Ramified rolling circle amplification for synthesis of nucleosomal DNA sequences.

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

Isolation of two plasmids, pRET1100 and pRET1200, from Rhodococcus erythropolis IAM1400 and construction of a Rhodococcus-Escherichia coli shuttle vector.

With the aim of being able to co-express multiple genes, I searched for novel compatible plasmids and isolated two plasmid species, pRET1100 and pRET1200, from Rhodococcus erythropolis IAM1400. Sequencing analysis revealed that the pRET1100 plasmid is a double-stranded DNA molecule of 5444 bp with two possible open reading frames (ORFs), repT and div, and three minor ORFs. The cryptic replication protein, RepT, is not highly homologous to those from other plasmids that have been reported. The Rhodococcus-Escherichia coli shuttle vector pRET1102 was transformed into R. erythropolis JCM2895 harboring the pRE2895 plasmid. The recombinant R. erythropolis JCM2895 harbored two plasmid species. These results suggest that plasmid derivatives of pRET1100 and pRE2895 are fully compatible in R. erythropolis. I determined the minimum region of pRET1100 required for autonomous replication in R. erythropolis and constructed a high-copy plasmid, pRET1129, in R. erythropolis.

j5 DNA assembly design automation software.

Recent advances in Synthetic Biology have yielded standardized and automatable DNA assembly protocols that enable a broad range of biotechnological research and development. Unfortunately, the experimental design required for modern scar-less multipart DNA assembly methods is frequently laborious, time-consuming, and error-prone. Here, we report the development and deployment of a web-based software tool, j5, which automates the design of scar-less multipart DNA assembly protocols including SLIC, Gibson, CPEC, and Golden Gate. The key innovations of the j5 design process include cost optimization, leveraging DNA synthesis when cost-effective to do so, the enforcement of design specification rules, hierarchical assembly strategies to mitigate likely assembly errors, and the instruction of manual or automated construction of scar-less combinatorial DNA libraries. Using a GFP expression testbed, we demonstrate that j5 designs can be executed with the SLIC, Gibson, or CPEC assembly methods, used to build combinatorial libraries with the Golden Gate assembly method, and applied to the preparation of linear gene deletion cassettes for E. coli. The DNA assembly design algorithms reported here are generally applicable to broad classes of DNA construction methodologies and could be implemented to supplement other DNA assembly design tools. Taken together, these innovations save researchers time and effort, reduce the frequency of user design errors and off-target assembly products, decrease research costs, and enable scar-less multipart and combinatorial DNA construction at scales unfeasible without computer-aided design.

[A novel vector for construction of a cDNA library].

A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at 3'-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector cyclization and synthesis of the second strand cDNA. This approach significantly simplifies cDNA library construction, it does not require PCR reaction (which can induce artifact mutations in cDNA sequences) and restrictase treatment.

Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3.

The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl- N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in vivo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.

A new generation of pPRIG-based retroviral vectors.

BACKGROUND: Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c). RESULTS: The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (<< modular >> PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (<< single color/resistance >> PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (<< dual color/selection >> PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells. CONCLUSION: These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (<< modular >> PRIGs), for multiple transductions and fluorescence analyses of transduced cells (<< single color/resistance >> PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (<< dual color/selection >> PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.

Design and switch of catalytic activity with the DNAzyme-RNAzyme combination.

Design and switch of catalytic activity in enzymology remains a subject of intense investigation. Here, we employ a DNAzyme-RNAzyme combination strategy for construction of a 10-23 deoxyribozyme-hammerhead ribozyme combination that targets different sites of the beta-lactamase mRNA. The 10-23 deoxyribozyme-hammerhead ribozyme combination gene was cloned into phagemid vector pBlue-scriptIIKS (+). In vitro the single-strand recombinant phagemid vector containing the combination sequence exhibited 10-23 deoxyribozyme activity, and the linear transcript displayed hammerhead ribozyme activity. In bacteria, the 10-23 deoxyribozyme-hammerhead ribozyme combination inhibited the beta-lactamase expression and repressed the growth of drug-resistant bacteria. Thus, we created a DNAzyme-RNAzyme combination strategy that provides a useful approach to design and switch of catalytic activities for nucleic acid enzymes.

Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK).

Molecular modeling studies led to the identification of LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) as a potent inhibitor of Polo-like kinase (Plk). LFM-A13 inhibited recombinant purified Plx1, the Xenopus homolog of Plk, in a concentration-dependent fashion, as measured by autophosphorylation and phosphorylation of a substrate Cdc25 peptide. LFM-A13 was a selective Plk inhibitor. While the human PLK3 kinase was also inhibited by LFM-A13 with an IC(50) value of 61 microM, none of the 7 other serine/threonine kinases, including CDK1, CDK2, CDK3, CHK1, IKK, MAPK1 or SAPK2a, none of the 10 tyrosine kinases, including ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET, or YES, or the lipid kinase PI3Kgamma were inhibited (IC(50) values >200-500 microM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K(i) value of 7.2 microM from Dixon plots. LFM-A13 blocked the cell division in a zebrafish (ZF) embryo model at the 16-cell stage of the embryonic development followed by total cell fusion and lysis. LFM-A13 prevented bipolar mitotic spindle assembly in human breast cancer cells and glioblastoma cells and when microinjected into living epithelial cells at the prometaphase stage of cell division, it caused a total mitotic arrest. Notably, LFM-A13-delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer at least as effectively as paclitaxel and gemcitabine. LFM-A13 showed a favorable toxicity profile in mice and rats. In particular there was no evidence of hematologic toxicity as documented by peripheral blood counts and bone marrow examinations. These results establish LFM-A13 as a small molecule inhibitor of Plk with in vitro and in vivo anti-proliferative activity against human breast cancer.

