats was inserted into a functional gene to allow changes to be observed when they occurred at the synthetic microsatellite. Restoration of the reading frame occurred through replication slippage in 15 of every million viable cells. Since only one-third of the potential insertion/deletion events would restore the reading frame, the frequency of change could be deduced to be 4.5 × 10−5. Analysis of the slippage events showed that template slippage was the primary event, resulting in deletions rather than duplications. These findings contrasted with events observed in Escherichia coli during maintenance of the plasmid, where duplications were the rule.

scholarly journals Deletion Formation Between the Two Salmonella typhimurium Flagellin Genes Encoded on the Mini F Plasmid: Escherichia coli ssb Alleles Enhance Deletion Rates and Change Hot-Spot Preference for Deletion Endpoints

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 563-572 ◽  
Author(s):  
Takafumi Mukaihara ◽  
Masatoshi Enomoto
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Hot Spots ◽  
Hot Spot ◽  
Direct Repeats ◽  
Replication Slippage ◽  
Detection Systems ◽  
Deletion Formation ◽  
Homologous Sequences ◽  
Deletion Detection

Deletion formation between the 5′-mostly homologous sequences and between the 3′-homeologous sequences of the two Salmonella typhimurium flagellin genes was examined using plasmid-based deletion-detection systems in various Escherichia coli genetic backgrounds. Deletions in plasmid pLC103 occur between the 5′ sequences, but not between the 3′ sequences, in both RecA-independent and RecA-dependent ways. Because the former is predominant, deletion formation in a recA background depends on the length of homologous sequences between the two genes. Deletion rates were enhanced 30- to 50-fold by the mismatch repair defects, mutS, mutL and uvrD, and 250-fold by the ssb-3 allele, but the effect of the mismatch defects was canceled by the ΔrecA allele. Rates of the deletion between the 3′ sequences in plasmid pLC107 were enhanced 17- to 130-fold by ssb alleles, but not by other alleles. For deletions in pLC107, 96% of the endpoints in the recA+ background and 88% in ΔrecA were in the two hot spots of the 60- and 33-nucleotide (nt) homologous sequences, whereas in the ssb-3 background >50% of the endpoints were in four- to 14-nt direct repeats dispersed in the entire 3′ sequences. The deletion formation between the homeologous sequences is RecA-independent but depends on the length of consecutive homologies. The mutant ssb allele lowers this dependency and results in the increase in deletion rates. Roles of mutant SSB are discussed with relation to misalignment in replication slippage.

scholarly journals To Divide or Not to Divide? How Deuterium Affects Growth and Division of Chlamydomonas reinhardtii

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 861
Author(s):  
Veronika Kselíková ◽  
Vilém Zachleder ◽  
Kateřina Bišová
Keyword(s):  
Cell Cycle ◽  
Chlamydomonas Reinhardtii ◽  
Cell Cycle Progression ◽  
Model Organism ◽  
Cycle Progression ◽  
Synchronous Cultures ◽  
Multiple Fission ◽  
First Choice ◽  
High Doses

Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including Chlamydomonas reinhardtii, a well-established model organism in cell cycle studies, are no exception. Chlamydomonas reinhardtii, a green unicellular alga of the Chlorophyceae class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in C. reinhardtii. Synchronous cultures of C. reinhardtii were cultivated in growth medium containing 70 or 90% D2O. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of C. reinhardtii, contradicting the role of the “sizer” in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.

scholarly journals Complex Regulation of the Organic Hydroperoxide Resistance Gene (ohr) from XanthomonasInvolves OhrR, a Novel Organic Peroxide-Inducible Negative Regulator, and Posttranscriptional Modifications

2001 ◽  
Vol 183 (15) ◽  
pp. 4405-4412 ◽  
Author(s):  
Rojana Sukchawalit ◽  
Suvit Loprasert ◽  
Sopapan Atichartpongkul ◽  
Skorn Mongkolsuk
Keyword(s):  
Secondary Structure ◽  
Organic Peroxide ◽  
Primer Extension ◽  
Negative Regulator ◽  
Rnase Iii ◽  
Stem Loop ◽  
Strong Activity ◽  
Reading Frame ◽  
Bicistronic Mrna

ABSTRACT Analysis of the sequence immediate upstream of ohrrevealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression ofohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohrpromoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5′ ends ofohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5′ ends ofohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

scholarly journals Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster.

1996 ◽  
Vol 16 (6) ◽  
pp. 2977-2986 ◽  
Author(s):  
C Antoniewski ◽  
B Mugat ◽  
F Delbac ◽  
J A Lepesant
Keyword(s):  
Fat Body ◽  
Cell Transformation ◽  
Direct Repeat ◽  
Transgenic Animals ◽  
Transcriptional Level ◽  
Regulatory Sequences ◽  
Direct Repeats ◽  
Fbp1 Gene

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.

scholarly journals Two related ARID family proteins are alternative subunits of human SWI/SNF complexes

