Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system

The nontoxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. We have investigated its potential use as an in vivo neurotropic carrier. In this work we show that a hybrid protein encoded by the lacZ–TTC gene fusion retains the biological functions of both proteins in vivo—i.e., retrograde transynaptic transport of the TTC fragment and β-galactosidase enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy could be used to deliver a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein could also be used to map synaptic connections between neural cells.

Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5′-GNN-3′ subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5′-(GNN)6-3′ family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5′-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16’s minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

Gene content phylogeny of herpesviruses

Clusters of orthologous groups [COGs; Tatusov, R. L., Koonin, E. V. & Lipman, D. J. (1997) Science 278, 631–637] were identified for a set of 13 completely sequenced herpesviruses. Each COG represented a family of gene products conserved across several herpes genomes. These families were defined without using an arbitrary threshold criterion based on sequence similarity. The COG technique was modified so that variable stringency in COG construction was possible. High stringencies identify a core set of highly conserved genes. Varying COG stringency reveals differences in the degree of conservation between functional classes of genes. The COG data were used to construct whole-genome phylogenetic trees based on gene content. These trees agree well with trees based on other methods and are robust when tested by bootstrap analysis. The COG data also were used to construct a reciprocal tree that clustered genes with similar phylogenetic profiles. This clustering may give clues to genes with related functions or with related histories of acquisition and loss during herpesvirus evolution.

Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 109 members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome

We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which the CAT gene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein σ2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens.

Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein–protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an α-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or α-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses.

We have developed improved procedures for the isolation of deletion mutant, point mutant, and recombinant herpesvirus saimiri. These procedures take advantage of the absence of NotI and AscI restriction enzyme sites within the viral genome and use reporter genes for the identification of recombinant viruses. Genes for secreted engineered alkaline phosphatase and green fluorescent protein were placed under simian virus 40 early promoter control and flanked by NotI and AscI restriction sites. When permissive cells were cotransfected with herpesvirus saimiri virion DNA and one of the engineered reporter genes cloned within herpesvirus saimiri sequences, recombinant viruses were readily identified and purified on the basis of expression of the reporter gene. Digestion of recombinant virion DNA with NotI or AscI was used to delete the reporter gene from the recombinant herpesvirus saimiri. Replacement of the reporter gene can be achieved by NotI or AscI digestion of virion DNA and ligation with a terminally matched fragment or, alternatively, by homologous recombination in cotransfected cells. Any gene can, in theory, be cloned directly into the virion DNA when flanked by the appropriate NotI or AscI sites. These procedures should be widely applicable in their general form to most or all herpesviruses that replicate permissively in cultured cells.

Construction of an improved mycoinsecticide overexpressing a toxic protease.

Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might he achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.

Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors.

Adenovirus (Ad) vectors have been extensively used to deliver recombinant genes to a great variety of cell types in vitro and in vivo. Ad-based vectors are available that replace the Ad early region 1 (E1) with recombinant foreign genes. The resultant E1-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that constitutively expresses the E1 genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and late proteins (despite the absence of E1 activity) resulting in immune recognition of virally infected cells. The infected cells are subsequently eliminated, resulting in only a transient expression of foreign genes in vivo. We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should block viral DNA replication and decrease viral protein production, resulting in a diminished immune response and extended duration of foreign gene expression in vivo. As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the E1 genes but also constitutively express the Ad serotype 2 140-kDa DNA polymerase protein, one of three virally encoded proteins essential for Ad genome replication. The Ad polymerase-expressing cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These packaging cell lines can be used to prepare Ad vectors deleted for the E1 and polymerase functions, which should facilitate development of viral vectors for gene therapy of human diseases.

Construction, purification, and functional incorporation on tumor cells of glycolipid-anchored human B7-1 (CD80).

