Stage-specific reprogramming of globin gene expression.
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Animals↗
Biomedical subjects
M H Baron
Publications and source records attributed to M H Baron.
Rapid reprogramming of globin gene expression in transient heterokaryons.
Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types. Analysis of total cellular RNA isolated 24 hr after fusion revealed that normally inactive globin genes can be activated in these "transient" heterokaryons, in which the nuclei do not fuse. In general, the types of globin genes expressed in the donor erythroid cell are activated in the nucleus of the recipient cell. Therefore, erythroid cells contain transacting regulatory factors that are capable of activating the expression of globin genes in a stage- and tissue-specific manner. These observations also indicate that globin genes are not irreversibly repressed in differentiated cells and that their expression can be rapidly reprogrammed in the presence of the appropriate regulatory factors.
Animals↗
Purification and properties of a host cell protein required for poliovirus replication in vitro.
A host cell protein required for poliovirus RNA-dependent RNA replicase activity in vitro has been purified several thousand-fold from an uninfected HeLa cell postmitochondrial supernatant. A single protein of apparent Mr = approximately 67,000 daltons and pI 6.3 is associated with this "host factor" activity. Poly(U)-Sepharose chromatography of the template-dependent replicase isolated from poliovirus-infected cells results in the complete loss of replicase activity if a salt gradient is used to develop the column. Host factor elutes early in the salt gradient and restores replicase activity to protein fractions eluted later in the gradient. The host factor, estimated to be present at 50,000-100,000 copies/cell, interacts physically with replicase.
HeLa Cells↗
In vitro copying of viral positive strand RNA by poliovirus replicase. Characterization of the reaction and its products.
Poliovirus replicase can be isolated in a form which depends on either oligo(U) or on a host cell protein for the initiation of copying of poliovirion (plus strand) RNA. The product of replicase reactions--initiated either with host factor or with oligo(U)--includes full length (35 S) RNA molecules, largely in double-stranded form, which contain the ribonuclease T1-resistant oligonucleotides of the poliovirus minus strand. For the oligo(U)-stimulated reaction, it is shown that the oligo(U) primer is covalently associated with full length product at its 5'-end. For either the host factor- or oligo(U)-dependent reactions, full length molecules appear only after 15 min of synthesis. The fraction of 35 S product is increased by raising the concentration of the limiting nucleoside triphosphate. The reaction is inhibited by as little as 100 mM salt, although it is stimulated by low (20 mM) salt concentrations. Zinc stimulates overall synthesis, but not the rate of appearance of full length molecules; the reaction is inhibited by agents which chelate zinc. Although synthesis of full length products occurs much more slowly than in the infected cell, this soluble system appears to mimic quite faithfully the initial steps of poliovirus replication.
Kinetics↗
Anti-VPg antibody inhibition of the poliovirus replicase reaction and production of covalent complexes of VPg-related proteins and RNA.
Anti-VPg antibodies inhibited host-factor-dependent RNA synthesis by the poliovirus replicase but not oligo(U)-primed synthesis, implicating VPg in the de novo initiation of replicase products. Complexes of VPg-related polypeptide and newly made RNA could be immunoprecipitated by anti-VPg antibody from the host-factor-stimulated products of the replicase reaction. The complexes appeared to be covalently linked and involved 50 to 150 nucleotide chains of RNA that were RNAase-T1-resistant and could be largely poly(U).
Poliovirus↗
Antibodies against the chemically synthesized genome-linked protein of poliovirus react with native virus-specific proteins.
The genome-linked protein (VPg) of poliovirus has been chemically synthesized, coupled to bovine serum albumin carrier and injected into rabbits. An antibody response was elicited not only by the full-length synthetic VPg peptide, but also by a synthetic 14-amino acid carboxy-terminal peptide. All antisera reacted with virus-specific proteins from HeLa cells infected with poliovirus. Three of these proteins have previously been implicated by others as precursors of VPg. No free cytoplasmic VPg could be detected, and the antibodies did not react with radiolabeled proteins from uninfected cells.
Amino Acid Sequence↗
Antibodies against a synthetic peptide of the poliovirus replicase protein: reaction with native, virus-encoded proteins and inhibition of virus-specific polymerase activities in vitro.
A carboxy-terminal peptide of the poliovirus replicase protein (p63) was chemically synthesized, coupled to bovine serum albumin carrier, and injected into rabbits. The resulting antisera reacted with six virus-specific proteins from HeLa cells infected with poliovirus: NCVP 0b, NCVP 1b, NCVP 2, a protein of about 60,000 daltons, p63, and NCVP 6b. The identity of the 60,000-dalton protein is not known, but the other results were consistent with previous experimental approaches which demonstrated that p63 and the other four polypeptides have common coding sequences. An amino-terminal peptide of p63 failed to elicit an immune response in rabbits. Antibodies raised against the p63 carboxy-terminal peptide inhibited poliovirus replicase and polyuridylic acid polymerase activities in vitro, providing strong support for earlier suggestions that these activities are a property of a single virus-specific polypeptide.
Animals↗
The location and local environment of the active sites of aspartate transcarbamylase.
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Aspartate Carbamoyltransferase↗
Poliovirus replicase: a soluble enzyme able to initiate copying of poliovirus RNA.
The soluble phase of the cytoplasm of poliovirus-infected cells contains an enzymatic activity able to copy RNA without an added primer. This replicase activity has been purified 60-fold; it is absent from uninfected cells. Poly(U) polymerase activity copurifies with replicase activity. Although less pure replicase fractions copy a variety of RNAs, purer fractions respond better to poliovirus RNA than to other viral RNAs. Even the less pure fractions make a specific copy of the added template, as shown by hybridization of the product to its template RNA but not to other RNAs. Among homopolymers only poly(A)-oligo(U) was copied by the replicase; other primed homopolymer templates were inactive.
HeLa Cells↗