Biomedical subjects

J Dunlop

Publications and source records attributed to J Dunlop.

At least 19 recordsLinked to original sources

PubMed2001

Human diseases: clues to cracking the connexin code?

The vertebrate gap junctions formed by the connexin family of transmembrane proteins came to the attention of geneticists in 1993 with the identification of mutations linked to a form of demyelinating neuropathy. Since then, several other genetic disorders have been linked to mutations in specific connexin genes. Also, different diseases can result from different mutations in the same connexin gene. In addition, specific connexin knockout mice have surprising phenotypes. This is leading cell biologists to look afresh at connexins and their involvement in intercellular communication through gap junctions, a process that seems central to coordinating cell function within tissues. Here, we comment on how genetic studies are giving a new impetus to the cell biology of gap junctions.

Animals↗

PubMed2001

Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems.

The glutamate analog (+/-) threo-3-methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high-affinity, Na(+)-dependent excitatory amino acid transporter (EAAT). We have examined the effects of T3MG on glutamate-elicited currents mediated by EAATs 1-4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian cell lines expressing EAATs 1-3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitively inhibited uptake of D-[(3)H]-aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate-like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded D-[(3)H]-aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 microM T3MG were required to elicit significant NMDA receptor-mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.

2-Amino-5-phosphonovalerate↗

PubMed2001

Hepatitis C virus among high and low risk pregnant women in Dundee: unlinked anonymous testing.

OBJECTIVE: To determine the prevalence of the hepatitis C virus among pregnant women, to gauge the non-injecting, particularly sexual, risk of them being hepatitis C virus infected and to assess the potential impact of selective antenatal screening. POPULATION: Antenatal clinic attenders and women undergoing termination of pregnancy in 1997. SETTING: Ninewells Hospital, Dundee. DESIGN: Unlinked anonymous hepatitis C virus antibody testing of residual sera from specimens sent to the virus laboratory for routine serological testing. The results were linked to non-identifying risk information. RESULTS: Overall anti-hepatitis C virus prevalence was 0.6% (23/3,548). Prevalences among injecting drug users, non-injectors who had a sexual partner who injected, and those with neither risk respectively were 41% (7/17), 15% (5/33) and 0.3% (11/3,498). Relative risks for being an injector and a sexual partner of an injector respectively were 131 (95% CI 58-297) and 48 (95% CI 5-32). It is estimated that one of the 18 antenatal clinic attenders gave birth to an infected child. CONCLUSION: Findings suggest that non-injecting partners of injectors may be at considerable risk of acquiring hepatitis C virus sexually. Efforts to promote the use of condoms among injectors and their sexual partners should be increased. Selective anti-hepatitis C virus screening of women who reported high risk behaviour would have failed to detect half the cases. Research to gauge the views of women of childbearing age on anti-hepatitis C virus testing is required.

Adult↗

PubMed2000

Synthesis and 5-hydroxytryptamine (5-HT) activity of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5-(6H)ones and 2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoxalines.

A series of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5-(6H)ones and 2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoxalines was shown to exhibit 5-HT2C agonist binding and functional activity. Compound 21R inhibited food intake over 2 h in fasted, male Sprague Dawley rats with ED50 values of 2 mg/kg (i.p.) and 10 mg/kg (p.o.).

Animals↗

PubMed2000

The novel gene glaikit, is expressed during neurogenesis in the Drosophila melanogaster embryo.

A novel gene glaikit (gkt) has been identified which is expressed in the delaminating neuroblasts of the D. melanogaster embryonic central nervous system. At the earliest stages of embryonic development the expression of glaikit was ubiquitous, but by the time the neuroblasts are delaminating gkt expression became restricted to neuroblasts and a few ganglion mother cells. The gkt gene has no characterized homologues and encodes no previously described protein motifs. There are, however, evolutionary conserved predicted genes present in S. pombe, S. cerevisiae and C. elegans. Ectopic neuroblasts induced in either Notch or Delta mutant backgrounds also showed expression of glaikit.

