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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 307 records · Page 17Linked to original sources
PubMed1973

Transcription of simian virus 40. II. Hybridization of RNA extracted from different lines of transformed cells to the separated strands of simian virus 40 DNA.

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and "revertants" isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and "revertant" cell lines. From 30 to 80% of the "early" strand and from 0 to 20% of the "late" strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.

Animals↗
PubMed1972

Electron microscope studies of heteroduplex DNA from a deletion mutant of bacteriophage phiX-174.

A population of double-stranded replicative form of DNA molecules from bacteriophage varphiX-174 carrying a deletion of about 9% of the wild-type DNA has been discovered in a sample cultivated under conditions where the phage lysozyme gene is nonessential. The structures of deleted monomers, dimers, and trimers were studied by the electron microscope heteroduplex method. The dimers and trimers are head-to-tail repeats of the deleted monomers. Some interesting examples of the dynamical phenomenon of branch migration in vitro have been observed in heteroduplexes of deleted dimer and trimer strands with undeleted monomer viral strands from the wild-type phage.

Coliphages↗
PubMed1972

Sequence heterogeneity in closed simian virus 40 deoxyribonucleic acid.

The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved. The undenatured DNA from viruses passaged twice at multiplicities of infection much less than 1 plaque-forming unit (PFU) per cell appeared to be homogeneous in size. The heteroduplexes formed by this DNA indicated that approximately 2% of the molecules carried deletions, but that substitutions were below the level of detection. In contrast, undenatured DNA from viruses grown by passaging undiluted lysates seven times or by infection with stock virus at a multiplicity of infection of 5 PFU per cell contained a large frequency of molecules shorter than the full length. The heteroduplex samples indicated that 12 and 7% of the undenatured material contained base substitutions, and 13 and 11% contained deletions. The deletions and substitutions appear to occur in separate molecules. Length measurements on heteroduplexes displaying the loop characteristic of substitutions have established that these molecules are from true sequence substitutions, and not from adjacent or overlapping deletions. More than 80% of the molecules carrying substitutions are shorter than the native SV40 length. On the average, the substituted sequence is about 20% of the length of SV40, but it replaces a sequence about 30% of the native length. The substituted sequences may be host cell nuclear DNA, possibly arising from integration of SV40 into the chromosome followed by excision of the SV40 DNA together with chromosomal DNA.

Animals↗
PubMedDate not supplied

Cofactor requirements of splicing of purified messenger RNA precursors.

The origin and functions of introns in protein coding genes is one of the enigmas of molecular biology. Splicing processes that remove intervening sequences from precursor RNAs must have either predated or co-evolved with introns. Inferences about the origin of introns and the possible modes of regulation of splicing should emerge from an understanding of the biochemical mechanisms of splicing. The biochemistry of splicing of tRNA and rRNA precursors has rapidly advanced with the development of in vitro reactions containing soluble components that duplicate in vivo reactions. We have recently shown that accurate splicing of an adenovirus mRNA precursor occurs during a coupled transcription/splicing reaction in a soluble whole cell extract. We now report that an exogenous RNA substrate containing the first and second leaders of adenovirus 2 is accurately spliced when added to an extract of HeLa cells. ATP and Mg2+ are essential cofactors for the reaction. The time course of splicing is unusual; a lag of 45 min is observed before the appearance of splicing product.

Adenosine Triphosphate↗
PubMedDate not supplied

Regulation of heat shock protein 70 gene expression by c-myc.

The myc gene seems to have a causal role in tumour formation in man, mouse and avian systems. The myc gene product has been localized to the nucleus, suggesting that it may be involved in the regulation of gene expression. The level of expression of the mammalian heat shock protein 70 (HSP70) gene is elevated in several tumour cell lines, implying that a cellular function expressed in these tumour lines can stimulate HSP70 production. We report here that the gene product of a rearranged mouse c-myc gene is capable of stimulating expression of chimaeric genes containing a Drosophila hsp70 promoter region and 5'-flanking sequences. This stimulation is dependent on sequences located more than 200 bases 5' of the normal start of hsp70 transcription.

Animals↗
PubMedDate not supplied

A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes.

