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C S Young

Publications and source records attributed to C S Young.

At least 55 records · Page 3Linked to original sources
PubMed1991

Transcription from the adenovirus major late promoter uses redundant activating elements.

The adenovirus major late promoter (MLP) has been analyzed by constructing recombinant viral genomes containing mutations in possible promoter elements. Single base pair changes in the TATA box had no effect on viral replication, and MLP expression, as measured by the accumulation of late mRNAs, was at wild type levels. However, a double mutation in the TATA box reduced viral replication and MLP expression, demonstrating that the TATA box is important, although not essential, for maximal activity in virus. Primer extension analysis showed that the mRNAs were initiated at the correct position. A mutation in the CAAT box was viable, and had only minor effects on MLP expression. However, this mutation when coupled to a single mutation in the TATA box, severely reduced viral replication and expression from the MLP. Similarly, a viable mutation in the UPE, shown previously to abolish binding of USF, coupled to a single mutation in the TATA box was lethal. These results suggest that both USF and the CAAT box binding factor CP1 can interact with TFIID to effect activation, and thus that the mechanism of activation is functionally redundant.

Adenoviridae↗
PubMed1991

End-joining of DNA fragments in adenovirus transfection of human cells.

Overlapping terminal fragments of adenovirus DNA transfected into human cells either recombine to form standard unit-length genomes, or can join end-to-end to produce internally redundant, viable, genomes. The end-joining reaction in human HeLa and A549 cells is almost as efficient as the recombination reaction, and is relatively insensitive to the nature of the ends, as pairs of fragments terminating in several different single strands or in blunt ends can join. In contrast to the results from transfection with SV40, the ends are usually modified, for example by the loss of 3' single strands or the repair of 5' single strands. The ability to recover viable redundant molecules is not confined to any one area of the adenovirus genome, but can occur in the E1 and L2 regions as well as in the E2b region. The redundant genomes contain extra splice signals and may have the capacity to encode fusion proteins.

Adenovirus Early Proteins↗
PubMed1991

Adenovirus homologous recombination does not require expression of the immediate-early E1a gene.

To investigate whether early genes other than those involved directly in DNA replication are required for efficient adenovirus recombination, pairs of viruses with deletions in E1a, E1b 496R, E1b 196R, or E4 and containing differing restriction site markers were used to infect both permissive and non- or semipermissive cells. Recombination was assayed among intracellular and extracellular genomes by restriction digestion and blot hybridization. Recombination was delayed in infections of nonpermissive cells with E1a- viruses until a time consistent with the late onset of DNA replication characteristic of the cell type. This shows that E1a expression is not absolutely required for adenovirus recombination. Similar tests with deletion mutations in E1b and E4 also show that these genes are not required for efficient recombination. Taken together with earlier results showing that recombination depends on DNA replication, it is likely that adenovirus recombination is a consequence of cellular repair functions acting on the substrates produced by replication.

Adenoviridae↗
PubMed1990

Transport and lairage times of lambs slaughtered commercially in the south of England.

The times spent in transport and lairage by 124,036 lambs slaughtered in two plants in the south of England were recorded. About half of them travelled for up to 120 km, taking up to four hours, and three-quarters travelled not more than 300 km, the journeys taking up to about six hours. Bimodal frequency distributions of lairage time were apparent; overall one third of the lambs spent up to four hours in lairage but more than 40 per cent were kept for longer than 14 hours, while they were held overnight. Early season lambs tended to travel shorter distances to slaughter and spent less time in lairage than late season lambs. The average total times spent in transit and lairage were 22.3 hours and 13.3 hours at the two plants.

Abattoirs↗
PubMed1990

Physical mapping of two temperature-sensitive adenovirus mutants affected in the DNA polymerase and DNA binding protein.

