Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients.

Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant beta-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of beta-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654/beta(E), IVS2-745/IVS2-745, and IVS2-745/IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was approximately 30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective beta-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.

Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake.

A novel, positive read-out assay that quantifies only sequence-specific nuclear activity of antisense oligonucleotides was used to evaluate morpholino and 2'-O-methyl sugar-phosphate oligonucleotides. The assay is based on modification of the splicing pathway of human beta-globin pre-mRNA. In addition, scrape-loading of cells with oligonucleotides allows the separate assessment of intracellular antisense activity of the oligonucleotides and their ability to penetrate the cell membrane barrier. The results show that, with scrape-loading, the morpholino oligonucleotides were approximately 3-fold more effective in their intrinsic antisense activity than alternating phosphodiester/phosphorothioate 2'-O-methyl-oligoribonucleotides and 6-9- and almost 200-fold more effective than the exclusively phosphorothioate and phosphodiester derivatives, respectively. The morpholino oligonucleotides were over 20-fold more effective than the phosphorothioate 2'-O-methyl-oligoribonucleotides in free uptake from the culture media. The antisense activity of the morpholino oligonucleotides was detectable not only in monolayer HeLa cells but also in suspension K562 cells. Time course experiments suggest that both the free uptake and efflux of morpholino oligonucleotides are slow.

Sensitivity of splice sites to antisense oligonucleotides in vivo.

A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.

A system for shuttling 200-kb BAC/PAC clones into human cells: stable extrachromosomal persistence and long-term ectopic gene activation.

A novel shuttle vector, pBH140, has been constructed that allows stable maintenance of large genomic inserts as human artificial episomal chromosomes (HAECs) in mammalian cells. The vector, essentially a hybrid BAC-HAEC, contains an F-based replication system as in a bacterial artificial chromosome (BAC) and the Epstein-Barr virus (EBV) latent origin of replication system, oriP, for replication in human cells. A 185-kb DNA insert containing the entire human beta-globin locus, including its locus control region (LCR), was retrofitted into this vector. The resulting beta-globin BAC-HAEC clone, p148BH, was transfected into human cells and analyzed for episomal maintenance and expression of the beta-globin gene. FISH revealed an association of the vector with different human chromosomes but no integration. The beta-globin BAC-HAECs were present at an average copy number of 11-15 per nucleus in the stably transformed human cells. After 1 year of continuous in vitro cultivation, the HAECs persisted as structurally intact 200-kb episomes. While no beta-globin transcription could be detected in the parental D98/Raji cells, correctly spliced RT-PCR products were produced at significant levels in long-term cultures of the BAC-HAEC-transduced cells. The wide availability of BAC and PAC libraries, the ease in manipulating cloned DNA in bacteria, and the episomal stability of the pBH140 vector make this system ideal for studies on gene expression and other genomic functions in human cells. The potential significance of large, functionally active episomes for gene therapy is discussed.

A common human beta globin splicing mutation modeled in mice.

The betaIVS-2-654 C-->T mutation accounts for approximately 20% of beta thalassemia mutations in southern China; it causes aberrant RNA splicing and leads to beta0 thalassemia. To provide an animal model for testing therapies for correcting splicing defects, we have used the "plug and socket" method of gene targeting in murine embryonic stem cells to replace the two (cis) murine adult beta globin genes with a single copy of the human betaIVS-2-654 gene. No homozygous mice survive postnatally. Heterozygous mice carrying this mutant gene produce reduced amounts of the mouse beta globin chains and no human beta globin, and have a moderate form of beta thalassemia. The heterozygotes show the same aberrant splicing as their human counterparts and provide an animal model for testing therapies to correct splicing defects at either the RNA or DNA level.

Repair of thalassemic human beta-globin mRNA in mammalian cells by antisense oligonucleotides.

In one form of beta-thalassemia, a genetic blood disorder, a mutation in intron 2 of the beta-globin gene (IVS2-654) causes aberrant splicing of beta-globin pre-mRNA and, consequently, beta-globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human beta-globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human beta-globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

A novel autoantibody to the putative oncoprotein DEK in pauciarticular onset juvenile rheumatoid arthritis.

OBJECTIVE: To define the frequency of a novel autoantibody reactive with a 45 kDa protein in children with juvenile rheumatoid arthritis (JRA). This protein is expressed by the putative oncogene DEK associated with a subtype of acute myeloid leukemia. METHODS: The sera of 158 children with JRA were analyzed for the presence of anti-DEK by immunoblotting using purified DEK protein and compared with sera of 109 children with other rheumatic diseases and 25 healthy controls with no connective tissue disease. RESULTS: Antibodies to DEK were found significantly more frequently among children with JRA than among children with other rheumatic diseases or controls (p < 0.001). Among children with JRA, anti-DEK was significantly more often associated with pauciarticular onset than with poly-articular and systemic onset subtypes (77 vs 29 and 8%, respectively, p < 0.001). Anti-DEK was no more frequent among children with pauciarticular JRA complicated by iritis than among those without iritis (88 vs 71%, respectively). The frequency of anti-DEK in other rheumatic diseases varied from 0 in children with spondyloarthritis to 31% in scleroderma. CONCLUSION: Antibodies to DEK are highly associated with pauciarticular onset JRA.

