Observation and Analysis of in vitro Expression of Mouse Heart Nuclear DNA Fragments by AFM

Using AFM,we observed linear chain-like complexes formed by some specific proteins and the multi-mRNAs during the in vitro expression of some active genes on the DNA fragments. The LDH mRNA in the multi-mRNA complex can in vitro translate LDH. Via AFM, we also discovered that nmRNA prepared from heart muscles, along with some specific proteins can form linear chain-like nmRNA complexes in which LDH mRNA can also translate LDH in vitro. Our work shows the prospective application of AFM in the research of the biological reaction of the active genes on the DNA fragments.

Polymorphism Analysis and In Vitro Expression of RANTES Genes

Objective: To clone, sequence and express the primate β-chemokine RANTES genes, hRANTES from H sapiens andmRANTES from M. Mulatta, in order to explore the possibilityof AIDS gene therapy. Methods: hRANTES and mRANTES were amplified byreverse transcription-polymerase chain reaction (RT-PCR)from RNAs extracted from phytoagglutinin (PHA)-activatedperipheral blood lymphocytes. hRANTES was cloned,sequenced and expressed in vitro, and mRANTES was directlysequenced for homology comparison. Results: An expected 276 bp fragment was obtained in bothamplincations, and sequence data demonstrated a relativelyhigh homology among different copies of hRANTES (97%),and hRANTES was up to 95.6% homologous to mRANTES.When compared with RANTES from other mammals,hRANTES gave rise to a homology ranging from 77% to 86%.The cloned hRANTES was expressed in vitro and a positivesignal of RANTES was detected by dot blotting. Conclusion: The full-length of hRANTES sequence wassubmitted to GenBank and had been released. Our mRANTESsequence is first reported and not yet appeared in GenBank.The successful cloning and expression of hRANTES willprovide a basis for AIDS gene therapy in the future.

Cloning and molecular characterization of△^(12)-fatty acid desaturase gene from Mortierella isabellina

AIM： To clone △^12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS： Reverse transcriptional polymerase chain reaction （RT-PCR） was used to clone the open reading frame of △^12-fatty acid desaturase gene （D12D） of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli（E.coli） strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain IN- VSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain IN- VSc1.RESULTS： Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E. coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of △^12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △^12-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.CONCLUSION： Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.

Molecular and functional characterization of three novel carboxylesterases in the detoxification of permethrin in the mosquito, Culex quinquefasciatus

Carboxylesterases (CarEs) belong to a super family of multifunctional enzymes associated with the degradation of endogenous and exogenous compounds. Many insect CarEs are known to play important roles in catalyzing the hydrolysis of organophosphates (OPs), carbamates, and synthetic pyrethroids (SPs). The elevation of esterase activity through gene amplification and overexpression of estα2 and estβ2 genes contributes to the development of resistance to OP insecticides in the mosquito Culex quinquefasciatus. Three additional CarE genes are upregulated in permethrin-resistant Cx. quinquefasciatus according to an RNA-seq analysis, but their function remains unknown. In this study, we, for the first time, characterized the function of these three novel genes using in vitro protein expression, an insecticide metabolism study and molecular docking analysis. All three CarE genes were significantly overexpressed in resistant mosquito larvae, but not adults, compared to susceptible strain. No gene copy differences in these three genes were found in the mosquitoes tested. In vitro high-performance liquid chromatography (HPLC) revealed that CPIJ018231, CPIJ018232, and CPIJ018233 metabolized 30.4% ± 2.9%, 34.7% ± 6.8%, and 23.2% ± 2.2% of the permethrin, respectively. No mutations in resistant strains might significantly affect their CarE hydrolysis ability. A docking analysis further confirmed that these three CarEs from resistant strain all potentially metabolize permethrin. Taken together, these three carboxylesterase genes could play important roles in the development of permethrin resistance in Cx. quinquefasciatus larvae through transcriptional overexpression, metabolism, and detoxification.