We have determined the exact nature of two thermosensitive (ts) adenovirus mutants, H5ts19 and H5ts149, which map to different genes in the E2 transcription unit. The H5ts19 mutation appears to stem from a single base-pair change of A-T to G-C at position 1840 (numbering as in ref. 1), corresponding to codon 154 of the gene coding for DBP. This results in a glutamine-to-arginine change in the amino-terminal domain of the protein. H5ts19 is defective in a late stage of infection, during virus assembly. This phenotype strongly differs from that described for the limited number of known DBP mutants, indicating that DBP is not only functional during DNA replication, but also plays a role in the late phase of the infection cycle. The defect of the (N group) mutant H5ts149 affects the initiation of viral DNA replication. Marker rescue experiments followed by nucleotide sequence analysis of H5ts149 DNA revealed a single point mutation in the gene coding for the Ad pol. A transition of C-G to A-T at position 7563 (numbering as in ref. 2) changes amino acid residue 411 of Ad pol, a leucine residue, to phenylalanine. This mutation is located in a region conserved among various DNA polymerases, which suggests an important role of this domain in DNA replication.

Adenoviridae↗
PubMed1990

Reductive biotransformations of organic compounds by cells or enzymes of yeast.

Saccharomyces cerevisiae catalyses the asymmetric reductive biotransformation of a variety of compounds containing a carbonyl group or carbon-carbon double bond. Oxidoreductases participating in these reactions which have commercial potential in biotransformation processes are likely to have relatively broad substrate specificity. Important carbonyl reductases falling into this category include YADH- and yeast NADP-dependent beta-ketoester reductases. The enoyl reductase component of the FAS complex may have a role in asymmetric yeast reduction of carbon-carbon double bonds of unnatural substrates. Other nicotinamide-requiring oxidoreductases of yeast are also surveyed to rationalize observed biotransformations of whole yeast cells in terms of specific enzymes. Genetic and protein engineering may enable enzymes to be tailored to accept new substrates. A greater understanding of the enzymes and reactions involved will facilitate further optimization and exploitation of these catalytic systems in industrial processes.

Biotransformation↗
PubMed1990

The upstream factor-binding site is not essential for activation of transcription from the adenovirus major late promoter.

An adenovirus major late promoter (MLP) has been constructed with a 4-bp alteration in the sequence which binds the transcription factor known as USF or MLTF. This upstream element has often been considered necessary and sufficient for maximal transcription of the MLP. A duplex oligonucleotide containing the mutant sequence was not capable of binding specific proteins in a band shift assay, nor was it capable of inhibiting such binding by the wild-type sequence. In an in vitro assay, the mutant sequence was incapable of inhibiting transcription from a duplex sequence containing the MLP, whereas the wild-type sequence could. These two pieces of evidence suggest that the sequence is functionally impaired. Surprisingly, a virus containing the mutant MLP had a normal replication phenotype. On more detailed examination however, we show that the mutant viral MLP was deficient in transcription at 9 h postinfection but that the rate of transcription was close to normal by 20 h postinfection. An inverted CAAT box located immediately upstream of the USF-binding element was not previously thought to be of importance to the functioning of the MLP. However, a single point mutation in the CAAT box, placed in the USF mutant background, had a marked effect upon transcription from the MLP. This result suggests that the MLP may exhibit functional redundancy in which either the USF-binding site or the CAAT box can serve as an upstream promoter element. Neither of the mutant viruses displayed any change in the levels of the divergent IVa2 transcription unit, suggesting that the levels of divergent transcription are not determined by competition for limiting transcription factors.

Adenoviridae↗
PubMed1990

Reactivation of latent herpes simplex virus by adenovirus recombinants encoding mutant IE-0 gene products.

