Interaction of the conserved meiotic regulators, BOULE (BOL) and PUMILIO-2 (PUM2)

Germ cell development is complex; it encompasses specification of germ cell fate, mitotic replication of early germ cell populations, and meiotic and postmeiotic development. Meiosis alone may require several hundred genes, including homologs of the BOULE (BOL) and PUMILIO (PUM) gene families. Both BOL and PUM homologs encode germ cell specific RNA binding proteins in diverse organisms where they are required for germ cell development. Here, we demonstrate that human BOL forms homodimers and is able to interact with a PUMILIO homolog, PUM2. We mapped the domain of BOL that is required for dimerization and for interaction with PUM2. We also show that BOL and PUM2 can form a complex on a subset of PUM2 RNA targets that is distinct from targets bound by PUM2 and another deleted in azoospermia (DAZ) family member, DAZ-like (DAZL). This suggests that RNA sequences bound by PUM2 may be determined by protein interactions. This data also suggests that although the BOL, DAZ, and DAZL proteins are all members of the same gene family, they may function in distinct molecular complexes during human germ cell development.     

Cloning of deleted sequences (CODE): A genomic subtraction method for enriching and cloning deleted sequences.

The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of d7TP After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.     

Identification of phage-displayed peptides which bind to the human HnRNPA1 protein-specific monoclonal antibody.

We have screened a peptide phage display library to examine if monoclonal antibody-binding phages could be isolated from the library and thereby predict the antigenic epitopes of the antibodies from the isolated phages. The library was screened for high-avidity binding to monoclonal antibodies by an affinity purification technique called biopanning. Among the monoclonal antibodies examined, the human hnRNPA1 protein-specific monoclonal antibody 9H10 showed selective binding of phages. After two rounds of the biopanning, twelve clones of high-avidity-binding phages were chosen and their inserts were sequenced. Nucleotide sequence comparison of the 12 clones showed that there were 5 different species, with two species containing four members, implying that they were predominantly selected by the biopanning. The amino acid sequences of the inserts of the 12 clones were compared with that of the human hnRNPA1 protein in order to find the putative epitope of the human hnRNPA1 protein for 9H10. The C-terminal region of the human hnRNPA1 protein shows significant homology with the peptide sequences of the selected phage clones. These results show that this peptide phage display library can be useful in defining the epitope of some monoclonal antibodies.     

Identification and characterization of peptides that bind human ErbB-2 selected from a bacteriophage display library

The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies. The overexpression of ErbB-2 in cancer cells as well as its extracellular accessibility makes it an attractive target for the development of tumor-specific agents. In this study, random peptide bacteriophage display technology was employed to identify peptides that bound the extracellular domain of human ErbB-2. The peptide KCCYSL, most frequently occurring in the affinity-selected phage population, was chemically synthesized and characterized for its binding activities to ErbB-2. The synthetic peptide exhibited high specificity for ErbB-2 and an equilibrium dissociation constant of 30 M. Peptide binding to ErbB-2 positive human breast and prostate carcinoma cells was visualized in direct cell binding assays. In conclusion, the peptide KCCYSL has the potential to be developed into a cancer imaging or therapeutic agent targeting malignant cells overexpressing the ErbB-2 receptor.     

Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin-binding protein B

Alignment of amino-acid sequences from the N-terminal and C-terminal halves of transferrin-binding protein B revealed an underlying bilobed nature with several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose-binding protein (Mbp) with intact TbpB, its N-terminal half or C-terminal half from the human pathogens Neisseria meningitidis and Moraxella catarrhalis were produced. Solid-phase binding assays and affinity isolation assays demonstrated that the N-terminal and C-terminal halves of TbpB could bind independently to human transferrin (hTf). A solid-phase overlapping synthetic peptide library representing the amino-acid sequence of hTf was probed with soluble, labelled Mbp-TbpB fusions to localize TbpB-binding regions on hTf. An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from both organisms, demonstrating a conserved TbpB-hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which included peptides from equivalent regions on the two hTf lobes, indicating that TbpB interacts with each lobe of hTf in a similar manner. Mapping of the peptide-binding regions on a molecular model of hTf revealed a series of nearly adjacent surface regions that nearly encircled each lobe. Binding studies with chimeric hTf/bTf transferrins demonstrated that regions in the C-lobe of hTf were preferentially recognized by the N-terminal half of TbpB. Collectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf-binding regions.     

