DNA Dot Blot Analysis Research Articles

Exercise Ameliorates Spinal Cord Injury by Changing DNA Methylation.

Exercise training is a traditional method to maximize remaining function in patients with spinal cord injury (SCI), but the exact mechanism by which exercise promotes recovery after SCI has not been identified; whether exercise truly has a beneficial effect on SCI also remains unclear. Previously, we showed that epigenetic changes in the brain motor cortex occur after SCI and that a treatment leading to epigenetic modulation effectively promotes functional recovery after SCI. We aimed to determine how exercise induces functional improvement in rats subjected to SCI and whether epigenetic changes are engaged in the effects of exercise. A spinal cord contusion model was established in rats, which were then subjected to treadmill exercise for 12 weeks. We found that the size of the lesion cavity and the number of macrophages were decreased more in the exercise group than in the control group after 12 weeks of injury. Immunofluorescence and DNA dot blot analysis revealed that levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the brain motor cortex were increased after exercise. Accordingly, the expression of ten-eleven translocation (Tet) family members (Tet1, Tet2, and Tet3) in the brain motor cortex also elevated. However, no macrophage polarization was induced by exercise. Locomotor function, including Basso, Beattie, and Bresnahan (BBB) and ladder scores, also improved in the exercise group compared to the control group. We concluded that treadmill exercise facilitates functional recovery in rats with SCI, and mechanistically epigenetic changes in the brain motor cortex may contribute to exercise-induced improvements.

Non‑invasive prenatal diagnosis of thalassemia through multiplex PCR, target capture and next‑generation sequencing

Prenatal clinical detection of thalassemia involves gap-PCR and reverse dot blot (RDB) analysis of fetal DNA acquired through invasive methods. The present study aimed to develop a non-invasive prenatal diagnostic method for thalassemia based on next-generation sequencing (NGS). A total of eight families with proband children with thalassemia were recruited for the study during a subsequent pregnancy. The sequence of the thalassemia genes of the parents and proband were determined using NGS, based on a thalassemia AmpliSeq panel. Cell-free plasma DNA from pregnant women related to the aforementioned proband was analyzed using an NGS panel, based on thalassemia-associated capture probes. Heterozygous single nucleotide polymorphisms within the 10 kb regions flanking exons of the targeted thalassemia genes were acquired using probes or AmpliSeq and employed for parental haplotype construction using Trio-based panel sequencing. The fetal haplotype was deduced from the parental haplotypes and relative haplotype dosage, and subsequently validated using gap-PCR and RDB, based on invasively sampled amniotic fluid. A non-invasive prenatal diagnosis procedure from maternal plasma fetal DNA was successfully developed based on haplotype analysis. The deduced haplotypes of eight fetuses were identical to the results of invasive prenatal diagnosis procedures, with an accuracy rate of 100%. Taken together, the present study demonstrated the potential for non-invasive prenatal diagnosis of α- and β-thalassemia using NGS and haplotype-assisted analysis.

Abstract 3834: Epigenetic modification of oncogenes or tumor suppressor genes by thymoquinone in triple negative breast cancer

Abstract Background and Objective: Triple negative breast cancer (TNBC) is a molecular subtype, and most aggressive and deadly among breast cancers. Different molecular subtypes of breast cancer have distinct molecular mechanisms, and in case of TNBC, the mechanisms are complex, and still obscure. Development of therapeutic strategies are thus also difficult against TNBC. Thymoquinone (TQ) is a natural compound, which has been found effective against TNBC cells, but the mechanism of TQ action in TNBC is not clear. In this study, we have focused on TQ’s genome-wide epigenetic effect on TNBC cell line BT549. Methods: The BT549 cells were treated with 1-10 µM of TQ for cellular growth analysis, and isolated DNA for dot blot analysis with anti-5-methyl cytosine (anti-5MC) antibody to observe whole genome methylation status affected by TQ. Also the chromatin immunoprecipitation (ChIP) was performed with anti-methyl binding domain protein (anti-MBD1) to identify DNA sections, of which methylation status is changed by TQ. Results: Cell viability assay showed that TQ dose-dependently reduced BT549 cell growth. DNA dot blot analysis results indicated no obvious changes in total methylation status change in genomic DNA. However, ChIP-seq confirmed that methylation level was affected (either increased or decreased) in certain genomic locations by TQ treatment. Some of the locations are proximal or close to some genes, indicating that this epigenetic modification might have influence on these genes’ expression pattern. Conclusion: Thus, here in this preliminary research study, we indicate some genes, which might be epigenetically targeted by TQ in TNBC cells. Funding support: National Natural Science Foundation of China (Grant No. 81850410555, 81672887, 81172049) Citation Format: Md. Asaduzzaman Khan, Meiling Zheng, Junjiang Fu. Epigenetic modification of oncogenes or tumor suppressor genes by thymoquinone in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3834.