We have previously shown that adenovirus recombinants expressing functional ICP0 reactivate latent herpes simplex virus type 2 (HSV-2) in an in vitro latency system. This study demonstrated that ICP0, independent of other HSV gene products, is sufficient to reactivate latent HSV-2 in this in vitro system. To assess the effects of defined mutations in the sequence encoding ICP0 (IE-0) on reactivation, seven in-frame insertion and three in-frame deletion mutants were moved into an adenovirus expression vector. Each recombinant directed the synthesis of stable ICP0 of the correct size. The transactivation activity of the mutated sequences in these recombinants was similar to that when they were tested in plasmids. When these recombinants were examined for their ability to reactivate in the in vitro latency system, mutants with dramatic defects in transactivation (Ad-0/125, Ad-0/89, Ad-0/2/7, and Ad-0/88/93) were unable to reactivate latent HSV-2 independent of the multiplicity of infection. An exception to this correlation was the finding that Ad-0/89, which transactivated poorly, was able to reactivate latent virus after prolonged incubation whereas other transactivation-deficient mutants could not. Moreover, the presence of ICP4 did not compensate for the inability of any of the recombinants tested to reactivate HSV-2. These results show that (i) the transactivation domains of ICP0 are also used in reactivation, (ii) the presence of another essential HSV regulatory protein ICP4 does not alter the pattern of reactivation by ICP0, and (iii) mutations in some regions of IE-0 previously shown to affect viral growth and plaque formation did not alter its ability to reactivate in this in vitro system.

Adenoviruses, Human↗
PubMed1989

A genetic investigation of the mechanism of adenovirus marker rescue.

We have developed quantitative and segregational methods for investigating the mechanism of genetic exchange in adenovirus marker rescue. Estimates of "marker rescue frequency" (m.r.f.) were used to show that marker rescue increases linearly with increasing dose of fragment up to equimolarity with the full-length genome. The m.r.f. is also affected by the size of the rescuing fragment and the position of the wild-type allele within it, regardless of whether the fragment is terminal or internal. This is compatible with marker rescue being based on homologous exchange between the recombining partners. Examination of individually transfected cells showed that there is very wide variability in the values of the m.r.f.'s. This suggests that marker transfer can occur after replication of the full-length genome has begun, and can occur late into the infectious cycle. Unselected markers on the rescuing fragment were shown to be co-inherited frequently. This suggests that physical linkage is accompanied by genetic linkage. To examine this more closely, a multifactorial marker rescue was performed. The data show unequivocally that markers resident on the same fragment as the selected allele are inherited at high frequency, with a gradient of transfer in which markers closest to the selected marker are transferred most frequently. Markers up to 13 and perhaps as many as 17 kb apart can be inherited together. There are very few examples of the inheritance of distal markers in the absence of proximal ones. These data suggest that large pieces of DNA are transferred in a concerted reaction during marker rescue.

Adenoviruses, Human↗
PubMed1988

Studies on the mechanism of histamine-induced release of noradrenaline and 5-hydroxytryptamine from slices of rat cerebral cortex.

The effect of histamine on the release of endogenous noradrenaline and 5-hydroxytryptamine (5-HT) has been examined in slices of rat cerebral cortex. Histamine was found to produce a marked release of both amines from rat cerebral cortex at concentrations between 0.1 and 1 mM. This response to histamine was relatively resistant to removal of calcium ions from the incubation medium when compared to the release evoked by depolarising potassium stimuli. The response to 1 mM histamine was not, however, significantly inhibited by the H1-antagonist mepyramine (1 microM) or the H2-antagonist cimetidine (100 microM). Furthermore, impromidine which is both a potent H2-agonist and a potent H3-antagonist was without effect on the basal and histamine-stimulated release of endogenous noradrenaline and 5-HT. The response to histamine was, however, significantly attenuated by nisoxetine, fluoxetine and imipramine which are inhibitors of the amine uptake systems. The results of this study show that high concentrations (0.1 to 1 mM) of histamine can produce a marked increase in the release of endogenous 5-HT and noradrenaline from rat cerebral cortex, apparently via a non-receptor mechanism. This effect will need to be borne in mind in interpreting biochemical and behavioral responses to histamine in this concentration range.

Animals↗
PubMed1988

Adenovirus vector expressing functional herpes simplex virus ICP0.

