A p21(Waf1/Cip1)carboxyl-terminal peptide exhibited cyclin-dependent kinase-inhibitory activity and cytotoxicity when introduced into human cells.

In the present study, we report the cyclin-dependent kinase (Cdk)-inhibitory activity of a series of p21waf1/cip1 (p21) peptide fragments spanning the whole protein against the cyclin D1/Cdk4 and cyclin E/Cdk2 enzymes. The most potent p21 peptide tested in our initial peptide series, designated W10, spanned amino acids 139 to 164, a region of p21 that has been found independently to bind to proliferating cell nuclear antigen and also to inhibit Cdk activity. We go on to report the importance of putative beta-strand and 3(10)-helix motifs in the W10 peptide for cyclin-dependent kinase-inhibitory activity. We also describe the cellular activity of W10 and derivatives that were chemically linked to an antennapedia peptide, the latter segment acting as a cell membrane carrier. We found that the W10AP peptide exhibited growth inhibition that resulted from necrosis in human lymphoma CA46 cells. Furthermore, regions in the W10 peptide responsible for Cdk-inhibition were also important for the degree of this cellular activity. These studies provide insights that may eventually, through further design, yield agents for the therapy of cancer.

Mutations associated with familial melanoma impair p16INK4 function.

Cell division is controlled by a series of positive and negative regulators which act at sequential points throughout the cell cycle. Disturbance of these checks could contribute to cancer by allowing excessive cell proliferation. The point in G1 at which cells irrevocably commit to DNA synthesis is controlled by protein complexes consisting of cyclin-dependent kinases (CDK4 or CDK6) and cyclins (D1, D2 or D3). These complexes are inhibited by low molecular weight proteins, such as p16INK4 (refs 1,2), p15INK4B (ref. 3) and p18 (ref. 4). Deletion or mutation of these CDK-inhibitors could lead to unchecked cell growth, suggesting that members of the p16INK4 family may be tumour suppressor genes. The recent detection of p16INK4 (MTS1) mutations in familial melanoma kindreds, many human tumour cell lines, and primary tumours is consistent with this idea. Previously, we described eight germline p16INK4 substitutions in 18 familial melanoma kindreds. Genetic analyses suggested that five mutations predisposed carriers to melanoma, whereas two missense mutations had no phenotypic effect. We now describe biochemical analyses of the missense germline mutations and a single somatic mutation detected in these families. Only the melanoma-predisposing mutants were impaired in their ability to inhibit the catalytic activity of the cyclin D1/CDK4 and cyclin D1/CDK6 complexes in vitro. Our data provide a biochemical rationale for the hypothesis that carriers of certain p16INK4 mutations are at increased risk of developing melanoma.

Allele shuffling: conjugational transfer, plasmid shuffling and suppressor analysis in Saccharomyces cerevisiae.

Trans-acting suppressor analysis represents a powerful genetic technique capable of revealing interactions among biochemical pathways in vivo. Suppressor characterization in Saccharomyces cerevisiae has traditionally utilized meiotic segregation for the requisite manipulation of strain genotypes. Meiotic segregation is not compatible with all yeast genotypes and can be prohibitively labor intensive when examining large collections of suppressors. To facilitate rapid phenotypic analysis of suppressor mutations, we have devised a novel genetic strategy called 'allele shuffling'. This plasmid-based method should in principle identify allele-specific, allele-dependent and bypass suppressors. A centromere vector (YCp) was developed that can be directly transferred from Escherichia coli to yeast via 'trans-kingdom' conjugation. Suppressors of a thermolabile cdc23 allele, cdc23-39, were isolated in the background of a yeast host strain harboring the mutant cdc23-39 gene positioned on a counterselectable plasmid. CDC23 or cdc23-39 genes cloned into a mobilizable YCp vector were then transferred directly from E. coli cultures to each suppressed yeast strain on the surfaces of agar plates. Plasmid shuffling of the cdc23-39 allele transconjugants segregated away the original cdc23-39 gene present during mutagenesis, allowing the intra- or extragenic nature of suppression to be determined. Phenotypes (if any) produced by suppressor mutations were revealed in those transconjugants receiving the wild-type CDC23-containing episome. The allele shuffling method should be generally applicable to the analysis of suppressors of any essential yeast gene.

Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis.

Cdc16p, Cdc23p and Cdc27p are all essential proteins required for cell cycle progression through mitosis in Saccharomyces cerevisiae. All three proteins contain multiple tandemly repeated 34 amino acid tetratricopeptide repeats (TPRs). Using two independent assays, two-hybrid analysis in vivo and co-immunoprecipitation in vitro, we demonstrate that Cdc16p, Cdc23p and Cdc27p self associate and interact with one another to form a macromolecular complex. A temperature sensitive mutation in the most highly conserved TPR domain of Cdc27p results in a greatly reduced ability to interact with Cdc23p, but has no effect on interactions with wild-type Cdc27p or Cdc16p. The specificity of this effect indicates that TPRs can mediate protein-protein interactions and that this mutation may define an essential interaction for cell cycle progression in yeast. The conservation of at least two of the three proteins from yeast to man suggests that this protein complex is essential for mitosis in a wide range of eukaryotes.

p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

Multifunctional yeast high-copy-number shuttle vectors.

A set of four yeast shuttle vectors that incorporate sequences from the Saccharomyces cerevisiae 2 mu endogenous plasmid has been constructed. These yeast episomal plasmid (YEp)-type vectors (pRS420 series) differ only in their yeast selectable markers, HIS3, TRP1, LEU2 or URA3. The pRS420 plasmids are based on the backbone of a multifunctional phagemid, pBluescript II SK+, and share its useful properties for growth in Escherichia coli and manipulation in vitro. The pRS420 plasmids have a copy number of about 20 per cell, equivalent to that of YEp24. During non-selective yeast growth, pRS420 plasmids are lost through mitotic segregation at rates similar to other YEp vectors and yeast centromeric plasmid (YCp) vectors, in the range of 1.5-5% of progeny per doubling. The pRS420 series provides high-copy-number counterparts to the current pRS vectors [Sikorski and Hieter, Genetics 122 (1989) 19-27].

A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis.

We have identified and characterized a novel, repeating 34 amino acid motif (the TPR motif) that is reiterated several times within the CDC23 gene product of S. cerevisiae. Multiple copies of this motif were discovered in five other proteins, three encoded by cell division cycle genes required to complete mitosis and two involved in RNA synthesis. Quantitative sequence analyses suggest the existence of a common underlying structure in each TPR unit that consists of amphipathic alpha-helical regions punctuated by proline-induced turns. The TPR motif defines a new family of genes and an important structural unit common to several proteins whose functions are required for mitosis and RNA synthesis.

A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.

Expression of the Rickettsia prowazekii citrate synthase gene in Escherichia coli.

Recombinant DNA techniques were used to isolate the Rickettsia prowazekii citrate synthase gene on the plasmid vector pBR322 by functional complementation of a gltA mutation of Escherichia coli K-12. Analysis of citrate synthase activity in crude extracts revealed that the enzyme expressed in E. coli retains the regulatory control mechanisms characteristic of the rickettsial enzyme.