scholarly journals Structure and transcription of the actin gene of Trypanosoma brucei.

1988 ◽  
Vol 8 (5) ◽  
pp. 2166-2176 ◽  
Author(s):  
M F Ben Amar ◽  
A Pays ◽  
P Tebabi ◽  
B Dero ◽  
T Seebeck ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Splice Site ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Actin Gene ◽  
S1 Nuclease ◽  
Actin Genes ◽  
Gene Copies ◽  
Actin Mrna

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

Structure and transcription of the actin gene of Trypanosoma brucei

1988 ◽  
Vol 8 (5) ◽  
pp. 2166-2176 ◽  
Author(s):  
M F Ben Amar ◽  
A Pays ◽  
P Tebabi ◽  
B Dero ◽  
T Seebeck ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Splice Site ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Actin Gene ◽  
S1 Nuclease ◽  
Actin Genes ◽  
Gene Copies ◽  
Actin Mrna

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

scholarly journals In vitro culture of freshly isolated Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes

2021 ◽  
Vol 15 (9) ◽  
pp. e0009738
Author(s):  
Julius Mulindwa ◽  
Geofrey Ssentamu ◽  
Enock Matovu ◽  
Kevin Kamanyi Marucha ◽  
Francisco Aresta-Branco ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Field Isolate ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Trypanosoma Brucei Brucei ◽  
High Coverage ◽  
Copy Numbers ◽  
Laboratory Rodents ◽  
Copy Number Changes

Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were obtained from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains. MAK65 and MAK98, which have undergone only 3 rodent passages since isolation from Ugandan cattle. High-coverage sequencing revealed that adaptation of the parasites to culture was accompanied by changes in gene copy numbers. T. brucei has so far been considered to be uniformly diploid, but we also found trisomy of chromosome 5 not only in one Lister 427 culture, but also in the MAK98 field isolate. Trisomy of chromosome 6, and increased copies of other chromosome segments, were also seen in established cultured lines. The two new T. brucei strains should be useful to researchers interested in trypanosome differentiation and pathogenicity. Initial results suggested that the two strains have differing infection patterns in rodents. MAK65 is uniformly diploid and grew more reproducibly in bloodstream-form culture than MAK98.

scholarly journals The effect of in vitro culture on unicellular eukaryotes: adaptation of Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes.

2021 ◽  
Author(s):  
Julius Mulindwa ◽  
Geoffrey Ssentamu ◽  
Enock Matovu ◽  
Kevin Kamanyi Marucha ◽  
Francisco Aresta-Branco ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Trypanosoma Brucei ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Trypanosoma Brucei Brucei ◽  
Unicellular Eukaryotes ◽  
Similar Work ◽  
Genes Encoding ◽  
Copy Number Changes

Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were taken from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains. MAK65 and MAK98, which have undergone only 3 rodent passages since isolation from Ugandan cattle. Adaptation of these strains to culture was accompanied by changes in gene copy numbers, some of which were also evident when other lab-adapted strains, field isolates of T. rhodesiense, and the genome strain TREU927 were compared. Reproducible increases were seen for genes encoding histones, enzymes of mRNA processing and degradation, the cytosolic chaperone HSP70, and two proteins required for the DNA damage response. These results indicate that similar work with other eukaryotic pathogens would be worthwhile. Meanwhile, the two new T. brucei strains should be useful to researchers interested in trypanosome differentiation and pathogenicity. They have differing pathogenicities in mice and may also differ in their propensity for stumpy-form differentiation, as judged by morphology and mRNA expression. MAK65 grows better than MAK98 in bloodstream-form culture, and is uniformly diploid, whereas MAK98 is triploid for chromosome 5. Genome sequence exceeding 100-fold coverage is available for both strains.

scholarly journals Members of the Arabidopsis Actin Gene Family Are Widely Dispersed in the Genome

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 663-675
Author(s):  
E C McKinney ◽  
R B Meagher
Keyword(s):  
Gene Family ◽  
Vascular Plant ◽  
Gene Copy Number ◽  
Rapid Expansion ◽  
Gene Copy ◽  
Actin Gene ◽  
Artificial Chromosomes ◽  
Plant Genomes ◽  
Actin Genes ◽  
Duplication Events