ICP0, one of the five immediate-early (IE) gene products of herpes simplex virus (HSV), is a potent activator of transcription. To assess the biological activities of ICP0 and to explore its mechanisms of action, two helper-independent recombinant adenoviruses were constructed. In each recombinant, the E1 region was substituted with the ICP0-encoding genomic segment under the control of either the adenovirus major late promoter (MLP-0) or the HSV IE-0 promoter (0PRO-0). Infection of HeLa cells or 293 cells (a human embryo kidney cell line expressing adenovirus 5 E1a and -b functions) with the MLP-0 recombinant results in the synthesis of more IE-0 mRNA and ICP0 protein than did infection with the 0PRO-0 recombinant. Although 293 cells infected with MLP-0 accumulate 5- to 10-fold more IE-0 mRNA late in the infection than cells infected with HSV, the level of the protein product, ICP0, increased only slightly. In 293 cells, both recombinants could replicate, albeit at a slower rate and with lower final yields than wild-type adenovirus. Neither virus replicates its DNA in HeLa cells, and thus ICP0 cannot substitute for adenovirus E1a; however, the level of ICP0 that accumulates in MLP-0-infected HeLa cells was comparable to that of HSV-infected HeLa cells. In a functional test, we demonstrated that the adeno-ICP0 recombinant viruses can transactivate a transfected TK-CAT cassette, indicating that the ICP0 is biologically active.

Adenovirus Early Proteins↗
PubMed1987

The creation of adenovirus genomes with viable, stable, internal redundancies centered about the E2b region.

During the course of constructing new adenoviral strains by overlap recombination, we have discovered that internally redundant viable genomes can be created by end-to-end joining of the input DNA molecules. The cellular functions responsible for the end-joining activity frequently ligated the overhanging single strands of the complementary ends to form a novel restriction site at the junction. In 2 of the 17 cases analyzed in detail by restriction digestion, and some sequence determinations, the cellular functions had repaired the ends, presumably prior to end-joining. Four of the isolates had suffered deletions at the junction ranging in size from 13 to 532 bp. The isolate with the largest deletion also had an insertion of 14 bp of unknown origin at the site of the deletion. All of the redundant isolates replicated as efficiently as isogenic unit length strains, and plaque dilution titrations obeyed one-hit kinetics, showing that the redundant genomes were nondefective. Nevertheless unit-length genomes were observed at a low level (some 5 to 10% of the total) in stocks of each isolate before and after plaque purification. They presumably arose by recombination between the redundant sequences either intra- or intermolecularly. Evidence from Southern blot analysis showed that molecules with three copies of the redundant sequences also arose and could be detected both in intracellular and in capsid viral DNA. These species would arise by unequal crossing-over between redundant genomes. The efficient replication of the redundant species demonstrates that the precise spatial relationships between splice donors and acceptors on either strand, in this region of the genome, do not have to be rigidly maintained. These data suggest that it may be possible to place other genetic information between the DNA polymerase and terminal protein precursor genes and have it expressed from the major late promoter in its normal location.

Adenoviruses, Human↗
PubMed1987

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.

Adenoviridae↗
PubMed1987

Submuscular breast reconstruction: a one-stage method of tissue expansion.

This article discusses the use of a permanent saline-filled expander for breast reconstruction incorporating on the anterior surface a filler valve that can be located by nonoperative means. The self-sealing filler valve has a well-marked design that is particularly amenable to ultrasound detection. This results in the capacity to inflate or deflate the prosthesis at will. Sixty patients were operated on and 123 submuscular custom-made prosthetic expanders were implanted. Seventy-four percutaneous volumetric changes were performed without significant complications. Follow-up studies for more than 6 years are presented. The primary advantage of this prosthesis is its capability for percutaneous access under local anesthetic to produce volumetric changes. The prosthesis is permanent and does not have to be replaced as normal tissue expanders do. Only 1 operation is necessary. In addition, capsule contracture and spontaneous deflation rates are within an acceptable range.

Breast↗
PubMed1985

A thermolabile mutant of adenovirus 5 resulting from a substitution mutation in the protein VIII gene.