The putative oncoprotein DEK, part of a chimera protein associated with acute myeloid leukaemia, is an autoantigen in juvenile rheumatoid arthritis.

The 45-kD autoantigen associated with juvenile rheumatoid arthritis (JRA) has been isolated from HeLa cell nuclei and purified about 2500-fold to near homogeneity in a five-step chromatographic procedure. Purification of the antigen was monitored by immunoblot assays using a nearly monospecific anti-45-kD serum from a child with JRA. Tryptic peptide mapping and partial amino acid sequencing of the purified 45-kD antigen demonstrated its identity with the DEK protein. DEK is a 43-kD protein of unknown function expressed by the putative oncogene dek located on chromosome 6. As a result of a (6;9) translocation offociated with a rare subtype of acute myeloid leukaemia a chimeric protein containing most of DEK amino acids at the N-terminus is found in leukaemic cells (von Linden et al., Mol Cell Biol. 1992; 12: 1687-97). The 43-kD DEK was detected by immunoblotting with serum from a patient with JRA in a variety of rat tissues, and was most abundant in the spleen and in bone marrow.

Antinuclear antibody profile in juvenile rheumatoid arthritis.

Immunoblot positive sera from children with juvenile rheumatoid arthritis detected from 1 to greater than or equal to 10 proteins in HeLa nuclear sonicates. Thirty percent of the sera reacted with histone H1. Antibodies to at least 1 of 6 most frequently detected nonhistone proteins were present in 85% of the sera. Using immunopurified antibodies to each of the 6 common antigens, we found that 4 of them were associated with mitotic chromosomes. Most sera detected at least 1 of these 4 nonhistone chromosomal proteins. Fifteen percent of the sera immunoprecipitated ribonucleoproteins; some exhibited a novel specificity, precipitating mature transcripts of RNA polymerase III. When present, antibodies to a 45 kDa protein occur only in sera from children without iritis and not in those with active iritis. Overall, the antibody profiles were highly individual and did not appear to correlate with disease subtype or activity.

Inhibition of pre-mRNA splicing by 5-fluoro-, 5-chloro-, and 5-bromouridine.

Pre-mRNA transcripts of the human beta-globin gene containing 5-fluoro-, 5-chloro-, and 5-bromouridine were tested for splicing in vitro. Pre-mRNA containing 5-fluorouridine was spliced accurately and efficiently in the nuclear extract from HeLa cells, whereas 5-chloro-, and 5-bromouridine containing transcripts were not spliced. Analysis of the splicing reactions by electrophoresis on nondenaturing polyacrylamide gels showed that the latter two transcripts were unable to form active splicing complexes. Treatment of HeLa cell cultures with 5-fluorouridine decreased the splicing activity of the nuclear extracts in a dose- and time-dependent fashion. The decrease in splicing activity of these extracts appears to be due in part to a decreased level of U-2 small nuclear RNA and the corresponding ribonucleoprotein particle, U2-snRNP.

Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells.

A protein of the C group, most likely C3 (Mr approximately 42,000, pI approximately 6, corresponding to IEF 48m,n of the HeLa protein catalogue (Celis, J. E., Bravo, R., Arenstorf, H. P., and LeStourgeon, W. M. (1986) FEBS Lett. 194, 101-109)), a minor hnRNP protein was purified to near homogeneity under nondenaturing conditions from 40 S heterogeneous nuclear ribonucleoprotein particles. Type C protein stoichiometrically disrupts the residual secondary structure of natural and synthetic RNAs, e.g. HeLa hnRNA, coliphage MS2 RNA, and poly(rU)-spermine, and decreases the Tm of duplex structures, e.g. poly[r(A + U)], by about 30 degrees C. Binding of the protein to polynucleotides is not highly cooperative and has a stoichiometry of one protein per about 10 nucleotides. Binding experiments with a variety of synthetic and natural poly- and oligonucleotides, including those containing consensus splice site sequences, indicate that the protein has a high affinity for G-rich and U-rich regions, G-rich regions being preferred. Base analogs I and T have affinities for the protein that are similar to G and U. There is little or no affinity for A- and C-rich regions. The presence of A residues in a G- or U-rich sequence does not interfere with binding while C-rich regions decrease or prevent the binding of the protein. The nucleotide specificity of type C protein, e.g. selective binding to an oligonucleotide from the 3' end of an intron, is discussed in relationship to the abundance of G and U and the relative scarcity of C residues in the processing signals in pre-mRNA.

Antibodies to hnRNP core proteins inhibit in vitro splicing of human beta-globin pre-mRNA.

In vitro splicing of human beta-globin pre-mRNA can be fully inhibited by treatment of the splicing extract with polyclonal antibodies against hnRNP core proteins prior to the addition of pre-mRNA. Inhibition of the first step in the splicing pathway, cleavage at the 5' splice site and lariat formation, requires more antibodies than inhibition of the second step, cleavage at the 3' splice site and exon ligation. The anti-hnRNP antibodies can also inhibit the splicing reaction after the formation of the active nucleoprotein splicing complex which is known to occur during the initial lag period. Thus, hnRNP core proteins appear to be present in the complex that performs pre-mRNA splicing.