A self-augmenting gene expression cassette for enhanced and sustained transgene expression in the presence of proinflammatory cytokines.

Viral promoters can yield high gene expression levels yet tend to be attenuated in vivo by host proinflammatory cytokines. Prolonged transgene expression can be obtained using constitutive cellular promoters. However, levels of transgene expression driven by cellular promoters are insufficient for effective therapy. We designed a novel self-augmenting gene expression cassette in which the transgene product can induce an endogenous transcription factor to enhance the activity of a weak cellular promoter driving its expression. Using the cellular major histocompatibility complex class I (H-2K(b)) promoter to drive the interferon (IFN-gamma) cytokine gene, we show that the H-2K(b) promoter, although exhibiting much lower basal activity, yields higher IFN-gamma production than the CMV promoter 2 days after transfection. IFN-gamma expression driven by the H-2K(b) promoter also lasts longer than that driven by the cytomegalovirus promoter. Our data demonstrate that the self-augmenting strategy provides a promising approach to achieve high and sustained transgene expression in vivo.

Refactoring bacteriophage T7.

Natural biological systems are selected by evolution to continue to exist and evolve. Evolution likely gives rise to complicated systems that are difficult to understand and manipulate. Here, we redesign the genome of a natural biological system, bacteriophage T7, in order to specify an engineered surrogate that, if viable, would be easier to study and extend. Our initial design goals were to physically separate and enable unique manipulation of primary genetic elements. Implicit in our design are the hypotheses that overlapping genetic elements are, in aggregate, nonessential for T7 viability and that our models for the functions encoded by elements are sufficient. To test our initial design, we replaced the left 11,515 base pairs (bp) of the 39,937 bp wild-type genome with 12,179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. The viability of our initial design suggests that the genomes encoding natural biological systems can be systematically redesigned and built anew in service of scientific understanding or human intention.

Chemical restriction: strand cleavage by ammonia treatment at 8-oxoguanine yields biologically active DNA.

Cleavage of DNA single and double strands at an 8-oxoguanine-containing nucleotide occurs in 90 % yield if the modified oligonucleotide is treated with NH(3) and O(2) at 60 degrees C. The mechanism of this oxidative cleavage reaction was studied, and the reaction was applied to the generation of single-stranded overhangs on PCR-amplified DNA that can be ligated. As an example, the lac Z' gene was amplified by PCR with 8-oxoguanine modified primers, restricted by ammonia treatment, ligated into a plasmid vector, transformed in Escherischia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure with restriction enzymes, and it allows the design of any 3'-overhang independent of the sequence of the cloned DNA.

Approaches for generating recombinant adenovirus vectors.

Various methods have been developed to facilitate the generation of recombinant adenovirus vectors, and three commercially available methods have been most widely used: the homologous recombination method in E1-complement cell lines, the homologous recombination method in bacteria, and an in vitro ligation method based on simple routine plasmid construction. These methods can insert foreign genes not only into the E1 deletion region, but also into the E3 deletion region, thereby permitting the construction of a binary transgene expression system in which heterologous genes can be inserted into both the E1 and E3 regions. By modifying the latter two methods, fiber-mutant adenovirus vectors can be also constructed in order to modify vector tropism. In this paper, we review recent advances in the construction of first generation adenovirus vectors and fiber-modified adenovirus vectors.

Catalytic acid-base groups in yeast pyruvate decarboxylase. 1. Site-directed mutagenesis and steady-state kinetic studies on the enzyme with the D28A, H114F, H115F, and E477Q substitutions.

The roles of four of the active center groups with potential acid-base properties in the region of pH optimum of pyruvate decarboxylase from Saccharomyces cerevisiae have been studied with the substitutions Asp28Ala, His114Phe, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis methods. The steady-state kinetic constants were determined in the pH range of activity for the enzyme. The substitutions result in large changes in k(cat) and k(cat)/S(0.5) (and related terms), indicating that all four groups have a role in transition state stabilization. Furthermore, these results also imply that all four are involved in some manner in stabilizing the rate-limiting transition state(s) both at low substrate (steps starting with substrate binding and culminating in decarboxylation) and at high substrate concentration (steps beginning with decarboxylation and culminating in product release). With the exception of some modest effects, the shapes of neither the bell-shaped k(cat)/S(0.5)-pH (and related functions) plots nor the k(cat)-pH plots are changed by the substitutions. Yet, the fractional activity still remaining after substitutions virtually rules out any of the four residues as being directly responsible for initiating the catalytic process by ionizing the C2H. There is no effect on the C2 H/D exchange rate exhibited by the D28A and E477Q substitutions. These results strongly imply that the base-induced deprotonation at C2 is carried out by the only remaining base, the iminopyrimidine tautomer of the coenzyme, via intramolecular proton abstraction. The first product is released as CO(2) rather than HCO(3)(-) by both wild-type and E477Q and D28A variants, ruling out several mechanistic alternatives.