PCR-based Assay for the Specific Detection of Pseudomonas syringae pv. tagetis using an AFLP-derived Marker

A PCR method has been developed for the pathovar-specific detection of Pseudomonas syringae pv. tagetis, which is the causal agent of bacterial leaf spots and apical chlorosis of several species within the Compositae family. One primer set, PSTF and PSTR, was designed using a genomic locus derived from an amplified fragment length polymorphism (AFLP) fragment produced a 554-bp amplicon from 4 isolates of P. syringae pv. tagetis. In DNA dot-blot analysis with the PCR product as probe, a positive signal was identified in only 4 isolates of P. syringae pv. tagetis. These results suggest that this PCR-based assay will be a useful method for the detection and identification of P. syringae pv. tagetis.

A Geminivirus-Satellite Complex is Associated with Leaf Deformity of Mentha (Mint) Plants in Punjab.

A widespread leaf deformity disease of mentha (mint), accompanied by whiteflies, the vectors of begomoviruses, was observed in Punjab in the last few years. The presence of begomovirus was indicated by DNA dot-blot analysis using the conserved coat protein and replication-associated protein genes of another begomovirus, Sri Lankan cassava mosaic virus (SLCMV). A DNA fragment (2.0kb), representing a partial genomic DNA of a begomovirus, amplified from the symptomatic mentha leaves was used to design end-primers and further amplify an additional 0.9kb fragment, representing the remaining portion of the resident viral DNA. The two sequences, assembled together (2.7kb), showed that they represented the complete sequence of an isolate of Tomato leaf curl Karnataka virus (ToLCKV) DNA. Using universal betasatellite primers, a 1.4kb fragment was amplified from the same sample. This cloned DNA fragment showed complete sequence identity with the previously reported Cotton leaf curl Multan betasatellite (CLCuMB). Majority of the symptomatic mentha leaf samples, collected from four districts of Punjab, showed cross-hybridization in DNA dot-blot using cloned SLCMV and CLCuMB DNA, indicating the presence of one or more begomoviruses related to SLCMV and the betasatellite, CLCuMB. The begomovirus and betasatellite could be mechanically transmitted to Nicotiana benthamiana. Whitefly transmission of the resident begomovirus could also be demonstrated on mentha. The evidence indicates the association of ToLCKV and CLCuMB, a hitherto new combination of a begomovirus and a betasatellite associated with a leaf deformity disease in mentha in Punjab.

Transcriptome analysis of cadmium response inGanoderma lucidum

Ganoderma species are white-rot fungi widespread throughout the world. In this study, a wild isolate of Ganoderma lucidum was first collected and its tolerance was tested in a medium containing 3.0 mM CdCl(2). The cDNA-amplified fragment length polymorphism method was conducted to analyze the transcription profiling of this Ganoderma species in response to Cd treatment. In total, 12 925 transcript-derived fragments (TDFs) were amplified using 256 primer combinations. Forty-nine differentially expressed TDFs were confirmed by DNA dot-blot analysis. Northern blot analysis was used to verify the transcription levels of 34 Cd-inducible TDFs. Sequence analysis indicated that genes involved in reactive oxygen species generation, synthesis of sulfur-containing metabolites, translation machinery, DNA repair, transporting system, proteolysis pathway, mitochondria function, and cell wall biosynthesis were upregulated by Cd treatment. Our results provide a genome-wide transcriptome profiling of Cd response in Ganoderma species.