Abstract Plant genomes are subjected to a variety of DNA turnover mechanisms that are thought to result in rapid expansion and presumable contraction of gene copy number. The evolutionary history of the 10 actin genes in Arabidopsis thaliana is well characterized and can be traced to the origin of vascular plant genomes. Knowledge about the genomic position of each actin gene may be the key to tracing landmark genomic duplication events that define plant families or genera and facilitate further mutant isolation. All 10 actin genes were mapped by following the segregation of cleaved amplified polymorphisms between two ecotypes and identifying actin gene locations among yeast artificial chromosomes. The Arabidopsis actin genes are widely dispersed on four different chromosomes (1, 2, 3, and 5). Even the members of three closely related and recently duplicated pairs of actin genes are unlinked. Several other cytoskeletal genes (profilins, tubulins) that might have evolved in concert with actins were also mapped, but showed few patterns consistent with that evoulutionary history. Thus, the events that gave rise to the actin gene family have been obscured either by the duplication of very small genic fragments or by extensive rearrangement of the genome.

scholarly journals Nosocomial Outbreak of Carbapenemase-Producing Proteus mirabilis With Two Novel Salmonella Genomic Island 1 Variants Carrying Different blaNDM–1 Gene Copies in China

2022 ◽  
Vol 12 ◽  
Author(s):  
Lang Yang ◽  
Hong He ◽  
Qichao Chen ◽  
Kaiying Wang ◽  
Yanfeng Lin ◽  
...  
Keyword(s):  
Proteus Mirabilis ◽  
Copy Number ◽  
Genomic Island ◽  
Gene Copy Number ◽  
Carbapenem Resistance ◽  
Multidrug Resistant ◽  
Gene Copy ◽  
Nosocomial Outbreak ◽  
Gene Copy Number Variation ◽  
Gene Copies

NDM-1-producing multidrug-resistant Proteus mirabilis brings formidable clinical challenges. We report a nosocomial outbreak of carbapenem-resistant P. mirabilis in China. Six P. mirabilis strains collected in the same ward showed close phylogenetic relatedness, indicating clonal expansion. Illumina and MinION sequencing revealed that three isolates harbored a novel Salmonella genomic island 1 carrying a blaNDM–1 gene (SGI1-1NDM), while three other isolates showed elevated carbapenem resistance and carried a similar SGI1 but with two blaNDM–1 gene copies (SGI1-2NDM). Four new single nucleotide mutations were present in the genomes of the two-blaNDM–1-harboring isolates, indicating later emergence of the SGI1-2NDM structure. Passage experiments indicated that both SGI variants were stably persistent in this clone without blaNDM–1 copy number changes. This study characterizes two novel blaNDM–1-harboring SGI1 variants in P. mirabilis and provides a new insight into resistance gene copy number variation in bacteria.

scholarly journals Effect of EPSPS Gene Copy Number and Glyphosate Selection on Fitness of Glyphosate-Resistant Bassia Scoparia in The Field

2021 ◽  
Author(s):  
Charlemagne Ajoc Lim ◽  
Prashant Jha ◽  
Vipan Kumar ◽  
Alan T. Dyer
Keyword(s):  
Sugar Beet ◽  
Seed Production ◽  
Great Plains ◽  
Copy Number ◽  
Gene Copy Number ◽  
Reproductive Traits ◽  
Gene Copy ◽  
Epsps Gene ◽  
Gene Copies

Abstract The widespread evolution of glyphosate-resistant (GR) Bassia scoparia in the U.S. Great Plains poses a serious threat to the long-term sustainability of GR sugar beet. Glyphosate resistance in B. scoparia is due to an increase in the EPSPS (5-enolpyruvyl-shikimate-3-phosphate) gene copy number. The variation in EPSPS gene copies among individuals from within a single GR B. scoparia population indicated a differential response to glyphosate selection. We tested the hypothesis of reduced GR B. scoparia fitness (reproductive traits) to increasing glyphosate rates (applied as single or sequential applications) potentially experienced within a GR sugar beet field. The variation in EPSPS gene copy number and total glyphosate rate (single or sequential applications) did not influence any of the reproductive traits of GR B. scoparia, except seed production. Sequential applications of glyphosate with a total rate of 2,214 g ae ha− 1 or higher prevented seed production in B. scoparia plants with 2–4 (low levels of resistance) and 5–6 (moderate levels of resistance) EPSPS gene copies. Timely sequential applications of glyphosate (full recommended rates) can potentially slow down the evolution of GR B. scoparia with low to moderate levels of resistance (2–6 EPSPS gene copies), but any survivors (highly-resistant individuals with ≥ 8 EPSPS gene copies) need to be mechanically removed before flowering from GR sugar beet fields. This research warrants the need to adopt ecologically based, multi-tactic strategies to reduce exposure of B. scoparia to glyphosate in GR sugar beet.