Amino Acid Sequence↗

PubMed2000

Human elastase 1: evidence for expression in the skin and the identification of a frequent frameshift polymorphism.

Human pancreatic elastase 1 is a serine protease which maps to the chromosomal region 12q13 close to a locus for an autosomal dominant skin disease, diffuse nonepidermolytic palmoplantar keratoderma, and was investigated as a possible candidate gene for this disorder. Expression of two elastase inhibitors, elafin and SLPI, has been related to several hyperproliferative skin conditions. elastase 1 is functionally silent in the human pancreas but elastase 1 expression at the mRNA level was detected in human cultured primary keratinocytes. Antibody staining localized the protein to the basal cell layer of the human epidermis at a number of sites including the palmoplanta. Sequencing of genomic DNA from individuals with/without the keratoderma revealed a sequence variant, which would result in a premature truncation of the protein. This sequence variant, however, did not segregate with the skin disease and, indeed, was found to occur at a relatively high frequency in the population. Individuals homozygous for the variant do not have any obvious skin abnormalities. Based on the analysis of the secondary structure of the translated putative protein, the truncation is unlikely to result in knock-out of the elastase, but may cause destabilization of the enzyme-inhibitor complex.

Amino Acid Sequence↗

PubMed2000

The E1E4 protein of human papillomavirus type 16 associates with a putative RNA helicase through sequences in its C terminus.

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1E4. L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1E4 (in vivo and in vitro) and could be immunoprecipitated as an E1E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1wedgeE4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1E4-associated protein indicates a possible role for E1E4 in virus synthesis.

Cell Nucleus↗

PubMed1999

Properties of excitatory amino acid transport in the human U373 astrocytoma cell line.

In this study, we describe the presence of Na(+)-dependent high-affinity L-glutamate transport activity in the human U373 astrocytoma cell line. U373 cells exhibited a robust accumulation of L-glutamate which was predominantly (85%) extracellular Na(+)-dependent. Kinetic analysis of this transport activity revealed that the uptake followed first-order Michaelis-Menten kinetics and was high-affinity in nature. The kinetic parameters estimated by Eadie-Hofstee transformation of the saturable uptake were 37.3 +/- 5.1 microM for K(m) and 0.13 +/- 0.02 nmol min-1 mg-1 protein for Vmax. A total of 14 known inhibitors of high-affinity L-glutamate transport were examined for their abilities to inhibit L-glutamate uptake by U373 cells. Three compounds, kainate (KA), dihydrokainate (DHK) and alpha-aminoadipic acid produced less than 30% inhibition at 1 mM. The lack of effect of both KA and DHK indicates that the predominant astroglial L-glutamate transporter EAAT2 (excitatory amino acid transporter 2) does not contribute to the uptake activity present in these cells. The rank order of inhibitory potency for the remaining 11 compounds tested was L-cysteine sulphinate = L-CCG-III = L-cysteate = L-aspartate = threo-beta-hydroxyaspartate > trans-PDC > D-aspartate = MPDC > beta-glutamate > L-CCG-IV = L-aspartate-beta-hydroxamate. Pre-treatment of U373 cells with phorbol ester for 30 min resulted in a 56% decrease in L-glutamate uptake and this effect was blocked in a concentration-dependent manner by the PKC inhibitor bisindolylmaleimide I. Expression of L-glutamate transporters by U373 cells was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western analysis. Transcripts for both the EAAT1 and EAAT3 transporter subtypes were detected but not for EAATs 2, 4, and 5. Immunoblot analysis confirmed the presence of EAAT3 protein, however, we were unable to detect EAAT1 protein. In conclusion, the Na(+)-dependent high-affinity L-glutamate transport into human U373 astrocytoma cells appears to be mediated predominantly by the EAAT3 subtype.

ATP-Binding Cassette Transporters↗

PubMed1999

Steroid hormone-inducible expression of the GLT-1 subtype of high-affinity l-glutamate transporter in human embryonic kidney cells.