Enzymatic corrections for cells derived from Fabry disease patients by a recombinant adenovirus vector.

Fabry disease is an X-linked inherited metabolic disorder caused by a deficiency of alpha-galactosidase (alpha-gal), resulting in the accumulation of ceramide trihexoside (CTH) in body fluids and in many organs and tissues. We constructed a recombinant adenovirus with a human alpha-gal cDNA (AxCAG alpha-gal), and transfected this vector to skin fibroblasts from Fabry patients. Transfected cells expressed high amounts of alpha-gal in their cytoplasm, and a high level of alpha-gal activity was detected in the medium. The accumulated CTH in the fibroblasts disappeared 3 days after infection. The secreted alpha-gal also eliminated the accumulated CTH from uninfected patient's cells. The enzyme may be taken up through mannose-6-phosphate receptors, as the addition of mannose-6-phosphate to the medium completely inhibited the uptake of the enzyme. The infected cells continued to express alpha-gal for more than 10 days. These results suggest that AxCAG alpha-gal could be used as enzyme replacement gene therapy for Fabry disease.

Delivery of avian cytokines by adenovirus vectors.

A fowl adenovirus serotype 8 (FAV-8) recombinant was constructed by inserting an expression cassette consisting of the FAV major late promoter/splice leader sequences (MLP/SL), the chicken interferon-gamma (ChIFN-gamma) gene and SV40 polyA into sites in the right hand end of the FAV-8 genome. One recombinant (A3-13) was constructed by an insertion of ChIFN-gamma into a 1.3 kilobase pair (kbp) deletion which removed a putative open reading frame (ORF) with identity to the CELO (FAV serotype 1) 36 kDa homologue. A second recombinant (S4) removed a further 0.9 kbp and a third recombinant (AA1) was constructed in a small 50 base pair (bp) SpeI deletion. The recombinants displayed differing growth characteristics in CK monolayers. A3-13 grew slowly and only attained a titre of 10(5) pfu/ml, S4 had intermediate growth and AA1 showed wild type growth kinetics. These differing growth properties indicated that removal of the 36 kDa homologue had an effect on growth in vitro. Supernatants from CK monolayers infected with the recombinant virus were assayed for the production of ChIFN-gamma. Detectable levels of ChIFN-gamma were observed in supernatants as early as 24 h post infection (p.i.), peaked at 48 h p.i. and this level was maintained for at least 10 days. The level of production of ChIFN-gamma correlated with each recombinant's growth characteristics in vitro. Chickens treated with rFAV-ChIFN-gamma showed increased weight gains compared to controls and suffered reduced weight loss when challenged with the coccidial parasite Eimeria acervulina.

Efficient recombination of Lym-1 scFv gene using multiple doubly-restricted DNA fragments.

In order to improve radioimmunotherapy of lymphoma, a Lym-1 single-chain antigen-binding (scFv) protein molecule was produced. Because the commonly used polymerase chain reaction (PCR) method frequently causes unexpected mutations, we developed a non-PCR method for scFv gene assembly. The method involved a stepwise linkage of doubly-restricted DNA fragments and re-digestion of the resultant concatamers. Using this strategy, the Lym-1 scFv expression gene was readily constructed without mutations. The recombinant gene was cloned into an expression vector and scFv protein was expressed. The method can be used for other genes or DNA recombination.

Retroviral vector targeting human cells via c-Kit-stem cell factor interaction.

Targeted gene transfer into hematopoietic stem cells by retroviral vectors would greatly facilitate the development of in vivo strategies for stem cell gene therapy. We engineered a recombinant retroviral vector that can target human cells expressing a c-Kit receptor via a ligand-receptor interaction. The ecotropic (Moloney murine leukemia virus) envelope protein was modified by insertion of a sequence encoding the N-terminal 161 amino acids of murine stem cell factor (mSCF), the ligand for murine c-Kit. The chimeric envelope protein was correctly processed and incorporated into viral particles as efficiently as the wild-type envelope protein. Virions pseudotyped with the chimeric envelope proteins bound to 293 cells expressing murine c-Kit (293KIT) preferentially; however, they could not transduce any c-Kit-positive cells under conventional conditions. They could transduce 293KIT cells in the presence of chloroquine, and HEL cells expressing human c-Kit on a fibronectin fragment (CH296)-coated dish. The fact that recombinant mSCF in the medium at the time of transduction greatly reduced the efficiency of both gene deliveries implies that the vector utilized the mSCF-c-Kit interaction for the initial step of transduction in either case. The vector may prove useful for targeting cells expressing c-Kit on their surface.

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments.

The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.