Burkholderia cenocepacia strains isolated from cystic fibrosis patients are apparently more invasive and more virulent than rhizosphere strains

Given the widespread presence of Burkholderia cenocepacia in the rhizosphere it is important to determine whether rhizosphere strains are pathogenic for cystic fibrosis patients or not. Eighteen B. cenocepacia strains of rhizosphere and clinical origin were typed by multi-locus sequence typing (MLST) analysis and compared for their ability to invade pulmonary epithelial cells and their virulence in a mouse model of airway infection. Although there was great variability, clinical strains were the most invasive in vitro. Almost all the rhizosphere and two clinical strains were defined as non-invasive, six clinical strains as invasive, and two strains of both clinical and environmental origin as indeterminate. Exposure of murine airways to clinical strains caused higher acute mortality than that seen after challenge with rhizosphere strains. Furthermore, both clinical and environmental strains were able to persist in the lungs of infected mice, with no significant differences in bacterial loads and localization 14 days after challenge. DNA dot blot analyses of AHL synthase, porin and amidase genes, which play a role in B. cenocepacia virulence, showed that they were present in B. cenocepacia strains irrespective of their origin. Overall, our results suggest that rhizosphere strains do not differ from clinical strains in some pathogenic traits.

Evaluation of Reuterin Production in Urogenital Probiotic Lactobacillus reuteri RC-14

Classified as a distinct species in 1980, Lactobacillus reuteri strains have been used in probiotic formulations for intestinal and urogenital applications. In the former, the primary mechanism of action of L. reuteri SD2112 (ATCC 55730) has been purported to be its ability to produce the antibiotic 3-hydroxypropionaldehyde (3-HPA), also known as reuterin. In the vagina, it has been postulated that probiotic Lactobacillus reuteri RC-14 does not require reuterin production but mediates a restoration of the normal microbiota via hydrogen peroxide, biosurfactant, lactic acid production, and immune modulation. The aim of the present study was to determine whether strain RC-14 produced reuterin. Using PCR and DNA dot blot analyses, numerous Lactobacillus species, including RC-14, were screened for the presence of the gene encoding the large subunit of glycerol dehydratase (gldC), the enzyme responsible for reuterin production. In addition, lactobacilli were grown in glycerol-based media and both high-performance liquid chromatography and a colorimetric assay were used to test for the presence of reuterin. L. reuteri RC-14 was determined to be negative for gldC sequences, as well as for the production of reuterin when cultured in the presence of glycerol. These findings support that the probiotic effects of L. reuteri RC-14, repeatedly demonstrated during numerous studies of the intestine and vagina, are independent of reuterin production.

Transcriptome analysis of auxin-regulated genes of Arabidopsis thaliana

Plant hormone auxin elicits diverse responses in plant growth and development. Accumulated data indicate that the ubiquitin-mediated proteolytic pathway plays a crucial role in transducing auxin signaling. To gain more understanding of the molecular mechanisms underlying auxin action, we performed a comparative transcriptome analysis of auxin responsive genes between Arabidopsis Columbia ecotype and the auxin insensitive mutant eta2 by cDNA-AFLP. Using 256 primer combinations, about 5900 transcript-derived fragments (TDFs) were amplified. Sixty-six differentially expressed TDFs were confirmed by DNA dot blot analysis. Sequence analysis indicated that, a large number of genes involved in transcription regulation or RNA metabolism were identified as auxin-regulated genes. Northern blot analyses confirmed transcription levels of 16 auxin-regulated genes. These genes include various forms of transcription regulators, defense related, RING-type ubiquitin ligases, and glycosyl hydrolase. This study demonstrates that auxin exerts its effect in complex transcriptional networks.