Nucleotide pyrophosphatase from potato tubers. Purification and properties.

Purification of potato tuber nucleotide pyrophosphatase (EC 3.6.1.9) has been modified to furnish a rapid and reproducible procedure yielding a preparation purified 1800-fold and homogeneous in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The Mr of the enzyme, from gel filtration or sucrose density gradient centrifugation, is 343000 or 346000 respectively; and SDS electrophoresis indicates an Mr for the subunit of 74000. Analytical isoelectrofocusing reveals a broad isoelectric range of pH 8.3-8.7. The enzyme is a glycoprotein. The purified enzyme exhibits the previously reported activities versus pyrophosphate linkages located at either the 5'-OH or 3'-OH of nucleosides, and phosphodiester linkages in: (a) aryl esters of nucleoside 3'- and 5'-phosphates, p-nitrophenylphosphate and orthophosphate, and (b) nucleoside cyclic 2',3'-phosphates. However, the relative rates of activity towards these substrates, and the corresponding V values, differ significantly. The enzyme exhibits additional novel activities, including ability to cleave dinucleoside polyphosphates such as A(5')p2(5')A-A(5')p5(5')A, and aryl phosphonates. Contrary to previous reports, there is no activity towards nucleoside cyclic 3',5'-phosphates. The present preparation is also devoid of endonucleolytic activity, so that it specifically cleaves m7GMP from the 5'-terminal m7G(5')p3(5')Gm of intact reovirus mRNA. NAD+ was found to be the most effective inhibitor of enzyme activity versus thymidine 5'-p-nitrophenylphosphate, with a Ki = 0.1 mM. Kinetic analyses demonstrated competitive inhibition between these two substrates. Both 2',3'-cAMP and thymidine 3'-p-nitrophenylphosphate inhibit hydrolysis of NAD+ noncompetitively and vice-versa.

Histochemical localization of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase in seeds and shoots of Triticum.

The activities of potato nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase against a common substrate, p-nitrophenyl thymidine 5'-phosphate and its histochemical analogue, AS-BI-naphthyl thymidine 5'-phosphate, were determined with the aid of relatively specific inhibitors, NAD and 2',3'-cAMP, respectively. These inhibitors were utilized to reexamine wheat (Triticum aestivum L. cv. Mironovska 808) seeds and 3-5-d old shoots for the occurrence and histochemical localization of nucleotide pyrophosphatase, and to establish the localization of cyclic nucleotide phosphodiesterase. Nucleotide pyrophosphatase is a cytoplasmic enzyme found to be particularly active in the coleoptile epidermis and hypodermis, leaf mesophyll, as well as in developing fibres and phloem. Cyclic nucleotide phosphodiesterase is also a cytoplasmic enzyme active in the shoot vascular bundles, particularly the xylem, and in the seed. Within the seed it is highly active in the crushed cell layer adjacent to the scutellum and in endosperm cells adjacent to the aleurone layer. Within the embryo, cyclic nucleotide phosphodiesterase is most active in epithelial cells adjacent to the crushed cell layer, the suspensor, radicle and root-cap, as well as in the pro-vascular tissues of the scutellum.

Nucleotide pyrophosphatase activity in dry and germinated seeds of Triticum, and its relationship to general acid phosphatase activity.

A cytochemical investigation has been made of nucleotide pyrophosphatase activity in dry and germinated seeds of Triticum, and its distribution compared to that of general acid phosphatase reactions seen with naphthol AS-BI phosphate and p-nitrophenylphosphate as substrates. Acid phosphatase activity was present in the cytoplasm and in channels through the walls of the aleurone cells in both dry and germinated seeds. The cytoplasmic activity was even more marked with nucleotide pyrophosphatase which was almost entirely absent from the cell walls. Nucleotide pyrophosphatase was active in all endosperm cells but particularly in some cells adjacent to the aleurone layer. In addition, all cells of the scutellum and embryo were positive for nucleotide pyrophosphatase activity, especially the developing fibres and xylem elements of leaves and coleoptiles, mature leaf xylem and phloem elements, scutellar provascular and vascular tissues and the epidermis of dark grown coleoptiles.

The cytochemical localization of nucleotide pyrophosphatase activity in plant tissues using naphthyl esters of thymidine-5'-phosphate.

A cytochemical method for the localization of nucleotide pyrophosphatase activity in plants employing naphthol AS-BI thymidine 5'-monophosphate and alpha-naphthyl thymidine 5'-monophosphate as specific substrates is reported. Biochemical evidence for the validity of this method is presented and the synthesis of the naphthol AS-BI ester is described. The application of this cytochemical technique to shoots of Triticum sp. and roots of Vicia faba has shown nucleotide pyrophosphatase to be ubiquitous in its distribution in these organs and to occur in a structurally-bound form in the cytoplasm. The highest activity was detected in developing fibres adjacent to the leaf vascular bundles, in the coleoptile epidermal and hypodermal cells and in the coleoptile and leaf xylem.