Trans-acting factors that mediate B-cell specific transcription of immunoglobulin genes have been postulated based on an analysis of the expression of exogenously introduced immunoglobulin gene recombinants in lymphoid and non-lymphoid cells. Two B-cell-specific, cis-acting transcriptional regulatory elements have been identified. One element is located in the intron between the variable (V) and constant (C) regions of both heavy and kappa light-chain genes and acts as a transcriptional enhancer. The second element is found upstream of both heavy and kappa light-chain gene promoters. This element directs lymphoid-specific transcription even in the presence of viral enhancers. We have sought nuclear factors that might bind specifically to these two regulatory elements by application of a modified gel electrophoresis DNA binding assay. We report here the identification of a human B-cell nuclear factor (IgNF-A) that binds to DNA sequences in the upstream regions of both the mouse heavy and kappa light-chain gene promoters and also to the mouse heavy-chain gene enhancer. This sequence-specific binding is probably mediated by a highly conserved sequence motif, ATTTGCAT, present in all three transcriptional elements. Interestingly, a factor showing similar binding specificity to IgNF-A is also present in human HeLa cells.

B-Lymphocytes↗
PubMedDate not supplied

Distinct factors bind to apparently homologous sequences in the immunoglobulin heavy-chain enhancer.

The intron separating the variable- and constant-region exons of the rearranged immunoglobulin heavy-chain locus contains a lymphocyte-specific transcriptional enhancer. The enhancer is a member of a class of cis-acting, tissue-specific, transcriptional control elements which are characterized by orientation-independent and relatively position-independent function. In vivo analysis of the position of DNA-binding factors, by assessing the availability of specific bases to chemical modification, has identified four sequence clusters within the heavy-chain enhancer, denoted E1 to E4 (refs 3, 4). These sites are protected (that is, occupied) only in B lymphocytes. A consensus sequence relationship (consensus CAGGTGGC) between these four sites was suggested where three of the sites conformed to the consensus in seven of eight positions while the other was homologus in six of eight positions. We proposed that a single trans-acting factor might recognize all four sites. Using an assay involving gel electrophoresis of DNA-protein complexes to detect sequence-specific DNA binding factors that recognize these related motifs, we have now identified a mouse B-cell nuclear factor (NF-muE1) which binds specifically to one such motif within the mouse heavy-chain gene enhancer. This factor binds poorly, if at all, to the other related motifs, and other factors have been identified which interact preferentially with some of these latter motifs. Dimethyl sulphate interference experiments suggest that the NF-muE1 factor is in contact with at least the guanine residues in the sequence GATGGCCGATC. This factor seems to be present in both lymphoid and non-lymphoid cell lines.

Animals↗
PubMedDate not supplied

A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes.

Immunoglobulin gene promoters are active only in lymphoid cells and this tissue-specific activity requires an octamer sequence, ATTTGCAT. Paradoxically, this same octamer motif seems to be a transcriptional control element in promoters which are active in all tissues. Using an electrophoretic mobility shift assay to identify DNA binding proteins, we have now detected two species of nuclear proteins which bind specifically to this octamer. One previously characterized form (NF-A1) was found in all cell lines tested while the other form (NF-A2) was restricted to lymphoid cell lines. NF-A2 was found in cell lines representing all stages of B-cell differentiation and in half of the T-lymphoma cell lines tested. The identification of a lymphoid-specific octamer binding protein may account for the lymphoid-specific activity of immunoglobulin promoters.

Animals↗
PubMedDate not supplied

A high-efficiency HeLa cell nuclear transcription extract.

A HeLa cell nuclear transcription extract that is approximately 20 times more efficient than standard HeLa cell transcription extracts was developed. Transcription of the strong adenovirus II major late promoter by this extract results in the synthesis of 1.5-4 molecules of product RNA per molecule of template, indicating that the extract is capable of multiple rounds of initiation. Standard HeLa cell nuclear extracts transcribe closed circular and linear adenovirus major late promoter templates with equal efficiency. In contrast, the new extract exhibits an increase of approximately twofold on transcription of a closed circular, as opposed to a linear, major late promoter template.

Adenoviridae↗
PubMedDate not supplied

Lymphocyte circulation to the middle ear.

The movement of lymphocytes into the tympanic cavity was studied during an immune response in the middle ear. Sensitized lymphocytes obtained from peripheral blood, peripheral lymph nodes, spleen, mesenteric lymph nodes and Peyer's patches of strain-13 inbred guinea pigs were labelled with 51Cr and injected intravenously into strain-13 recipients undergoing a middle ear immune response. The middle ears and immune organs of the recipients were assayed for radioactivity to detect the infiltration of labelled cells. Lymphocytes from all sources entered the middle ear mucosa in response to immune stimulation at equal levels, suggesting that the middle ear is seeded non-specifically by circulating lymphocytes in response to antigenic stimulation. The infiltration of lymphocytes of mucosal origin into the middle ear cavity is consistent with the participation of the middle ear in a defense system in common with other mucosal organs.

Animals↗