Isolation of erythropoietin receptor agonist peptides using evolved phage libraries.

Cyclic peptides capable of activating the erythropoietin receptor (EPOR) were isolated from phage display libraries by screening with a novel EPOR-IgG fusion protein reagent. A parental clone ERB1 (EPO Receptor Binder 1) was first isolated from a phage display library displaying 38 random amino acids as an N-terminal fusion with the M13 minor capsid protein, pill. An evolved library was then produced from the parental sequence using an oligonucleotide saturation mutagenesis strategy which yielded EPOR binding sequences with 20 times the relative affinity of ERB1. Two synthetic peptides were constructed from these sequences both of which bind the EPO receptor in specific ELISA, and act as full agonists in EPO dependent cell proliferation assays. These peptides are 18 amino acids in length, disulfide-bonded, and have a minimum consensus sequence of CXXGWVGXCXXW, where X represents positions tolerant of several amino acids.

Expression and ligand binding assays of soluble cytokine receptor-immunoglobulin fusion proteins.

We have developed a cloning vector for the expression of type I cytokine receptor, NO, extracellular domain (ECD)-mouse IgG1 Fc fusion proteins. The vector has a versatile polylinker that allows in-frame cloning of the receptor ECD with the mouse IgG1 sequence to encode a receptor ECD-IgG1 fusion construct. The receptor-IgG1 fusion proteins are transiently expressed in useful amounts following transfection of the expression vector into COS7 cells and G418 selection. The mouse IgG1 portion of the fusion protein provides a universal handle for purification on an affinity matrix and detection by anti-mouse IgG antibodies in ELISA or Western blot formats. The expressed receptor ECD-IgG1 fusion proteins bind their cognate ligands. In order to demonstrate that the fusion proteins have similar ligand binding affinities as the native receptors, the affinity constants (Kd) for EPOR, TNFR, IL-4R, and IL-6R-IgG1 fusion proteins were measured by surface plasmon resonance and shown to be in good agreement with published values. The TNFR-IgG1 fusion protein was employed in a demonstration of a novel ELISA format for detecting cytokine receptor binding to cytokine.

Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast.

pep12/vps6 mutants of Saccharomyces cerevisiae are defective in delivery of soluble vacuolar hydrolases to the vacuole. Morphological analysis by electron microscopy revealed that pep12 cells accumulate 40- to 50-nm vesicles. Furthermore, pep12 cells have enlarged vacuoles characteristic of class D pep/vps mutants. PEP12 encodes a protein of 288 amino acids that has a C-terminal hydrophobic region and shares significant sequence similarity with members of the syntaxin protein family. These proteins appear to participate in the docking and fusion of intracellular transport vesicles. Pep12p is the first member of the syntaxin family to be implicated in transport between the Golgi and the vacuole/lysosome. Pep12p-specific polyclonal antisera detected a 35-kDa protein that fractionated as an integral membrane protein. Subcellular fractionation experiments revealed that Pep12p was associated with membrane fractions of two different densities; the major pool (approximately 90%) of pep12p may associate with the endosome, while a minor pool (approximately 10%) cofractionated with the late Golgi marker Kex2p. These observations suggest that Pep12p may mediate the docking of Golgi-derived transport vesicles at the endosome.

The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit.

The retinoblastoma protein (p110RB) interacts with many cellular proteins in complexes potentially important for its growth-suppressing function. We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind p110RB. One clone encodes a novel type 1 protein phosphatase catalytic subunit (PP-1 alpha 2), which differs from the originally defined PP-1 alpha by an amino-terminal 11-amino-acid insert. In vitro-binding assays demonstrated that PP-1 alpha isoforms preferentially bind the hypophosphorylated form of p110RB. Moreover, similar p110RB sequences are required for binding PP-1 alpha 2 and SV40 large T antigen. Cell cycle synchrony experiments revealed that this association occurs from mitosis to early G1. The implications of these findings on the regulation of both proteins are discussed.

Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.

Genomic footprinting in mammalian cells with ultraviolet light.

A simple and accurate genomic primer extension method has been developed to detect ultraviolet footprinting patterns of regulatory protein-DNA interactions in mammalian genomic DNA. The technique can also detect footprinting or sequencing patterns introduced into genomic DNA by other methods. Purified genomic DNA, containing either damaged bases or strand breaks introduced by footprinting or sequencing reactions, is first cut with a convenient restriction enzyme to reduce its molecular weight. A highly radioactive single-stranded DNA primer that is complementary to a region of genomic DNA whose sequence or footprint one wishes to examine is then mixed with 50 micrograms of restriction enzyme-cut genomic DNA. The primer is approximately 100 bases long and contains 85 radioactive phosphates, each of specific activity 3000 Ci/mmol (1 Ci = 37 GBq). A simple and fast method for preparing such primers is described. Following brief heat denaturation at 100 degrees C, the solution of genomic DNA and primer is cooled to 74 degrees C and a second solution containing Taq polymerase (Thermus aquaticus DNA polymerase) and the four deoxynucleotide triphosphates is added to initiate primer extension of genomic DNA. Taq polymerase extends genomic hybridized primer until its polymerization reaction is terminated either by a damaged base or strand break in genomic DNA or by the addition of dideoxynucleotide triphosphates in the polymerization reaction. The concurrent primer hybridization-extension reaction is terminated after 5 hr and unhybridized primer is digested away by mung bean nuclease. Primer-extended genomic DNA is then denatured and electrophoresed on a polyacrylamide sequencing gel, and radioactive primer extension products are revealed by autoradiography. By using this method we demonstrate that it is possible to footprint with ultraviolet light, in intact monkey cells, regulatory protein--DNA interactions along a single copy of a simian virus 40 viral genome integrated into the monkey genome.

Cloning of the gene topA encoding for DNA topoisomerase I and the physical mapping of the cysB-topA-trp region of Escherichia coli.

The gene topA of Escherichia coli that encodes for DNA topoisomerase I has been cloned by a combination of genetic and radioimmunal screening. The gene has been mapped to be within a 3.4 Kb segment of the bacterial genome. The intracellular level of the enzyme in strains harboring extrachromosomal copies of topA gene increases with increasing copy number of the gene and the introduction of extrachromosomal copies of the topA gene truncated at its 3' side into a topA strain of E. coli does not significantly influence the expression of the chromosomal copy of topA. These results suggest that the expression of topA is not tightly regulated. Strains in which DNA topoisomerase I is overproduced grow significantly slower in broth and give smaller size colonies on agar plates. Physical mapping of a 20 Kb region containing cysB; topA and trp has also been carried out with a number of restriction enzymes; topA is found to be immediately adjacent to cysB and is separated from trp by a 7 Kb segment where no known gene resides.

Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition.

Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome, near cysB. Strains carrying deletions of the top gene are viable. The top mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerase I have a more rapid rate of induction and a higher level of catabolite-sensitive enzymes including tryptophanase and beta-galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished. The frequency of transposition of Tn5, a transposon carrying kanamycin resistance, is decreased by a factor of 40 or more in top mutants. A direct or indirect role of the topoisomerase in transposition is discussed. The transposition frequency of Tn3, however, is not dependent on top. Based on the studies of the E. coli top mutants, it appears that the supX gene, which was originally studied in Salmonella typhimurium [Dubnau, E. & Margolin, P. (1972) Mol. Gen. Genet. 117, 91-112] is likely to be the structural gene for DNA topoisomerase I.