High prevalence of treponemes in bovine digital dermatitis-A molecular epidemiology

To validate the epidemiology of Treponema spp. associated with digital dermatitis (DD) a large number of DD samples ( n = 56) were examined by DNA–DNA dot blot analyses using oligonucleotide probes specific for phylogenetic group I–VII of oral treponemes and DD-associated phylotypes DDKL-4, DDKL-12 and DDKL-20 as well as for T. brennaborense and T. socranskii. Positive hybridisation results were obtained for phylogenetic groups I, II and IV and phylotypes DDKL-4 and DDKL-12. While phylotype DDKL-4 was detected in 100% of the samples treponemes belonging to phylogenetic group TRE I, TRE II and TRE IV were prevalent in nearly 80% of the samples and phylotype DDKL-12 was detected in 66.1% of the samples. Analysis of Treponema groups present concurrently in the same sample revealed that a combination of TRE I–TRE II–TRE IV–DDKL-4 was most prevalent and could be detected in up to 71% of the samples. These data indicate that this combination of different Treponema spp. seems to be the most important one in the pathogenesis of DD. In contrast, T. brennaborense originally isolated from DD material this treponeme was not detected in any of the samples clearly indicating that this species is not absolutely associated with DD and therefore may represent only an incidental treponeme. Fluorescence in situ hybridisation (FISH) obviously highlights the invasive character of DD-associated treponemes. Mainly treponemes belonging to phylogenetic group TRE I and phylotype DDKL-4 were detected in high numbers compared to the total number of bacteria and also in deeper layers of the epithelium at the transition of unaffected and affected tissue. Our results confirm a high prevalence and diversity of Treponema spp. in DD lesions. In addition, our data indicate that certain combinations of Treponema spp. are detected much more frequently than others. Furthermore, Treponema spp. appears at the interface between healthy and diseased tissue underlining their importance for the pathogenesis of DD.

Enteric adenoviruses.

The 41 serotypes of human adenoviruses are classified into six subgenera (A-F) with different tropisms. Enteric infections are caused in children by serotypes Ad40 and Ad41 of subgenus F. Serotypes Ad40 and Ad41 transform embryonic cells but cannot induce tumours in newborn hamsters. They differ from all other (established) human adenoviruses by being unable to replicate in conventional cell cultures. Ad40 and Ad41 grow in 293 cells (human embryonic kidney cells immortalized by transfection with the E1A, E1B regions of Ad5). In spite of the difficulty of isolating Ad40 and Ad41 they can be directly identified in stools by enzyme-linked immunosorbent assay (ELISA) and solid-phase immuno-electron microscopy. The amount of viral DNA in stool preparations is sufficient for identification by DNA restriction or dot-blot analysis. Adenoviruses have been associated with 7-17% of cases of diarrhoea in children. Ad40 and Ad41 cause diarrhoea throughout the year. Clinical features are watery stools, vomiting and moderately elevated temperature; respiratory symptoms are infrequent. The diarrhoea is protracted (mean 8.6 and 12.2 days for Ad40 and Ad41 respectively). Children with rotavirus diarrhoea vomited more frequently and had a higher temperature and diarrhoea of shorter duration. The impact of enteric adenoviruses in the aetiology of diarrhoea world-wide is not known but is accessible to investigation.

Transformation of strawberry (Fragaria ananassa Duch.) with late embryogenesis abundant protein gene

SummaryCalli from anthers of strawberry (Fragaria ananassa Duch.), Toyonaka, were induced on MS medium plus 6-benzylamino-purine and naphthaleneacetic acid and transformed by particle bombardment with plasmid pBY520 containing late embryogenesis abundant protein gene, LEA3, from barley (Hordeum vulgare L.). Hybridization signals were detected by DNA dot blot analysis using a probe labelled with digoxigenin; it indicated foreign gene integration into the strawberry genome. The difference in salt tolerance between transgenic strawberry and controls was proved by salt stress test. Under 50 mmol l–1 and 100 mmol l–1 NaCl salt stress conditions, the wilting rates of controls were 62.2% and 96.0% respectively, compared with 18.6% and 42.8% for transgenic shoots. The results showed that the salt tolerance of the transgenic strawberry had been increased.

