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1.
EMBO J ; 20(4): 864-71, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11179230

ABSTRACT

SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation.


Subject(s)
Adenovirus E4 Proteins/metabolism , Neoplasm Proteins/metabolism , RNA Splicing , HeLa Cells , Humans , Open Reading Frames , Phosphorylation , Protein Binding
2.
Comb Chem High Throughput Screen ; 3(3): 185-96, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10903378

ABSTRACT

We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen 'escape variants', and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review here our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.


Subject(s)
Antibodies/genetics , Gene Library , Recombinant Proteins/immunology , Animals , Breast Neoplasms/immunology , Cryptosporidium parvum/immunology , DNA Primers , Female , Genetic Vectors , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Variable Region/genetics , Ovarian Neoplasms/immunology , Peptide Library , Recombinant Proteins/genetics
3.
Comb Chem High Throughput Screen ; 3(1): 51-7, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10702614

ABSTRACT

We have previously described a vector system for generating recombinant polyclonal antibody libraries. This system uses bidirectional phagemid and mammalian expression vectors to facilitate mass transfer of selected variable light and variable heavy (VL-VH) region gene pairs from the phagemid to the mammalian vector, to express polyclonal libraries of whole IgG antibodies. We report here the first stage of generating a polyclonal antibody library to the human breast carcinoma cell line BT-20, using this vector system. VL and VH region gene pairs were obtained from a mouse immunized with BT-20 cells, and cloned, in opposite transcriptional orientations, in the bidirectional phagemid vector, to produce an Fab phage display library. This library was selected by panning on BT-20 cells and shown to bind specifically to BT-20 cells. Such libraries, after suitable negative selection to eliminate major reactivities against normal tissue, could be transferred in mass to our bidirectional mammalian expression vector for production of libraries of chimeric antibodies with mouse V regions and human constant (C) regions. These polyclonal antibody libraries will mediate effector functions and are expected to be useful for breast cancer therapy, as well as diagnosis.


Subject(s)
Bacteriophages/genetics , Breast Neoplasms/immunology , Immunoglobulin Fab Fragments/genetics , Animals , Antibody Specificity , Base Sequence , Cloning, Molecular , DNA, Complementary , Female , Humans , Immunoglobulin Fab Fragments/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Sequence Homology, Nucleic Acid , Tumor Cells, Cultured
4.
Mol Cell Biol ; 20(7): 2317-25, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10713155

ABSTRACT

Splicing of the adenovirus IIIa pre-mRNA is subjected to a temporal regulation, such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that IIIa pre-mRNA splicing is activated more than 200-fold in nuclear extracts prepared from late adenovirus-infected cells (Ad-NE) compared to uninfected HeLa cell nuclear extracts (HeLa-NE). In contrast, splicing of the beta-globin pre-mRNA is repressed in Ad-NE. We constructed hybrid pre-mRNAs between IIIa and beta-globin in order to identify the minimal IIIa sequence element conferring enhanced splicing in Ad-NE. Using this approach, we show that the IIIa branch site/pyrimidine tract functions as a Janus element: it blocks splicing in HeLa-NE and functions as a splicing enhancer in Ad-NE. Therefore, we named this sequence the IIIa virus infection-dependent splicing enhancer (3VDE). This element is essential for regulated IIIa pre-mRNA splicing in Ad-NE and sufficient to confer an enhanced splicing phenotype to the beta-globin pre-mRNA in Ad-NE. We further show that the increase in IIIa splicing observed in Ad-NE is not accompanied by a similar increase in U2AF binding to the IIIa pyrimidine tract. This finding suggests that splicing activation by the 3VDE may operate without efficient U2AF interaction with the pre-mRNA. Importantly, this report represents the first description of a splicing enhancer that has evolved to function selectively in the context of a virus infection, a finding that adds a new level at which viruses may subvert the host cell RNA biosynthetic machinery to facilitate their own replication.


Subject(s)
Adenoviridae/genetics , Gene Expression Regulation, Viral/genetics , RNA Precursors/metabolism , RNA Splicing , Globins/genetics , HeLa Cells , Humans , Mutation , Nuclear Proteins , RNA, Viral/metabolism , RNA-Binding Proteins/genetics , Ribonuclease H/metabolism , Ribonucleoprotein, U2 Small Nuclear/metabolism , Ribonucleoproteins/genetics , Splicing Factor U2AF
5.
EMBO J ; 18(4): 1014-24, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-10022843

ABSTRACT

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.


Subject(s)
Hyaluronan Receptors , Nuclear Proteins/genetics , Proteins/genetics , RNA Splicing/genetics , RNA-Binding Proteins/genetics , Carrier Proteins , Cytoplasm/genetics , Gene Expression Regulation/genetics , Globins/genetics , HeLa Cells , Humans , Mitochondrial Proteins , Phosphoproteins/genetics , Phosphorylation , Recombinant Proteins/genetics , Serine-Arginine Splicing Factors , Transfection/genetics
6.
J Eukaryot Microbiol ; 45(6): 661-7, 1998.
Article in English | MEDLINE | ID: mdl-9864857

ABSTRACT

The full length coding sequence of the Euglena gracilis actin gene was determined by RT-PCR of Euglena gracilis mRNA. Conserved regions in the actin amino acid sequence were used as guides for the synthesis of degenerate primers. Sequence was obtained for 1,238 nucleotides, of which 1,131 were coding for 377 amino acids. Sequence comparisons showed a similarity with other actins of 56% to 80%. Even though most of the actin amino acid sequence was conserved, some regions showed high divergence, i.e. the DNase I-binding loop at the N-terminal region. The construction of a phylogenetic tree based on actin sequences from different organisms placed Euglena gracilis in a cluster with Trypanosoma brucei and Leishmania major.


Subject(s)
Actins/genetics , Euglena gracilis/genetics , Genes, Protozoan , Amino Acid Sequence , Animals , Base Sequence , DNA, Protozoan , Molecular Sequence Data , Sequence Homology, Amino Acid
7.
Nature ; 393(6681): 185-7, 1998 May 14.
Article in English | MEDLINE | ID: mdl-9603524

ABSTRACT

SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. Here we show that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. We further show that the virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into that of SR proteins purified from late adenovirus-infected cells. Taken together, our results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. We conclude that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation.


Subject(s)
Adenoviridae/genetics , Adenovirus E4 Proteins/genetics , RNA Splicing , RNA-Binding Proteins/metabolism , Viral Proteins/metabolism , Escherichia coli , HeLa Cells , Humans , Open Reading Frames , Phosphorylation , Transfection , Viral Proteins/genetics
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