Joseph Sambrook

HSP70 Binding Sites in the Tumor Suppressor Protein p53

Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures. In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined. We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK. We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly β-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found. Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation. We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule.

Rapid isolation of yeast DNA.

: This method is used to isolate yeast genomic DNA or shuttle plasmids that replicated both in Saccharomyces cerevisiae and in E. coli.

Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol

: This procedure is the method of choice for purification of genomic DNA from mammalian blood when large amounts of DNA are required, for example, for Southern blotting. The usual yield of DNA from buffy coat lymphocytes isolated from 20 mL of normal blood is ∼250 µg.

Agarose Gel Electrophoresis

Agarose gel electrophoresis is used to resolve DNA fragments on the basis of their molecular weight. Smaller fragments migrate faster than the larger ones; the distance migrated on the gel varies inversely with the logarithm of the molecular weight. The size of fragments is determined by calibrating the gel, using known size standards, and comparing the distance that the unknown fragment has migrated. In the method described in the chapter, this technique is used to resolve complex DNAs for Southern blot analysis or to resolve simpler digests of bacteriophage and plasmid clones for RE site mapping and blotting. The time required to carry out this procedure is two days. . Reagents such as agarose, ethidium bromide, and gel loading buffer are required. DNA is prepared and analyzed in advance including RE digest.

The Yeast Inositol Polyphosphate 5-Phosphatases Inp52p and Inp53p Translocate to Actin Patches following Hyperosmotic Stress: Mechanism for Regulating Phosphatidylinositol 4,5-Bisphosphate at Plasma Membrane Invaginations

ABSTRACT The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and PtdIns(3,5)P2. Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P2 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P2 and PtdIns(4,5)P2 at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.

SDS-Polyacrylamide Gel Electrophoresis of Proteins

INTRODUCTIONThis protocol describes the separation of proteins by SDS-polyacrylamide gel electrophoresis. SDS is used with a reducing agent and heat to dissociate the proteins. SDS-polypeptide complexes form and migrate through the gels according to the size of the polypeptide. By using markers of known molecular weight, the molecular weight of the polypeptide chain(s) can be estimated.

Rejection of intraocular tumors from transgenic mice by tumor-infiltrating lymphocytes

The present study examined the role of tumor infiltrating lymphocytes (TIL) in the rejection of intraocular tumors from SV40 transgenic mice. Tumor cells from an intraocular tumor arising in an SV40 transgenic FVB/N mouse were transplanted into the eyes of syngeneic FVB/N mice and the TIL isolated. TIL were assessed for direct cytolytic activity in vitro. TIL were also transferred passively to immunosuppressed FVB/N mice to determine if they could mediate intraocular tumor rejection. The role of CD4+ and CD8+ T cells in intraocular tumor rejection was evaluated by depleting the respective cell populations in FVB/N hosts prior to intraocular tumor challenge. The results showed that intraocular tumors undergoing rejection in immunocompetent syngeneic hosts became infiltrated with T cells, with the CD8+ subset predominating at the time of rejection. By contrast, athymic nude mice did not reject the intraocular tumors nor did the tumors become infiltrated with TIL. TIL displayed direct, tumor-specific cytolytic activity immediately after isolation from the tumor-containing eyes. FVB/N hosts depleted of CD4+ T cells were unable to reject their intraocular tumors. In vivo depletion of CD8+ T cells delayed, but did not prevent tumor rejection. Adoptively transferred TIL mediated swift rejection of intraocular tumors in immunoincompetent recipients. Recipients of TIL, but not recipients of normal spleen cells, acquired significant tumor-specific CTL activity that was demonstrable in vitro. The results strongly suggest, but do not prove, that TIL mediate rejection of intraocular tumors from transgenic mice by direct cytolysis. Although CD4+ T cells are necessary for tumor rejection and are capable of direct cytolysis, the predominant effector cells are CD8+ CTL.

Fragmentation of DNA by sonication.

INTRODUCTIONDNA fragmentation is often necessary prior to library construction or subcloning for DNA sequencing. This protocol describes a method for DNA fragmentation by sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonication. DNA that has been sonicated for excessive periods of time is extremely difficult to clone. Most sonicators will not shear DNA to a size of less than 300-500 bp, and it is tempting to continue sonication until the entire DNA population has been reduced in size. However, the yield of subclones is usually greater if sonication is stopped when the fragments of the target DNA first reach a size of ~700 bp.

Preparation of genomic DNA from mouse tails and other small samples.

: This protocol is used for extracting DNA from fragments of tissue for genotyping transgenic and knockout mice. Digestions of mouse tail fragments are performed overnight at 55°C. Each tail snippet generates 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and, more expediently, as a template in polymerase chain reactions (PCRs).

Isolation of High-Molecular-Weight DNA Using Organic Solvents

Perhaps the most basic of all procedures in molecular cloning is the purification of nucleic acids. The key step, the removal of proteins, can often be performed simply by extracting aqueous solutions of nucleic acids with phenol:chloroform and chloroform.