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I H Maxwell

Publications and source records attributed to I H Maxwell.

At least 55 records · Page 3Linked to original sources
PubMed1987

Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTH1-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTH1, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15- to 30-fold more pTH1-176 than pTH1 was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.

Amino Acid Sequence↗
PubMed1986

Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide.

As an alternative to directing plant or bacterial toxins to surface receptors, we are investigating the possibility of killing tumor cells by the expression of an exogenously introduced toxin gene (i.e., cell suicide). Tissue-specific gene regulatory elements might thus be exploited to achieve selective killing. To assess the feasibility of such an approach, we have transfected human cells (HeLa, B-lymphoblastoid, and 293 cells) with plasmids containing the diphtheria toxin A-chain (DT-A) coding sequence. The presence of the DT-A sequence lowered the level of transient expression of chloramphenicol acetyltransferase from a cotransfected plasmid, pSV2cat. This expression level in B-cells was further diminished by the inclusion of an immunoglobulin enhancer in the DT-A plasmid. In cotransfection experiments with a DT-A plasmid lacking an enhancer, chloramphenicol acetyltransferase expression was much more strongly inhibited in 293 cells (which express adenovirus E1A and E1B products) than in the other cell types; furthermore, the presence of the DT-A sequence eliminated recovery of G418-resistant 293 cell transformants after transfection with a plasmid containing the neo selectable marker. These results suggest that cell-specific regulatory mechanisms can be exploited to achieve selective cell killing by expression of an introduced toxin gene.

Acetyltransferases↗
PubMed1982

Herpesvirus infection alters the steady-state levels of cellular polyadenylated RNA in polyoma virus-transformed BHK cells.

Polyoma-transformed BHK cells are permissive for the replication of herpes simplex virus type 1. The effect of herpes infection on the steady-state levels of bulk mRNA sequences in these cells was studied by using cDNA to polyadenylated cytoplasmic RNA from uninfected cells. The principal findings were: (i) herpes simplex virus type 1 infection caused a pronounced reduction in the cytoplasmic levels of moderately abundant mRNAs' (ii) after infection, increased amounts of RNA complementary to the cDNA were isolated as part of the nonadenylated cytoplasmic RNA fraction.

Animals↗
PubMed1980

General occurrence and transcription of intervening sequences in mouse genes expressed via polyadenylated mRNA.

cDNA of modal size approximately 1600 nucleotides, transcribed from mouse brain polyadenylated mRNA, was annealed with excess of high molecular weight (approximately kb) genomic DNA. The S1 nuclease method was then applied to determine possible sequence discontinuity between the cDNA and genomic DNA. A substantial reduction in the average size of the annealed cDNA was observed following S1 nuclease treatment. Large single copy genomic DNA, annealed with excess high molecular weight DNA, and cDNA, hybridized with its template mRNA, were resistant to cleavage by S1 nuclease. We interpret these results to indicate a high frequency of discontinuous coding sequences in the genomic DNA that annealed with the cDNA. The same result was obtained using fractionated cDNA, enriched in transcripts of relatively infrequent or abundant mRNA species. The result obtained with the infrequent sequence class cDNA indicates that tens of thousands of split genes exist in the mouse genome. Extensive cleavage of the cDNA by S1 nuclease was also observed after hybridization with greater than 30S nuclear RNA, indicating that intervening sequences are generally transcribed.

Animals↗
PubMed1979

Complex population of nonpolyadenylated messenger RNA in mouse brain.

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.

Animals↗
PubMed1978

Complex population of mRNA sequences in large polyadenylylated nuclear RNA molecules.

Polyadenylylated heterogeneous nuclear RNA [poly(A)-hnRNA] from mouse brain was subjected to electrophoresis in agarose gels containing CH3HgOH, and molecules larger than 8 kilobases or 13 kilobases were recovered. cDNA was then transcribed from polyadenylylated RNA fragments cleaved from these large molecules. The resulting cDNA hybridized almost to completion with poly(A)-mRNA isolated from mouse brain polysomes. From the hybridization kinetics of this cDNA with its template RNA, it was estimated that the sequence complexity of the 3'-proximal sequences (of the same average size as mRNA) of the greater than 8 kilobase poly(A)-hnRNA was about 57,000 kilobases. The sequence complexity of poly(A)-mRNA, estimated from the template-driven hybridization kinetics of its respective cDNA, was about 110,000 kilobases. It is concluded that most, if not all, of the 3'-proximal sequences of large poly(A)-hnRNA molecules are homologous with mRNA in the mouse brain and that at least 40,000 different mRNA sequences (or portions of mRNA sequences) are represented in the 3'-proximal sequences of greater than 8 kilobase poly(A)-hnRNA.

Animals↗
PubMed1978

Assay of DNA-RNA hybrids by S1 nuclease digestion and adsorption to DEAE-cellulose filters.

A fast and accurate assay procedure for DNA-RNA hybrids is described in which exhaustive digestion of unhybridized DNA with S1 nuclease is followed by binding of hybrids to filter discs of DEAE-cellulose. The digested DNA can be efficiently washed from the filters so that background levels of 0.1-0.2% of input tracer DNA can be achieved, in contrast to the much higher (approximately 1-5%) backgrounds obtained using TCA precipitation procedures. Short duplexes, as small as 36 nucleotides in length, which are inefficiently bound to hydroxyapatite, are quantitatively bound to the DEAE-cellulose filters.