Identification of Plasmodium falciparum reticulocyte binding protein RBP-2 homologue a and b (PfRBP-2-Ha and -Hb) sequences that specifically bind to erythrocytes

Plasmodium falciparum reticulocyte binding protein RBP-2 homologues a and b (PfRBP-2-Ha and -Hb) have been described as being high molecular weight proteins, expressed at the P. falciparum merozoite apical extreme, belonging to a family of proteins found in other Plasmodium involved in the search for erythrocyte populations before being invaded by merozoites. 185, 20-mer-long non-overlapping peptides, spanning the entire PfRBP-2-Ha and -Hb sequences, were synthesised, radiolabelled and tested in erythrocyte binding assays. Fifteen PfRBP-2-Ha and -Hb high binding activity peptides (HBAPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants were between 70 and 300 nM and Hill coefficients were 1 approximately. HBAPs residues critical for binding to erythrocytes were determined. Cross-linking was performed allowing possible receptors for PfRBP-2-Ha and -Hb to be identified on the surface of the erythrocytes. Some of the HABPs showed merozoite invasion inhibition greater than 90% in in vitro assays.     

Identification and characterization of peptides that bind to cyanovirin-N, a potent human immunodeficiency virus-inactivating protein

Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein-protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X3CX6(W/F)(Y/F)CX2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a KD value of 1.9 microM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.     

Identification and polymorphism of Plasmodium vivax RBP-1 peptides which bind specifically to reticulocytes

Plasmodium vivax merozoite preferentially invades reticulocytes probably using PvRBP-1 as ligand. One hundred and ninety-five, 15-mer peptides has been synthesised from PvRBP-1 sequence; tested in reticulocyte- or erythrocyte-binding assays. Twenty-five peptides (K_d=76–380 nM) specifically defined four reticulocyte-binding regions. It has been reported that a highly conserved Region-I recombinant fragment binds specifically to reticulocytes. HABP-critical residues for reticulocyte-binding were highly conserved in 20 Colombian P. vivax clinical isolates, suggesting an important biological function. There were six overlapping reticulocyte-binding sites for these peptides according to enzyme sensitivity and mutual competition-binding assays; located on 26- and 41-kDa reticulocyte membrane surface proteins.     

Identification of mimotope peptides which bind to the mycotoxin deoxynivalenol-specific monoclonal antibody.

Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.     

Internal micronuclear DNA regions which include sequences homologous to macronuclear telomeres are deleted during development in Tetrahymena

C4A2 repeats are present in multiple clusters in both the macronucleus and micronucleus of Tetrahymena. Although the macronucleus is generated from the micronucleus after sexual conjugation, the repeats are telomeric sequences in the macronucleus but are internally located in the micronucleus (1). This study investigates the fate of the sequences adjacent to the micronuclear C4A2 repeats. Southern blot analyses of 21 C4A2-containing micronuclear clones show that extensive elimination of the adjacent sequences occurs during the formation of the macronucleus. Comparison of one C4A2-containing micronuclear clone with its derived macronuclear segment indicates that approximately 4.5 kb of DNA, which includes the C4A2 repeats and adjacent sequences on both sides is deleted from the macronucleus. The two regions adjoining the deletion are joined together to form a contiguous segment in the macronucleus. This excision of C4A2 repeats and surrounding sequences and the rejoining of the retained segments is probably the mechanism by which all or most of the other C4A2 adjacent sequences are eliminated.     

The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the alpha 1(IV) and alpha 2(IV) which preferentially bind heparin

Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 greater than 100 nm greater than 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the alpha 1(IV) and alpha 2(IV) chains. Three peptides from the known sequence of the alpha 1(IV) and alpha 2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the alpha 1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the alpha 2 (NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the alpha 1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.