The mutant adenoviruses H5sub304 and H5RIr were isolated sequentially from adenovirus 5 wild type by selection for the loss of EcoRI restriction endonuclease sites by Jones and Shenk (Cell 13:181-188, 1978). sub304 lacks the site at 84.0 map units (m.u.), and RIr lacks both that and the site at 75.9 m.u. A set of derivatives of RIr that lack the site at 75.9 m.u. accumulated virus more slowly at 38.8 or 39.5 degrees C than those with the site present, as measured by low-multiplicity passage or single-step replication cycles, respectively. Since the EcoRI site at 75.9 m.u. is predicted to lie in the gene encoding the precursor to virion polypeptide VIII (pVIII), the failure to accumulate virus rapidly could lie either in some step in processing and assembly of virions or in an increased virion thermolability. The latter possibility was shown to be the case, as all strains mutated at the EcoRI 75.9 m.u. site were extremely thermolabile in vitro, even at 37 degrees C. CsCl equilibrium density centrifugation of heated crude stocks of RIr and sub304 demonstrated that loss of infectivity in RIr was accompanied by physical disruption of virions. Polyacrylamide gel electrophoresis of infected cell extracts or of purified virions showed that pVIII of RIr had an apparent molecular weight that was slightly greater than that of sub304, and mature RIr and sub304 virions displayed polypeptide VIIIs which appeared to be of identical molecular weights. Nucleotide sequence analysis of RIr demonstrated that it contained a 9-base-pair (bp) substitution for 6 bp found in sub304, leading to a loss of the EcoRI site and a predicted insertion of a single amino acid. Comparison of the sequence of sub304 with the published sequence of adenovirus 2 revealed two changes, a single transversion at bp 1,722 and a bp deletion at 1,749, leading to the loss of a TaqI site. The predicted reading frame change would lead to a stop codon at bp 1,885. This raises the question of whether adenovirus 2 and adenovirus 5 use the same reading fame for pVIII.

Adenoviridae↗
PubMed1984

Polarity in adenovirus recombination.

The distributions of the crossovers necessary to generate ts+ genomes have been examined in a collection of clonally unrelated ts+ recombinants from a set of ts X ts adenovirus crosses. In a cross between two parents that are grossly heterologous between map units 80.2 and 91.5, the distribution of crossovers was significantly skewed toward the left-hand end of the genome, with a declining frequency proceeding rightward. This gradient of recombination was modified by the removal of the right-hand heterology and by the presence of another region of heterology between map units 3.67 and 10.11. In a cross where the ts markers were flanked by both heterologies, no gradient was observed and ts+ recombinants were characterized by a higher rate of supernumerary crossovers. In a cross designed so that one ts marker was internal to two heterologies, crossovers were found disproportionately between the second ts marker and the nearby heterology. In addition, ts+ recombinants formed by crossing over internal to the heterologies again were accompanied by a high frequency of supernumerary crossovers. Finally, ts+ recombinant frequencies in crosses identical except for the presence of either one or two flanking heterologies were markedly lower in the latter case. These data, taken together, suggest that a major pathway of adenovirus recombination initiates at, or near, the molecular termini and is perhaps driven by the displaced single strands produced during DNA replication. Internal initiation, on the other hand, may employ these single strands to form genetic "patches."

Adenoviridae↗
PubMed1984

Replication and recombination in adenovirus-infected cells are temporally and functionally related.

We have studied the temporal and functional relationships between DNA replication and recombination in adenovirus-infected cells by using Southern blot hybridization to detect recombinant products among intracellular viral genomes. The data show that recombination can be detected soon after DNA replication has commenced and that the proportion of recombinant products increases thereafter. To determine the functional relationship between DNA replication and recombination, replication was blocked with the protein synthesis inhibitor anisomycin, the replication inhibitor cytosine arabinoside, and conditionally lethal mutations in either the virus-specified DNA-binding protein or the DNA polymerase. All treatments that directly or indirectly blocked DNA replication caused a delay in the appearance of recombinant products and a marked decline in their abundance relative to products of parental genotype. These data strongly suggest that DNA replication and recombination are interrelated, either because both processes share functions or because DNA structures produced by replication are suitable substrates for recombination. In addition, we have shown that some recombination function(s) is intrinsically thermolabile at 40.9 degrees C, even in wild-type crosses, since the appearance of recombinant products is delayed and their extent is reduced compared with that from crosses performed at 39.9 degrees C.

Adenoviruses, Human↗