Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates ...observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic contexts of the measurement. Furthermore, in vivo measurement methods must accommodate variation in translation, protein folding, and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered to be extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents, and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method to quantitatively describe promoter properties.
Leishmania species of the subgenus
Viannia are responsible for a large proportion of New World leishmaniasis. Here we report the development of a set of microsatellite markers which are able to ...discriminate between all species within the subgenus
Viannia, including the closely related species pairs:
Leishmania (V.)
braziliensis and
Leishmania (V.)
peruviana;
Leishmania (V.)
panamensis and
Leishmania (V.)
guyanensis. Potential species hybrids were uncovered in the analysis. These markers are sufficiently polymorphic such that within-species epidemiological, population and genetic studies are theoretically possible for all species analyzed.
Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad ...distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity.
Tubulin expression has been analysed as the insect stage of the protozoan parasite
Leishmania major differentiates from a non-infective to an infective form. This transformation of the promastigote ...stage occurs in vitro and analysis of β-tubulin mRNA expression in axenically grown promastigotes showed that a 2200 nt transcript is predominately expressed in non-infective promastigotes. The message contains a motif associated with mRNA intracellular localisation and its level is reduced by an order of magnitude in infective promastigotes through a mechanism involving RNA stability. A 3200 nt RNA, the major β-tubulin transcript in the infective stage, is encoded by a single copy gene at the 3′ end of the array that encodes the 2200 nt RNA. These RNAs, as well as a gene encoding a β-tubulin transcript highly up-regulated in the mammalian stage of the parasite, encode polypeptides that are apparently functionally equivalent but have highly diverged 3′ untranslated regions. This differential regulation of the dispersed isogenes may reflect the involvement of a mechanism altering tubulin synthesis during the
Leishmania life cycle. The analysis of α-tubulin RNA levels revealed the abundance of this message falls as promastigotes differentiate into an infectious stage and the transcript is destabilised in infective promastigotes. These data demonstrate that the regulation of mRNA half-life contributes to controlling gene expression as promastigotes differentiate into an infectious form.
Summary
Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in ...combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease‐mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.
Toxoplasma gondii is a common parasite of animals that also causes a zoonotic infection in humans. Previous studies have revealed a strongly clonal population structure that is shared between North ...America and Europe, while South American strains show greater genetic diversity and evidence of sexual recombination. The common inheritance of a monomorphic version of chromosome Ia (referred to as ChrIa*) among three clonal lineages from North America and Europe suggests that inheritance of this chromosome might underlie their recent clonal expansion. To further examine the diversity and distribution of ChrIa, we have analyzed additional strains with greater geographic diversity. Our findings reveal that the same haplotype of ChrIa* is found in the clonal lineages from North America and Europe and in older lineages in South America, where sexual recombination is more common. Although lineages from all three continents harbor the same conserved ChrIa* haplotype, strains from North America and Europe are genetically separate from those in South America, and these respective geographic regions show limited evidence of recent mixing. Genome-wide, array-based profiling of polymorphisms provided evidence for an ancestral flow from particular older southern lineages that gave rise to the clonal lineages now dominant in the north. Collectively, these data indicate that ChrIa* is widespread among nonclonal strains in South America and has more recently been associated with clonal expansion of specific lineages in North America and Europe. These findings have significant implications for the spread of genetic loci influencing transmission and virulence in pathogen populations.
Understanding parasite population structure is important for evaluating the potential spread of pathogenicity determinants between different geographic regions. Examining the genetic makeup of different isolates of Toxoplasma gondii from around the world revealed that chromosome Ia is highly homogeneous among lineages that predominate on different continents and within genomes that were otherwise quite divergent. This pattern of recent shared ancestry is highly unusual and suggests that some gene(s) found on this chromosome imparts an unusual fitness advantage that has resulted in its recent spread. Although the basis for the conservation of this particularly homogeneous chromosome is unknown, it may have implications for the transmission of infection and spread of human disease.
The expressed sequence tag (EST) effort in Toxoplasma gondii has generated a substantial amount of gene information. To exploit this valuable resource, we chose to study tgd057, a novel gene ...identified by a large number of ESTs that otherwise show no significant match to known sequences in the database. Northern analysis showed that tgd057 is transcribed in this tachyzoite. The complete cDNA sequence of tgd057 is 1169 bp in length. Sequence analysis revealed that tgd057 possibly adopts two polyadenylation sites, utilizes the fourth in-frame ATG for translation initiation, and codes for a secretory protein. The longest open reading frame for the tgd057 gene was cloned and expressed as a recombinant protein (rd57) in Escherichia coli. Western analysis revealed that serum against rd57 recognized a molecule of ~21 kDa in the tachyzoite protein extract. This suggests that the tgd057 gene is expressed in vivo in the parasite.
A more sensitive screen for
Leishmania major genes differentially expressed as the insect stage develops into an infectious form (metacyclogenesis) has been devised. The screen exploits the ...observation that in kinetoplastid protozoa differentially expressed genes are often associated with unique 3′ untranslated regions (
UTRs). To obtain probes encoding this region, cDNA is synthesised using an oligo-dT primer containing the universal vectorette sequence in the first strand reaction and an oligonucleotide comprising the spliced leader sequence in the second strand reaction. The cDNAs are then cleaved with
Sau3AI, ligated to the vectorette and the 3′
UTRs polymerase chain reaction (PCR) amplified using the universal vectorette sequence as the primer. Differential screening with PCR-amplified 3′
UTRs uncovered: (1) previously identified metacyclic-specific expressed genes; (2) cloned genes which had not been shown to be differentially regulated; and (3) a new gene identified only as a match to two identical
L. major expressed sequence tags (ESTs) that is upregulated in the infectious stage.
This paper describes functional and genetic studies on the macrophage resistance gene Lsh/Ity/Bcg first described almost two decades ago. Working in vitro with resident peritoneal, liver (Kupffer ...cells) and bone marrow derived macrophages from congenic B10 (LshS) and B10.L-LshR mice it has been possible to demonstrate that the final effector mechanism for the gene in regulating antileishmanial activity involves production of reactive nitrogen rather than reactive oxygen intermediates. This in turn is dependent upon priming/activation of macrophages for enhanced TNF-alpha release which acts back on the macrophage in an autocrine manner to increase nitric oxide production. The precise point at which Lsh acts to control macrophage priming/activation has not been identified, but studies of early response gene expression show differences in KC mRNA levels at 2 h after LPS stimulation, and in c-fos mRNA as early as 20 min after stimulation with PMA plus ionophore, in peritoneal macrophages from congenic LshS and LshR mice. Data available suggest that both negative and positive signals may be involved in macrophage priming/activation, with LshS macrophages down-regulating their capacity for continued response to the autocrine loop. Work in progress will examine the role of TPA and cAMP response element-binding proteins in regulating gene expression in Lsh congenic mice. A major new initiative has also commenced to clone the Lsh gene by reverse genetics using yeast artificial chromosomes to walk towards Lsh from the closet proximal and distal markers on mouse chromosome 1.
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