Adaptation of Musca domestica L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

Background

The housefly, Musca domestica, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain.

Results

We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F₁, F₁₃ and F₂₉ to test how gene expression changes following laboratory adaption. Four (CYP6A1, CYP6A36, CYP6D3, CYP6G4) of five investigated P450 genes adapted to breeding by decreasing expression. CYP6D1 showed higher female expression in F₂₉ than in F₁. For males, about half of the genes accessed in the global gene expression were up-regulated in F₁₃ and F₂₉ in comparison with the F₁ population. In females, 60% of the genes were up-regulated in F₁₃ in comparison with F₁, while 33% were up-regulated in F₂₉. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F₁₃ flies in comparison with F₁. Gene expression then increased from F₁₃ to F₂₉ in males and decreased further in females.

Conclusion

The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains.     

Oxidative stresses elevate the expression of cytochrome c peroxidase in Saccharomyces cerevisiae

Cytochrome c peroxidase (CcP) uses hydrogen peroxide as an electron acceptor to oxidize cytochrome c (Cc) in the mitochondrial intermembrane space. A null allele of yeast CCP1 gene encoding CcP was created by one-step gene disruption method in a diploid yeast strain. Haploid yeast cells with the disrupted CCP1 gene were viable and able to grow in a medium containing lactic acid or glycerol as an energy source, indicating that CcP is not essential for both cell viability and respiration. However, CCP1-disrupted cells were more sensitive to H₂O₂ than wild-type cells. We also constructed a CCP1–lacZ fused gene and integrated this gene into yeast chromosomal DNA to monitor the expression of CCP1 gene. We found that expression of CCP1 gene increases under respiratory culture conditions and by treatments with H₂O₂. These results hint that the biological function of CcP is to reduce H₂O₂ generated during aerobic respiratory process. Moreover, expression of CCP1 gene increased by treatments with peroxynitrite, indicating that CcP may act as a peroxynitrite scavenger.     

The effect of duplications in the T-DNA on the stability of manifestation of heterologous genes in transgenic tobacco plants

The duplication of uidA gene within T-DNA was shown to disturb stability of expression of another marker gene, nptII, in the second generation (T₂) of selfed initial transformants and in F₁ hybrids of the crosses with nontransgenic tobacco. Hybridological analysis of the progeny resulting from various crosses involving T₁ plants demonstrated that the expression of nptII gene was impaired in the hybrids that were hemizygous for the inactivated copy of uidA gene.     

Induction of gene expression in bacteria at optimal growth temperatures

Traditional temperature-sensitive systems use either heat shock (40–42 °C) or cold shock (15–23 °C) to induce gene expression at temperatures that are not the optimal temperature for host cell growth (37 °C). This impacts the overall productivity and yield by disturbing cell growth and cellular metabolism. Here, we have developed a new system which controls gene expression in Escherichia coli at more permissive temperatures. The temperature-sensitive cI857-P _L system and the classic lacI-P _lacO system were connected in series to control the gene of interest. When the culture temperature was lowered, the thermolabile cI857 repressor was activated and blocked the expression of lacI from P _L. Subsequently, the decrease of LacI derepressed the expression of gene of interest from P _lacO . Using a green fluorescent protein marker, we demonstrated that (1) gene expression was tightly regulated at 42 °C and strongly induced by lowering temperature to 25–37 °C; (2) different levels of gene expression can be induced by varying culture temperature; and (3) gene expression after induction was sustained until the end of the log phase. We then applied this system in the biosynthesis of acetoin and demonstrated that high yield and production could be achieved using temperature induction. The ability to express proteins at optimal growth temperatures without chemical inducers is advantageous for large-scale and industrial fermentations.     

Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp.

Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC _Re from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC _Re was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established.     

Gene expression during the intra-puparial stage of Chrysomya megacephala: Implications for postmortem interval estimation

Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) is one of the most predominant calliphorid species which arrives and colonizes a cadaver first in its native range of the Australian and Oriental regions, and the intra-puparial stage of this species accounts for about half of its immature stage. Therefore, establishing a reliable aging method of the intra-puparial stage of C. megacephala is very important to accurately estimate the minimum postmortem interval (PMI_min). In this study, actin was used as an internal reference gene to study the expression of three genes, ecdysone receptor gene ecr, white-eye gene white, and heat shock protein gene hsp70, at different time points during intra-puparial development of C. megacephala. Quantification through real-time PCR revealed that these genes can be used to age the intra-puparial period of C. megacephala as they exhibit regular changes and gene expression level was temperature-dependent. The overall gene expression profile of ecr showed a downward trend throughout the intra-puparial stage. White expression increased steadily until it peaked when intra-puparial development reached 45%, and the expression began to decrease when the intra-puparial development reached 55%. hsp70 was first down-regulated at 0–15% of intra-puparial development, and then slightly up-regulated at 15–40%, and finally down-regulated again until the end of intra-puparial development. This study provides molecular indicators of age during the intra-puparial stage of C. megacephala, and combining gene expression with the morphological methods can lead to more accurate estimation for PMI_min.     