Two apple repetitive sequence elements: characterisation and potential use as genetic markers

Within the 5′ flanking regions of two ripening-related genes from apple (Malus pumila), two short repeat sequences were identified and named Ars1 andArs2 (Apple repetitivesequences). Both elements were present in the promoter of the Md-ACS1-1 ACC synthase gene and Ars2was also found in the promoter of a gene homologous to the ABG1 β-galactosidase gene. Database searches revealed other examples of each element in apple genomic sequences and a partial Ars2element in a sequence from Asian pear (Pyrus pyrifolia). DNA dot blot analysis demonstrated that both elements are present in high numbers in the apple genome. Ars1 is thought to represent a MITEminiature inverted-repeat transposable element) related to the MITE Alien, while the identity ofArs2, though it is undoubtedly a transposable element, remains undetermined. DNA dot blot analysis and PCR amplification using primers designed from Ars1and Ars2 showed that elements similar to Ars1 and Ars2 are present across the Rosaceae. The amplification patterns obtained using Ars1- and Ars2-specific primers with genomic DNA from different cultivars or mapping populations suggest that these elements could be used for finger printing and segregation analysis in Rosaceae. Evidence from DNA database searches suggests that they may be applicable to genetic studies in a wide range of other organisms.

A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI.

We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxy-terminus of a scFv fragment that binds a scorpion toxin (AahI). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by single-step affinity chromatography on streptavidin-agarose with a yield close to 1 mg/l. DNA sequencing, dot blot and mass spectrometric analyses demonstrated the integrity of the soluble immunoconjugate. Fusion to the streptavidin-binding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3 x 10(-10) M). Similarly, the streptavidin-binding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifunctional and had a low molecular mass of 28 kDa. This enabled us to develop rapid and sensitive immunoassays for the specific detection of the toxin AahI accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of envenomations.

Somatic gene alteration of AIB1 gene in patients with breast cancer.

AIB1 (amplified in breast cancer 1) is a coactivator which stimulates the transcriptional activity of the liganded-estrogen receptor (ER). It has been reported that AIBI gene is amplified and overexpressed in some breast cancer cell lines. AIB1 contains a stretch of homopolymeric glutamines (poly-Q). We reported that the poly-Q shows polymorphism, which provides an opportunity to study somatic gene alteration such as loss of heterozygosity (LOH). In the present study, we aimed to investigate the frequency of somatic gene alteration in Japanese patients with breast cancer. Amplification of AIB1 gene was detected only in 1 (2.6%) of 39 breast cancer tissues by DNA dot blot analysis. On the other hand, LOH was found in 2 (8%) breast cancer tissues out of 25 patients showing heterozygosity in peripheral blood mononuclear cells (PBMC). Taken together, both LOH and amplification of AIB1 gene were identified in breast cancer patients, raising the possibility that AIB1 have oncogenic as well as antioncogenic potential for the pathogenesis of breast cancer.

Narrow Host Range Nucleopolyhedrovirus for Control of the Browntail Moth (Lepidoptera: Lymantriidae): Table 1.

The browntail moth, Euproctis chrysorrhoea (L.), is periodically a major urban pest in the southern United Kingdom. High populations cause severe defoliation of a range of host plants, often in urban areas; and urticating hairs of larvae are highly irritating to humans. Control of outbreak populations is therefore desired. As an alternative to chemical insecticide sprays and labor intensive nest removal, nucleopolyhedrovirus (NPV) has been considered as a biological control. To evaluate if use of this spray would affect other lepidopterans we carried out detailed host range testing using a number of wild-caught lepidopteran species. Seventy-three species of Lepidoptera from 14 families, including four species of Lymantriidae and two species of hymenopteran sawfly, were found to be nonpermissive to E. chrysorrhoea NPV at a dose of 106 occlusion bodies per second instar. Some individuals from 11 species of Lepidoptera and one hymenopteran sawfly died of baculovirus infection, but none of these were shown, by dot blot analysis of DNA, to be infected with E. chrysorrhoea NPV. In two of these species, uninfected control larvae also died of infection, indicating that they carried an overt infection in the field. However, in eight species of Lepidoptera and one sawfly, there were no control deaths, providing possible evidence that an inapparent or latent infection had been stressed out of the wild-caught insects by inoculation with a high dose of E. chrysorrhoea NPV. Our results suggest this NPV may be monospecific, and that it is unlikely to present a risk to any nontarget species. These characteristics make it highly suitable for use as a bioinsecticide, particularly in urban areas and nature reserves.

