Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

BackgroundVarious DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis.ResultsTo demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed Escherichia coli (E. coli) with the plasmids obtained. We observed that the over 90% of resulting colonies possessed the plasmids containing the reverted GFPuv gene and exhibited fluorescence. We extended the test to introduce up to nine mutations in Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) by assembling 11 DNA fragments using MUPAC. Analysis of the cloned plasmid by electrophoresis and DNA sequencing revealed that approximately 30% of colonies had the objective mutant M-MLV RT gene. Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact.ConclusionsMUPAC could efficiently introduce various mutations at multiple specific sites within a gene. Furthermore, it could facilitate the preparation of experimental gene materials important to molecular and synthetic biology research.

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

Treatment of murine leukaemia virus reverse transcriptase with benzophenone 4-maleimide inactivates DNA polymerase activity, but has no effect on the RNAase H function. Kinetic measurements indicated that benzophenone 4-maleimide is a competitive inhibitor with respect to template-primer binding, but is non-competitive with respect to dNTP binding. Enzyme modified with benzophenone 4-maleimide cannot bind template-primer or primer alone, as judged by u.v.-mediated cross-linking of radiolabelled substrates. Of the eight cysteine residues in murine leukaemia virus reverse transcriptase, only two were modified by benzophenone 4-maleimide, which were identified as Cys-90 and Cys-310 by comparative tryptic-peptide mapping and amino acid composition analysis. Inclusion of template-primer or primer alone in the modification mixture protected only Cys-90 from modification by benzophenone 4-maleimide. To investigate the role of Cys-90 in detail, we converted it to alanine by site-directed mutagenesis. The mutant enzyme, however, exhibited no loss either of DNA polymerase or of RNAase H activity. These results indicate that Cys-90 is located in a domain of murine leukaemia virus reverse transcriptase that binds template-primer, but may not have a direct role in the enzymic function of the enzyme. Ala-90 mutant murine leukaemia virus reverse transcriptase is at least 10-fold more susceptible to heat inactivation than is the wild-type enzyme, which suggests that Cys-90 in murine leukaemia virus reverse transcriptase may play a role in maintaining structural integrity.

BackgroundMultiple approaches for the site-directed mutagenesis (SDM) have been developed. However, only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. In addition, many of the existing multiple SDM methods have technical limitations associated with type and number of mutations that can be introduced, or are technically demanding and require special chemical reagents.ResultsIn this study we developed a quick and efficient strategy for introduction of multiple complex mutations in a target DNA without intermediate subcloning by using a combination of connecting SDM and suppression PCR. The procedure consists of sequential rounds, with each individual round including PCR amplification of target DNA with two non-overlapping pairs of oligonucleotides. The desired mutation is incorporated at the 5' end of one or both internal oligonucleotides. DNA fragments obtained during amplification are mixed and ligated. The resulting DNA mixture is amplified with external oligonucleotides that act as suppression adapters. Suppression PCR limits amplification to DNA molecules representing full length target DNA, while amplification of other types of molecules formed during ligation is suppressed. To create additional mutations, an aliquot of the ligation mixture is then used directly for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA.ConclusionA wide variety of complex multiple mutations can be generated in a short period of time. The procedure is rapid, highly efficient and does not require special chemical reagents. Thus, MALS represents a powerful alternative to the existing methods for multiple SDM.

Retroviruses exhibit high levels of variation primarily due to high rates of mutation and recombination during viral replication. The high levels of variation present in retroviral populations affect the genetic diversity and evolution of the virus, and play a significant role in viral pathogenesis as well as resistance to antiviral drugs and vaccines. The virally encoded reverse transcriptase (RT) is responsible for the majority of mutations that are introduced into the viral genome. The low processivity of RT also contributes to the high frequency of recombination that is observed. We have developed a powerful in vivo system utilizing directly repeated sequences within the green fluorescent protein (GFP) gene to analyze properties of RT that are involved in template switching and recombination. Mutations were introduced into various domains of RT and analyzed for their effects on template switching. Based on these results, we proposed a dynamic copy-choice model for RT template switching in which the dynamic equilibrium between DNA polymerization and RNase H degradation determines the rate of template switching. In the second study, we determined whether polymerase-dependent and/or polymerase-independent RNase H activity was important for template switching. Using a trans-complementation system, we were able to determine that the dynamic equilibrium between DNA polymerization and polymerase-dependent RNase H activity is responsible for high rates of RT template switching. We also demonstrated for the first time, the existence of polymerase-dependent RNase H activity in vivo. Finally, we determined the frequency of direct repeat deletion for short direct repeats and identified an RNA secondary structure that formed a hotspot for RT template switching. Analysis of the template switching hotspot revealed that the majority of template switching events occurred within 19 base pairs 3′ of the predicted RNA structure. We also showed that base pairing between the newly synthesized DNA and RNA acceptor template is essential for efficient RT template switching.