2004 ◽  
Vol 383 (2) ◽  
pp. 319-325 ◽  
Author(s):  
Xiaomei WANG ◽  
Norman G. NAGL ◽  
Deborah WILSKER ◽  
Michael VAN SCOY ◽  
Stephen PACCHIONE ◽  
...  
Keyword(s):  
Dna Binding ◽  
Direct Evidence ◽  
Potential Interaction ◽  
Atpase Subunit ◽  
Reading Frame ◽  
Atpase Subunits ◽  
A Subunit ◽  
Distinct Subunit ◽  
Binding Behaviour

p270 (ARID1A) is a member of the ARID family of DNA-binding proteins and a subunit of human SWI/SNF-related complexes, which use the energy generated by an integral ATPase subunit to remodel chromatin. ARID1B is an independent gene product with an open reading frame that is more than 60% identical with p270. We have generated monoclonal antibodies specific for either p270 or ARID1B to facilitate the investigation of ARID1B and its potential interaction with human SWI/SNF complexes in vivo. Immunocomplex analysis provides direct evidence that endogenous ARID1B is associated with SWI/SNF-related complexes and indicates that p270 and ARID1B, similar to the ATPase subunits BRG1 and hBRM, are alternative, mutually exclusive subunits of the complexes. The ARID-containing subunits are not specific to the ATPases. Each associates with both BRG1 and hBRM, thus increasing the number of distinct subunit combinations known to be present in cells. Analysis of the panels of cell lines indicates that ARID1B, similar to p270, has a broad tissue distribution. The ratio of p270/ARID1B in typical cells is approx. 3.5:1, and BRG1 is distributed proportionally between the two ARID subunits. Analysis of DNA-binding behaviour indicates that ARID1B binds DNA in a non-sequence-specific manner similar to p270.

scholarly journals Human Cytomegalovirus Open Reading Frame TRL11/IRL11 Encodes an Immunoglobulin G Fc-Binding Protein

2001 ◽  
Vol 75 (22) ◽  
pp. 11218-11221 ◽  
Author(s):  
Brendan N. Lilley ◽  
Hidde L. Ploegh ◽  
Rebecca S. Tirabassi
Keyword(s):  
Human Cytomegalovirus ◽  
Immunoglobulin G ◽  
Binding Protein ◽  
Fc Receptors ◽  
Binding Activity ◽  
Open Reading Frame ◽  
Membrane Glycoprotein ◽  
Type I ◽  
Reading Frame

ABSTRACT Several herpesviruses encode Fc receptors that may play a role in preventing antibody-mediated clearance of the virus in vivo. Human cytomegalovirus (HCMV) induces an Fc-binding activity in cells upon infection, but the gene that encodes this Fc-binding protein has not been identified. Here, we demonstrate that the HCMV AD169 open reading frame TRL11 and its identical copy, IRL11, encode a type I membrane glycoprotein that possesses IgG Fc-binding capabilities.

scholarly journals Regulation of Metalloprotease Gene Expression in Vibrio vulnificus by a Vibrio harveyi LuxR Homologue

2001 ◽  
Vol 183 (4) ◽  
pp. 1369-1375 ◽  
Author(s):  
Chung-Ping Shao ◽  
Lien-I Hor
Keyword(s):  
Parent Strain ◽  
Vibrio Vulnificus ◽  
Vibrio Harveyi ◽  
Negative Regulation ◽  
Open Reading Frame ◽  
Protease Gene ◽  
Reading Frame ◽  
Allelic Exchange ◽  
Exchange Technique

ABSTRACT Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyiLuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene ofV. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificusvirulence in mice.

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

scholarly journals In Vivo Interactions between Photosynthesis, Mitorespiration, and Chlororespiration in Chlamydomonas reinhardtii

2002 ◽  
Vol 129 (4) ◽  
pp. 1921-1928 ◽  
Author(s):  
Laurent Cournac ◽  
Gwendal Latouche ◽  
Zoran Cerovic ◽  
Kevin Redding ◽  
Jacques Ravenel ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii

scholarly journals Novel TRIM5 Isoforms Expressed by Macaca nemestrina

2007 ◽  
Vol 81 (22) ◽  
pp. 12210-12217 ◽  
Author(s):  
Greg Brennan ◽  
Yury Kozyrev ◽  
Toshiaki Kodama ◽  
Shiu-Lok Hu
Keyword(s):  
Coiled Coil ◽  
Macaca Nemestrina ◽  
Cdna Clones ◽  
Reading Frame ◽  
Spry Domain ◽  
Immunodeficiency Virus ◽  
Hiv Type 1 ◽  
Hiv 1

ABSTRACT The TRIM5 family of proteins contains a RING domain, one or two B boxes, and a coiled-coil domain. The TRIM5α isoform also encodes a C-terminal B30.2(SPRY) domain, differences within which define the breadth and potency of TRIM5α-mediated retroviral restriction. Because Macaca nemestrina animals are susceptible to some human immunodeficiency virus (HIV) isolates, we sought to determine if differences exist in the TRIM5 gene and transcripts of these animals. We identified a two-nucleotide deletion (Δ2) in the transcript at the 5′ terminus of exon 7 in all M. nemestrina TRIM5 cDNA clones examined. This frameshift results in a truncated protein of 300 amino acids lacking the B30.2(SPRY) domain, which we have named TRIM5θ. This deletion is likely due to a single nucleotide polymorphism that alters the 3′ splice site between intron 6 and exon 7. In some clones, a deletion of the entire 27-nucleotide exon 7 (Δexon7) resulted in the restoration of the TRIM5 open reading frame and the generation of another novel isoform, TRIM5η. There are 18 amino acid differences between M. nemestrina TRIM5η and Macaca mulatta TRIM5α, some of which are at or near locations previously shown to affect the breadth and potency of TRIM5α-mediated restriction. Infectivity assays performed on permissive CrFK cells stably transduced with TRIM5η or TRIM5θ show that these isoforms are incapable of restricting either HIV type 1 (HIV-1) or simian immunodeficiency virus infection. The expression of TRIM5 alleles incapable of restricting HIV-1 infection may contribute to the previously reported increased susceptibility of M. nemestrina to HIV-1 infection in vivo.

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