To generate a potent cell-mediated immune response, at least two signals are required by T cells. One is engagement of the T-cell receptor with peptide-bearing major histocompatibility complex molecules. The other signal can be delivered by various molecules on the antigen-presenting cell, such as B7-1 (CD80). Many tumor cells escape immune recognition by failing to express these costimulatory molecules. Transfection of the B7 gene into some murine tumor cells allows for immune recognition and subsequent rejection of the parental tumor. We have studied an alternative approach for the introduction of B7-1 onto the surface of tumor cells. This method involves purified glycosyl-phosphatidylinositol (GPI)-anchored proteins which can spontaneously incorporate their lipid tail into cell membranes. We have created and purified a GPI-anchored B7-1 molecule (called GPI-B7) which is able to bind its cognate ligand, CD28, and incorporate itself into tumor cell membranes after a short incubation. Tumor cells that have been reconstituted with GPI-B7 can provide the costimulatory signal needed to stimulate T cells. These findings suggest an approach for the introduction of new proteins onto cell membranes to create an effective tumor vaccine for potential use in human immunotherapy.

Construction and characterization of a highly complex retroviral library for lineage analysis.

Replication-incompetent retroviral vectors encoding histochemical reporter genes have been used for studying lineal relationships in a variety of species. A crucial element in the interpretation of data generated by this method is the identification of sibling relationships, or clonal boundaries. The use of a library of viruses in which each member is unique can greatly facilitate this aspect of the analysis. A previously reported murine retroviral library containing about 80 members demonstrated the utility of the library approach. However, the relatively low number of tags in the murine library necessitated using low infection rates in order to give confidence in clonal assignments. To obviate the need for low infection rates, a far more complex library was created and characterized. The CHAPOL library was constructed such that each member encodes a histochemical reporter gene and has a DNA tag derived from a degenerate oligonucleotide pool synthesized to have a complexity of > 1 x 10(7). The library was tested after infection of cells in vitro or in vivo. The DNA tag from each histochemically labeled cell or clone of cells was recovered by PCR and sequenced for unambiguous identification. Three hundred and twenty tags have been identified after infection, and so far no tag has been seen to result from more than one independent infection. Thus, an equal distribution of inserts is suggested, and Monte Carlo analysis predicts a complexity of > 10(4) members.

Targeting a complex transcriptome: The construction of the mouse full-length cDNA encyclopedia

We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large. numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, S'-end clusters identify regions that are potential promoters for 8637 known genes and S'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Insertion of an E-coli lacZ gene in Acetobacter xylinus for the production of cellulose in whey

A mini-Tn10:lacZ: kan was inserted into a wild-type strain of Acetobacter xylinus by random transposon mutagenesis, generating a lactose-utilising and cellulose-producing mutant strain designated ITz3. Antibiotic selection plate assays and Southern hybridisation revealed that the lacZ gene was inserted once into the chromosome of strain ITz3 and was stably maintained in non-selective medium after more than 60 generations. The modified strain had, on the average, a 28-fold increase in cellulose production and a 160-fold increase in beta-galactosidase activity when grown in lactose medium. beta-Galactosidase activity is present in either lactose or sucrose medium indicating that the gene is constitutively expressed. Cellulose and beta-galactosidase production by the modified strain was also evaluated in pure and enriched whey substrates. Utilisation of lactose in whey substrate by ITz3 reached 17 g l(-1) after 4 days incubation. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mungbean (Vigna radiata L. Wilczek)

Bacterial artificial chromosome (BAC) libraries have been widely used in different aspects of genome research. In this paper we report the construction of the first mungbean (Vigna radiata L. Wilczek) BAC libraries. These BAC clones were obtained from two ligations and represent an estimated 3.5 genome equivalents. This correlated well with the screening of nine random single-copy restriction fragment length polymorphism probes, which detected on average three BACs each. These mungbean clones were successfully used in the development of two PCR-based markers linked closely with a major locus conditioning bruchid (Callosobruchus chinesis) resistance. These markers will be invaluable in facilitating the introgression of bruchid resistance into breeding programmes as well as the further characterisation of the resistance locus.