Molecular cloning and characterization of mutant and wild-type human beta-actin genes

1984 ◽  
Vol 4 (10) ◽  
pp. 1961-1969
Author(s):  
J Leavitt ◽  
P Gunning ◽  
P Porreca ◽  
S Y Ng ◽  
C S Lin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Human Fibroblasts ◽  
Actin Gene ◽  
Actin Genes ◽  
Beta Actin ◽  
Gene Libraries ◽  
Actin Mrna ◽  
Dna Fragment ◽  
Specific Sequences

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.

Variable Inheritance of AmplifiedEPSPSGene Copies in Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri)

Weed Science ◽  
2018 ◽  
Vol 67 (2) ◽  
pp. 176-182 ◽  
Author(s):  
Darci A. Giacomini ◽  
Philip Westra ◽  
Sarah M. Ward
Keyword(s):  
Copy Number ◽  
Single Gene ◽  
Gene Copy Number ◽  
Transgressive Segregation ◽  
Palmer Amaranth ◽  
Gene Copy ◽  
Amaranthus Palmeri ◽  
Gene Copies ◽  
Central United States ◽  
Free Environment

AbstractGlyphosate-resistant (GR) Palmer amaranth (Amaranthus palmeriS. Watson) is considered one of the most troublesome weeds in the southern and central United States, but results of previous research to determine the mode of inheritance of this trait have been conflicting and inconclusive. In this study, we examined segregation patterns ofEPSPSgene-copy numbers in F1and F2generations ofA. palmeriand found no evidence of a Mendelian single-gene pattern of inheritance. Transgressive segregation for copy number was exhibited by several F1and all of the F2families, most likely the product ofEPSPScopy-number variation within each plant. This variation was confirmed by assaying gene-copy number across clonal generations and among individual shoots on the same plant, demonstrating thatEPSPSamplification levels vary significantly within a single plant. Increases and decreases in copy number occurred in a controlled, stress-free environment in the absence of glyphosate, indicating thatEPSPSgene amplification is a random and variable process within the plant. The ability ofA. palmerito gain or loseEPSPSgene copies is a valuable adaptive trait, allowing this species to respond rapidly to selection pressures and changing environments.

scholarly journals Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

1992 ◽  
Vol 117 (4) ◽  
pp. 787-797 ◽  
Author(s):  
C Lloyd ◽  
G Schevzov ◽  
P Gunning
Keyword(s):  
Actin Cytoskeleton ◽  
Feedback Regulation ◽  
Cytochalasin D ◽  
Actin Gene ◽  
Down Regulation ◽  
Actin Genes ◽  
Beta Actin ◽  
Actin Mrna ◽  
Actin Protein

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.

scholarly journals Influence of Promoter, Gene Copy Number, and Preexisting Immunity on Humoral and Cellular Responses to a Vectored Antigen Delivered by a Salmonella enterica Vaccine

2008 ◽  
Vol 16 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Manvendra Saxena ◽  
Peter J. Coloe ◽  
Peter M. Smooker
Keyword(s):  
Salmonella Enterica ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Heterologous Antigen ◽  
Cell Responses ◽  
Vaccine Trials ◽  
Serovar Typhimurium ◽  
Promoter Gene ◽  
Dna Plasmid

ABSTRACT Attenuated Salmonella strains are currently in production as vaccines for protection of animals against salmonellosis. Such commercial strains offer the potential to deliver heterologous antigen to protect animals against other diseases. One vaccine strain, attenuated Salmonella enterica serovar Typhimurium (STM-1), was tested for the ability to deliver ovalbumin and to induce immune responses in mice. Two vaccine trials were performed testing the influence of promoter choice, the location of the encoding DNA (plasmid or chromosome), and the effect of preexisting homologous or heterologous immunity. The results demonstrated that humoral and T-cell responses were induced from either of two promoters, from either the plasmid or the chromosome, and that preexposure to the empty homologous vector, STM-1, or the heterologous vector, S. enterica serovar Enteritidis, had no detrimental effect on subsequent antigen-specific responses. In the case of homologous preexposure, responses were generally greater, and this was correlated with an increased uptake of Salmonella by macrophages in vitro after opsonization with immune sera.

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