Animals↗
PubMed1977

Removal of RNase activity from DNase by affinity chromatography on agarose coupled aminophenylphosphoryl-uridine-2' (3')-phosphate.

Severe degradation of high molecular weight RNA was shown to occur during incubation with commercially purified DNase. Most of the RNase activity could be removed by passage of the DNase through a column of agarose-coupled amino phenylphosphoryl-uridine-2' (3')-phosphate. Incubation with the treated DNase caused only minimal alteration of the sedimentation pattern of high molecular weight nuclear RNA, determined under partially denturing conditions. No impairment of DNase activity was detected.

Chromatography, Affinity↗
PubMed1976

Polyadenylated virus-specific RNA in baby hamster kidney cells, transformed by polyoma virus.

RNA from a clone of polyoma virus-transformed hamster cells was fractionated by chromatography on oligo(dT)-cellulose. The proportion of virus-specific RNA in the polyadenylated and non-polyadenylated fractions was determined by hybridization of the labelled RNA with excess purified polyoma DNA, immobilized on filters. Seventy to 80% of the virus-specific RNA in both polysomal and total cell RNA was found in the polyadenylated fraction. Since it has been shown previously that more than 65% of the total virus-specific RNA is restricted to the nucleus in this cell line, these results indicate that a high proportion (at least 53%) of the nuclear virus-specific RNA is polyadenylated. The sedimentation profile of total polyadenylated virus-specific RNA in dimethyl sulphoxide was comprised mainly of a broad band with a median sedimentation coefficient about 26S (relative to 28S rRNA). This profile was similar to that of total nuclear, and not cytoplasmic, virus-specific RNA. To estimate the intramolecular proximity of virus-specific sequences to poly(A), total RNA was subjected to limited thermal scission to an average mol. wt. similar to that of mRNA. The RNA remaining attached to poly(A) was then isolated, using oligo(dT)-cellulose. It was found that 65% of the virus-specific RNA that was originally attached to poly(A) was released by the thermal scission. Most of the virus-specific sequence within polyadenylated RNA molecules therefore must have been located at some distance from the polyadenylated 3'-terminus. This observation, together with the results of sedimentation analysis, can most simply be explained by postulating the existence of 'hybrid' RNA molecules containing a host-specified sequence located between a virus-specific sequence and the 3'-terminal poly(A).

Cell Line↗
PubMed1976

Intracellular distribution and sedimentation properties of virus-specific RNA in two clones of BHK cells transformed by polyoma virus.

The virus-specific RNA in two independently derived clones of polyoma virus-transformed hamster cells was studied by hybridizing labeled RNA, with excess purified polyoma DNA, immoblized on filters. In one clone (PyBHK1), less than 25% of the total labeled virus-specific RNA was found in the cytoplasm, irrespective of the labeling time. In the other clone (PyBHK2), it was estimated that 39% of the total virus-specific RNA was present inthe cytoplasm after labeling for 3 h. Both the proportion of radioactive label incorporated into virus-specific RNA and the sedimentation pattern of total virus-specific RNA differed markedly between PyBHK and PyBHK2. Most of the virus-specific RNA of PyBHK1 sedimented in the range 25S-35S, whereas a prominent 18S component was present in PyBHK2. Most of the cytoplasmic virus-specific RNA in both clones sedimented at 18S-19S. The sedimentation patterns of virus-specific RNA from whole cells and from washed nuclei of PyBHK1 were closely similar: it was estimated from sedimentation analysis in dimethyl sulfoxide that the molecular weight of 50% of this RNA was within the range 1.1 X10(6) to 2.9 X 10(6). These results, demonstrating the accumulation of virus-specific RNA within the nucleus in at least one virus-transformed cell line, indicate that the large virus-specific RNA previously described in the nuclei of transformed cells may not have represented precursors of virus-specific mRNA.

Cell Line↗
PubMed1975

Strand-specific transcription of polyoma virus DNA-early in productive infection and in transformed cells.

The DNA strand origin of nuclear and cytoplasmic polyoma-specific RNA in productively infected mouse cells and in a line of polyoma-transformed hamster cells was determined by hybridization of unlabeled RNA with radioactively labeled separated strands of polyoma DNA. Early in the productive cycle (10 h postinfection) nuclear viral RNA is complementary to only about 40% of the E strand of viral CNA. No RNA complementary to the L strand was detected even when the RNA was first self-annealed to enrich for possible minor species. Early cytoplasmic RNA is complementary to the same 40% of the E strand. Thus, only that part of the poloma genome which codes for early virual messenger RNA appears to be transcribed. Late in infection, nuclear viral RNA is complementary to most or all of the L strand and to at least 60% of the E strand. Late cytoplasmic viral RNA hybridizes to 40 to 45% of the E strand and 50 to 55% of the L strand. The transformed cell nuclear viral RNA is complementary to 60% of the E strand, whereas cytoplasmic RNA is complementary to 40% of the E strand and comprises the same polyoma-specific sequences as are found in RNA early in productive infection. No L strand transcripts could be detected. Thus, in the transformed cells and late in productive infection, viral RNA sequences in the cytoplasm are a specific subset of those in the nucleus.

Cell Line↗