The cDNA encoding the predominant rat brain high-affinity l-glutamate transporter GLT-1 was isolated and subcloned into the pIND expression vector for the establishment of steroid hormone inducible expression in vitro using the ecdysone-inducible mammalian expression system. Steroid hormone-inducible expression was demonstrable in a stable cell line designated HEK/GLT-1. Treatment of HEK/GLT-1 cells with 10 microM ponasterone A for 24 hincreased the maximum velocity (V(max)) of Na(+)-dependent l-glutamate uptake by greater than 10-fold, as compared with the uninduced cells. Equivalent levels of l-glutamate transport capacity were observed in the uninduced GLT-1 cell line and the host cell line indicating that the expression of GLT-1 was tightly regulated. To confirm that the increased l-glutamate uptake observed in HEK/GLT-1 cells following induction was attributable to the expression of GLT-1, rather than the up-regulation of the endogenously expressed EAAT3 subtype present in the host cells, we evaluated the effects of the selective GLT-1 inhibitors dihydrokainate (DHK) and kainate. Both DHK and kainate produced concentration-dependent inhibition of the l-glutamate uptake into HEK/GLT-1 cells, and the estimated IC(50) values were consistent with those described for the cloned GLT-1. These results demonstrate that the expression of GLT-1 can be tightly regulated in vitro using the ecdysone system.

ATP-Binding Cassette Transporters↗

PubMed1999

Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L-glutamate transporter.

1. In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. 3. Na+-dependent L-glutamate uptake activity (3.2 pmol min-1 mg-1) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/EAAT2 cells to the ecdysone analogue Ponasterone A (10 microM for 24 h) resulted in a > or = 10 fold increase in the Na+-dependent activity. 4. L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5 microM, Vmax 3.8+/-0.9 nmol min-1 mg-1 protein. 5. The pharmacological profile of the EAAT2 subtype was characterized using a series of L-glutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+/-5% control in the presence of 30 microM) of the L-glutamate transport capacity in the induced HEK/EAAT2 cells. 7. This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone-inducible mammalian expression system.

ATP-Binding Cassette Transporters↗

PubMed1998

The pharmacological profile of L-glutamate transport in human NT2 neurones is consistent with excitatory amino acid transporter 2.

The human teratocarcinoma cell line NTera2/D1 can be differentiated to produce post-mitotic neurones (NT2-N cells) by prolonged (> 3 week) exposure to retinoic acid. In this study, we describe the characterisation of high-affinity Na+-dependent L-glutamate transport activity in post-mitotic differentiated NT2-N cells. NT2-N cells, but not the undifferentiated precursor cells, transported L-glutamate in a Na+-dependent manner, as determined by equimolar replacement of Na+ with choline. L-glutamate uptake was saturable and Eadie-Hofstee transformation of the saturation data revealed a Km of 10.6+/-0.8 microM, and a maximum transport capacity (Vmax) of 100.3+/-12.3 pmol min(-1) mg(-1) protein. Pharmacological characterisation of the transport activity in NT2-N cells produced a rank order of inhibitory activity which was identical to that determined for the human excitatory amino acid transporter 2 which we have analysed in a stable mammalian cell line (Madin Darby Canine Kidney (MDCK) cells). Of particular note, L-glutamate transport by NT2-N cells was sensitive to both dihydrokainate and kainate. The expression of human excitatory amino acid transporter mRNAs was studied using reverse transcriptase polymerase chain reaction. NT2-N cells expressed transcripts for excitatory amino acid transporters 2 and 3, but not for the subtypes 1, 4 and 5. We conclude that although the mRNA expression studies suggest the presence of transcripts for both excitatory amino acid transporter 2 and 3 in NT2-N cells, the sensitivity to dihydrokainate and kainate determined in the pharmacological analysis indicates that, of the known transporter subtypes, excitatory amino acid transporter 2 contributes to the bulk of the L-glutamate transport activity present in these cells.