Glucuronidation, a major metabolic pathway for a large variety of endobiotics and xenobiotics, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT2B4 conjugates a large variety of endogenous and exogenous molecules and is considered to be the major bile acid conjugating UGT enzyme in human liver. In the present study, we identify UGT2B4 as a novel target gene of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha), which mediates the hypolipidemic action of fibrates. Incubation of human hepatocytes or hepatoblastoma HepG2 and Huh7 cells with synthetic PPAR alpha agonists, fenofibric acid, or Wy 14643 resulted in an increase of UGT2B4 mRNA levels. Furthermore, treatment of HepG2 cells with Wy 14643 induced the glucuronidation of hyodeoxycholic acid, a specific bile acid UGT2B4 substrate. Analysis of UGT2B mRNA and protein levels in PPAR alpha wild type and null mice revealed that PPAR alpha regulates both basal and fibrate-induced expression of these enzymes in rodents also. Finally, a PPAR response element was identified in the UGT2B4 promoter by site-directed mutagenesis and electromobility shift assays. These results demonstrate that PPAR alpha agonists may control the catabolism of cytotoxic bile acids and reinforce recent data indicating that PPAR alpha, which has been largely implicated in the control of lipid and cholesterol metabolism, is also an important modulator of the metabolism of endobiotics and xenobiotics in human hepatocytes.

The efficiency of three commonly used reverse transcriptases, namely Moloney Murine Leukaemia Virus (M-MuLV) reverse transcriptase, RNase H--M-MuLV reverse transcriptase and Avian Myeloblastoma Leukaemia Virus (AMV) reverse transcriptase (RT), have been compared in RT-PCR amplification of antibody genes. These enzymes differed significantly in their efficiency of cDNA synthesis as indicated by the degree of V gene amplification by PCR; Moloney Murine Leukaemia Virus (M-MuLV) reverse transcriptase and RNase H--M-MuLV reverse transcriptase produced greater amplification of the human V-gene repertoire using RT-PCR than that of the Avian Myeloblastoma Leukaemia Virus (AMV) reverse transcriptase.

Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors

Complexation with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase offers a novel method of obtaining crystal structures of nucleic acid duplexes, which can be phased by molecular replacement. This method is somewhat similar to the method of using a monoclonal antibody Fab fragment complexed to the molecule of interest in order to obtain crystals suitable for X-ray crystallographic analysis. Here a novel DNA structure including two G-A mispairs in a pseudo-hexadecamer determined at 2.3 A resolution in a complex with the N-terminal fragment is reported. This structure has an asymmetric unit consisting of the protein molecule bound to the blunt end of a DNA 6/10-mer, which is composed of a six-base strand (5'-CTCGTG-3') and a ten-base strand (3'-GAGCACGGCA-5'). The 6/10-mer is thus composed of a six-base-pair duplex with a four-base single-stranded overhang. In the crystal structure, the bases of the overhang are reciprocally paired (symmetry element -x - 1, -y, z), yielding a doubly nicked pseudo-hexadecamer primarily B-form DNA molecule, which has some interesting A-like structural features. The pairing between the single strands results in two standard (G-C) Watson-Crick pairs and two G-A mispairs. The structural DNA model can accommodate either a standard syn or a standard anti conformation for the 5'-terminal adenine of the ten-base strand of the DNA based on analysis of simulated-annealing omit maps. Although the DNA model here includes nicks in the phosphodiester backbone, modeling of an intact phosphodiester backbone results in a very similar DNA model and indicates that the structure is biologically relevant.