Transduction of human pancreatic tumor cells with vesicular stomatitis virus G-pseudotyped retroviral vectors containing a herpes simplex virus thymidine kinase mutant gene enhances bystander effects and sensitivity to ganciclovir.

We examined the suitability of Moloney murine leukemia virus (MLV) 4070A-, cat endogenous virus (CEV) RD114-, or vesicular stomatitis virus G (VSV-G)-pseudotyped retroviruses containing the humanized enhanced green fluorescent protein (hEGFP) or one of two herpes simplex virus thymidine kinase (HSV-TK) genes to transduce and provide gene expression in human pancreatic tumor cells. Fluorescence-activated cell sorter analysis demonstrated that VSV-G-pseudotyped hEGFP vector infected a greater percentage of cells and generated more robust gene expression than MLV 4070A- or CEV RD114-pseudotyped vectors. Dot blot and Southern blot analysis of genomic DNA revealed up to 10-fold more gene copies in G418-selected VSV-G hEGFP vector-transduced cells compared with genomic DNA from cells transduced with MLV 4070A or CEV RD114 pseudotypes. Cells transduced with VSV-G pseudotypes of HSV-TK(WT) or the HSV-TK30 vectors were 5- to 10-fold more sensitive to ganciclovir (GCV) than other pseudotype-transduced cells. A 40- to 61-fold difference in sensitivity to GCV was observed between cells transduced with VSV-G HSV-TK30 vector and cells transduced with MLV 4070A HSV-TK(WT) vector in vitro. A 13-fold reduction in tumor volume was observed in severe combined immunodeficient mice inoculated with PancTuITK30 cells compared with mice inoculated with PancTuITK(WT) cells during GCV treatment. We conclude that the choice of glycoprotein envelope and the potency of a particular suicide gene were therapeutically additive and increased the number of HSV-TK-positive cells and sensitivity toward GCV in human pancreatic tumors cells for prodrug gene therapy.

Population characteristics of Irish Helicobacter pylori isolates: a tRNA-associated locus.

A strain-variable transfer RNA-associated-locus (trl) was present in 50% of Irish Helicobacter pylori (H. pylori), isolates and did not correlate with the origin of the isolates. To associate a particular genotype or phenotype to trl status in H. pylori by further screening the isolates from the original study for the presence of known genotypic and phenotypic characteristics. Forty two clinical isolates were screened for the presence of the cagA, vacA, iceA1 and vapD genes by Southern or DNA dot blot analysis. Western blot analysis was performed using antibodies to CagA, VacA, Lewis X (Le(x)) and Lewis Y (Le(y)). Plasmids were identified by the alkaline lysis method. The cagA gene was present in 29 (69%) of isolates screened and 21 (50%) produced the CagA protein. The vacA gene was detected in all of the isolates while VacA was expressed in 71.4%. The iceA1 and vapD loci were detected in 73.8% and 71.4% respectively. Le(x) was expressed in 42.9% and Le(y) in 38.1% of the isolates. Expression of both Lewis antigens was detected in 7.1% while in 30.9% neither antigen was detected. Plasmids were present in 47.6%. There was no association between the trl status of isolates and any of the above. There were no significant associations between the phenotypic and genotypic characteristics studied and peptic ulcer disease or non-ulcer dyspepsia. The strain-variable tRNA-associated locus is independent of the vacA/VacA, cagA/CagA, Lewis X, Lewis Y, iceA1, vapD and plasmid status in the population of Irish H. pylori isolates studied.

Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estα21 and estβ21 genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estα21 and estβ21 are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estβ21 gene from this amplicon has greater transcription than estα21 in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.

Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estalpha2(1) and estbeta2(1) genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estalpha2(1) and estbeta2(1) are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estbeta2(1) gene from this amplicon has greater transcription than estalpha2(1) in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.