Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.

A novel genetic system to detect protein-protein interactions.

'Bottom-up fabrication', which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton molecular complex).

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Folding DNA to create nanoscale shapes and patterns

Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.

A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments.

Publisher Summary This chapter discusses the production of single-stranded plasmid DNA. The chapter focuses on M13KO7 and its uses. The chapter reviews M13 biology; M13 mutants play a vital role in the functioning of M13KO7. M13 is a phage that contains a circular single-stranded DNA (ssDNA) molecule of 6407 bases packaged in a filamentous virion, which is extruded from the cell without lysis. It can infect only cells having F-pili to which it binds for entering the cell. The phage genome consists of nine genes encoding 10 proteins and contains an intergenic region of 508 bases. Phage replication consists of three phases: (1) ss-double strand (ds), (2) ds-ds, and (3) ds-ss. The intergenic region (IG) structure contains regions important for four phage processes: (1) the sequences necessary for the recognition of an ssDNA by phage proteins for its efficient packaging into viral particles; (2) the site of synthesis of an RNA primer that is used to initiate strand synthesis; (3) the initiation; and (4) the termination of (+) strand synthesis. In the chapter, the IG, which has the potential to form five hairpin structures, is represented schematically and important regions designated. The origin of replication of the (+) strand is stated most important to the functioning of M 13KO7.

Jeffrey Vieira

Papers

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.

A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments.

Production of Single-Stranded Plasmid DNA

New pUC-derived cloning vectors with different selectable markers and DNA replication origins.