Animals↗

PubMed1998

The cholecystokinin-B receptor antagonist L-740,093 produces an insurmountable antagonism of CCK-4 stimulated functional response in cells expressing the human CCK-B receptor.

A stable cell line expressing the human cholecystokinin-B receptor gene (hCCK-B.CHO) has been employed in an evaluation of the recently developed CCK-B receptor antagonist L-740,093. L-740,093 exhibited high affinity (IC50 0.49 nM) and selectivity (<50% displacement at CCK-A sites at 1 microM) for the human CCK-B receptor subtype as estimated from [125I]-CCK-8S displacement studies with membranes prepared from hCCK-B.CHO cells. The elevation of intracellular free Ca2+ in hCCK-B.CHO cells in response to stimulation with CCK-4 was used to evaluate the antagonist activity of L-740,093 in vitro. L-740,093 potently (IC50 5.4 nM) antagonized the 30 nM CCK-4-stimulated Ca2+ mobilization in hCCK-B.CHO cells. Further studies were performed to investigate the nature of the antagonist activity of L-740,093. When tested at 10 nM L-740,093 produced a modest rightward shift in the CCK-4 dose response curve, an effect which was accompanied by a small reduction (13%) in the maximum response to CCK-4. In the presence of 30 nM L-740,093 the maximum functional response to CCK-4 was further reduced by 45% indicating that L-740,093 behaves as an insurmountable antagonist of the human CCK-B receptor subtype.

Animals↗

PubMed1998

CCK receptor antagonists.

1. The peptide hormone and neurotransmitter, cholecystokinin, is widely distributed throughout the gastrointestinal tract and central nervous system and mediates a diverse number of biological functions. 2. Two receptor subtypes, CCK-A and CCK-B, have been identified by both pharmacological characterization and molecular cloning. The CCK-A receptor is the predominant peripheral CCK receptor subtype and the CCK-B receptor is the predominant central CCK receptor. In addition, there are discrete populations of CCK-A receptors in the brain and CCK-B receptors are present in gastric mucosa. 3. Subtype selective antagonists have been developed which discriminate between the two receptor subtypes. One of the major chemical classes has exploited a benzodiazepine template present in asperlicin which was initially discovered in a natural product screen for CCK receptor antagonists. 4. The structurally related benzodiazepines L-365,260, L-740,093, and YM022 are selective antagonists of the CCK-B receptor subtype. Their in vitro pharmacological profiles have been characterized using the human CCK-B receptor expressed in CHO cells. 5. L-365,260 behaves in a manner consistent with that of a competitive antagonist and both L-740,093 and YM022 behave as insurmountable CCK-B receptor antagonists in vitro.

Animals↗

PubMed1998

Characterization of 5-HT1A receptor functional coupling in cells expressing the human 5-HT1A receptor as assessed with the cytosensor microphysiometer.

The functional activity of a series of 5-HT1A receptor ligands has been evaluated in a cell line expressing the human 5-HT1A receptor (h5-HT1A x CHO) using the agonist-stimulated increase in extracellular acidification rate, measured with the microphysiometer, as a functional assay. Both 5-CT and 8-OH-DPAT were potent agonists in stimulating an increase in extracellular acidification rate in h5-HT1A x CHO cells with estimated EC50 values of 1.2 and 7.8 nM, respectively. Additionally, these two 5-HT1A receptor agonists elicited a similar maximum response. Concentration-dependent agonist activity was also observed in the presence of buspirone, ipsapirone, BMY7378, NAN-190 and WAY100135, and each of these compounds behaved as partial 5-HT1A receptor agonists. The selective 5-HT1A receptor antagonist WAY100635 produced a potent (IC50, 2.3 nM) and complete block of the 8-OH-DPAT-stimulated response. An evaluation of the inhibitory activity of a series of 5-HT1A receptor antagonists produced the following rank order of potency; WAY100635 > LY206130 (IC50, 7.1 nM) > WAY100135 (30.8 nM) > pindolol (76.2 nM) > (-)UH-301 (92.8 nM). Parallel studies on the inhibition of forskolin-stimulated adenylyl cyclase activity in hS-HT1A x CHO cells revealed that agonist potencies were generally similar between the two functional assays and were in good agreement with the estimated 5-HT1A receptor binding affinities. However, the relative efficacies determined for the partial agonists in the cAMP assay were substantially greater than those observed with the microphysiometer. Finally, antagonists were considerably weaker in the cAMP assay compared with the microphysiometer. The evaluation of 5-HT1A ligands using the microphysiometer, which represents a very distinct indice of 5-HT1A receptor function compared with the cAMP assay, results in a different profile of functional activity.