The petunia homologue of tomato gast1: transcript accumulation coincides with gibberellin-induced corolla cell elongation

Gibberellins (GAs) regulate petunia corolla pigmentation and elongation. To study this hormone's effect at the molecular level, we used the tomato gast1 gene as a probe to isolate a gibberellin-induced gene (gip) from petunia corollas. The deduced sequence of gip exhibited 82% identity with GAST1 protein and contained a short, highly hydrophobic N-terminal region. High levels of gip expression were detected in elongating corollas and young stem internodes. When detached corollas were grown in vitro in sucrose medium, gip expression was strongly induced by gibberellic acid (GA₃). GA₃-induced gip expression in corollas was inhibited by abscisic acid (ABA). The expression of the gene was also induced by GA₃ in detached young stem segments. Sucrose was not essential for GA-induced gip expression in corollas but enhanced its effect. In stems, on the other hand, sucrose inhibited the effect of the hormone. The results of the present work support the possible role of gip in GA-induced corolla and stem elongation.     

Effect of conceptus secretions on HOXA10 and PTGS2 gene expression, and PGE2 release in co-cultured luminal epithelial and stromal cells of the porcine endometrium at the time of early implantation

Homeobox A10 (HOXA10) gene expression was demonstrated in the endometrium of adult porcine uteri, however there is little information concerning the role of this gene in the pig. Objectives of the present study were to examine: 1) the expression of HOXA10 in the endometrium of cyclic and early pregnant gilts; 2) the effect of estradiol (E₂) and progesterone (P₄) on HOXA10 expression in porcine luminal epithelial (LE) and stromal (ST) cells in vitro; 3) the effect of E₂ and conceptus-exposed medium (CEM) on HOXA10 and prostaglandin endoperoxide synthase (PTGS2) gene expression and prostaglandin (PG) E₂ secretion from LE and ST cells in a co-culture model. The abundance of HOXA10 mRNA was increased on day 15 of pregnancy in comparison to day 15 of the estrous cycle. Moreover, increased HOXA10 mRNA level was detected in ST cells after E₂ and P₄ treatment. E₂ stimulated the expression of HOXA10 in LE cells cultured on collagen and pre-treated with steroids, but not in LE on plastic surfaces. Addition of CEM to LE cells cultured in collagen-coated inserts of the co-culture system resulted in elevated HOXA10 and PTGS2 gene expression and PGE₂ secretion in these cells, but not in ST cells cultured in basal compartments. ST cells directly treated with E₂ or CEM showed higher levels of HOXA10 and PTGS2 expression. Blocking of estrogen receptors with ICI-182,780 did not influence the stimulatory effect of CEM. We conclude that HOXA10 expression in the porcine endometrium is closely related to the implantation process and stimulated by conceptus products. Moreover, the co-culture system of LE and ST cells is a promising model for the study of endometrial response to conceptus-derived factors.     

Engineering a platform for photosynthetic isoprene production in cyanobacteria,using Synechocystis as the model organism

The concept of “photosynthetic biofuels” envisions application of a single organism, acting both as photo-catalyst and producer of ready-made fuel. This concept was applied upon genetic engineering of the cyanobacterium Synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from CO₂ and H₂O. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light-intensity-dependent isoprene synthase expression. Results showed that oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO₂, without a prior requirement for the harvesting, dewatering and processing of the respective biomass.     

Bradyrhizobium japonicum FixK2, a Crucial Distributor in the FixLJ-Dependent Regulatory Cascade for Control of Genes Inducible by Low Oxygen Levels

Bradyrhizobium japonicum possesses a second fixK-like gene, fixK₂, in addition to the previously identified fixK₁ gene. The expression of both genes depends in a hierarchical fashion on the low-oxygen-responsive two-component regulatory system FixLJ, whereby FixJ first activates fixK₂, whose product then activates fixK₁. While the target genes for control by FixK₁ are unknown, there is evidence for activation of the fixNOQP, fixGHIS, and rpoN₁ genes and some heme biosynthesis and nitrate respiration genes by FixK₂. FixK₂ also regulates its own structural gene, directly or indirectly, in a negative way.     

Analysis of Promoter Activity for the Gene Encoding Pyruvate Orthophosphate Dikinase in Stably Transformed C4 Flaveria Species

The C₄ enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C₄ plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C₄ photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C₄ cycle. We fused 1.5 kb of a 5′ flanking region from the Pdk gene, including the entire 5′ untranslated region, to the uidA reporter gene and stably transformed the closely related C₄ species Flaveria bidentis. β-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C₄ form in mesophyll cells and the chloroplast form in other cells and organs are co-located within the same 5′ region of the Pdk gene.