The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondria-rich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network.

Type II alveolar epithelial cells (AEC II) proliferate and transdifferentiate into type I alveolar epithelial cells (AEC I) when the normal AEC I population is damaged in the lung alveoli. We hypothesized that signaling by transforming growth factor beta1 (TGF beta1), through its downstream Smad proteins, is involved in keeping AEC II quiescent in normal cells and its altered signaling may be involved in the trans-differentiation of AEC II to AEC I. In the normal lung, TGF beta1 and Smad4 were highly expressed in AEC II. Using an in vitro cell culture model, we demonstrated that the trans-differentiation of AEC II into AEC I-like cells began with a proliferative phase, followed by a differentiation phase. The expression of TGF beta1, Smad2, and Samd3 and their phosphorylated protein forms, and cell cycle inhibitors, p15(Ink4b) and p21(Cip1), was lower during the proliferative phase but higher during the differentiation phase. Furthermore, cyclin-dependent kinases 2, 4, and 6 showed an opposite trend of expression. TGF beta1 secretion into the media increased during the differentiation phase, indicating an autocrine regulation. The addition of TGF beta1 neutralizing antibody after the proliferative phase and silencing of Smad4 by RNA interference inhibited the trans-differentiation process. In summary, our results suggest that the trans-differentiation of AEC II to AEC I is modulated by signaling through the Smad-dependent TGF beta1 pathway by altering the expression of proteins that control the G1 to S phase entry in the cell cycle.

A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20-base region encompassing the PPT, within this region there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilize the M-MuLV PPT as an origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in M-MuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV-1, the -2G and -4G residues play key roles in determining the specificity of priming.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are genetic elements that function with CRISPR-Associated (Cas) proteins as an adaptive immune system to foreign genetic material in prokaryotic organisms. The CRISPR-dCas9 system is modified to suppress gene transcription. Methicillin-Resistant Staphylococcus aureus (MRSA) is a dangerous human pathogen that is resistant to beta-lactam antibiotics. This is due to the mecA methicillin resistance gene coding for penicillin binding protein 2A (PBP 2A), which inhibits the activity of beta-lactam antibiotics. Two CRISPR-dCas9 systems were designed to target the promoter region of mecA in MRSA to suppress transcription of the gene. A cefoxitin disk diffusion test showed that the target on the coding strand significantly reduced antibiotic resistance in MRSA, whereas the target on the noncoding strand did not. An oxacillin microbroth serial dilution was used to confirm disk diffusion results. The CRISPR system targeting the coding strand was the only one to reduce antibiotic resistance and thus was chosen for continued testing. mecA gene expression levels were analyzed using Reverse Transcriptase Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). Results showed that mecA gene expression in the CRISPR-treated sample was reduced to 0.230 fold of the value in the control, representing a 77% decrease in gene transcription. The 77% decrease in gene expression was not enough to make MRSA clinically susceptible to betalactam antibiotics. ABBREVIATIONS:BLAST - basic local alignment search tool, bp - basepairs, Cas - CRISPR-associated, cDNA - complementary DNA, CLSI - Clinical Laboratory Standards Institute, Cq - quantification cycle, CRISPRs - clustered regularly spaced short palindromic repeats, E.coli - Escherichia coli,M-MLV RT - Moloney Murine Leukemia Virus Reverse Transcriptase, MIC - minimum inhibitory concentration, MRSA - methicillin-resistant Staphylococcus aureus,NCBI - National Center for Biotechnology Information, PAM - protospacer adjacent motif, PBP 2A - penicillin binding protein 2A, RT-qPCR - reverse transcriptase quantitative real-time polymerase chain reaction, tracr - trans-activating CRISPR, UDG - uracil DNA glycosylase

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

DNA methylation inhibits transcription driven by the collagen alpha2(I) promoter and the 5' end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha2(I) gene. Complex formation increased when the CpG site at +7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at +7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen alpha2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region.