Animals↗

PubMed1997

YM022 [(R)-1-[2,3-dihydro-1-(2'-methylphenacyl)-2-oxo-5-phenyl-1H-1,4- benzodiazepin-3-yl]-3-(3-methylphenyl)urea]: an irreversible cholecystokinin type-B receptor antagonist.

A functional evaluation of the recently developed cholecystokinin type-B (CCK-B) receptor antagonist YM022 [(R)-1-[2,3-dihydro-1-(2'-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-++ +benzodiazepin-3-yl]-3-(3-methylphenyl)urea] was undertaken in Chinese hamster ovary cells stably expressing the human CCK-B receptor gene (hCCK-B.CHO). YM022 exhibited high affinity and selectivity for the CCK-B receptor subtype as estimated from [125I]CCK8S displacement studies using membranes derived from hCCK-B.CHO and hCCK-A.CHO cells. Functional antagonist activity of YM022 was demonstrated employing CCK-4-stimulated Ca2+ mobilization in hCCK-B.CHO cells. In the presence of 30 nM YM022, the maximum effect of CCK-4 was suppressed to 48 +/- 11% of control, an effect that was accompanied by a modest rightward shift in the CCK-4 concentration-response curve. In contrast, the structurally similar CCK-B receptor antagonist L-365,260 [3R(+)-N-[2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin -3-yl]-N'-(methylphenyl)urea; 30 nM-10 microM] produced progressive rightward shifts in the CCK-4 concentration-response curve, with no effect observed on the CCK-4 maximum response. Further characterization using the technique of microphysiometry revealed that the agonist activity of CCK-4 was not restored following washout after exposure to YM022. The antagonist activity of L-365,260, however, was found to be fully reversible in this system. Thus, YM022 behaves as an irreversible antagonist, whilst its structural analogue L-365,260 exhibits properties consistent with a competitive antagonist.

Animals↗

PubMed1997

Connexin 26 mutations in hereditary non-syndromic sensorineural deafness.

Severe deafness or hearing impairment is the most prevalent inherited sensory disorder, affecting about 1 in 1,000 children. Most deafness results from peripheral auditory defects that occur as a consequence of either conductive (outer or middle ear) or sensorineuronal (cochlea) abnormalities. Although a number of mutant genes have been identified that are responsible for syndromic (multiple phenotypic disease) deafness such as Waardenburg syndrome and Usher 1B syndrome, little is known about the genetic basis of non-syndromic (single phenotypic disease) deafness. Here we study a pedigree containing cases of autosomal dominant deafness and have identified a mutation in the gene encoding the gap-junction protein connexin 26 (Cx26) that segregates with the profound deafness in the family. Cx26 mutations resulting in premature stop codons were also found in three autosomal recessive non-syndromic sensorineuronal deafness pedigrees, genetically linked to chromosome 13q11-12 (DFNB1), where the Cx26 gene is localized. Immunohistochemical staining of human cochlear cells for Cx26 demonstrated high levels of expression. To our knowledge, this is the first non-syndromic sensorineural autosomal deafness susceptibility gene to be identified, which implicates Cx26 as an important component of the human cochlea.

Aged↗

PubMed1997

Full and partial agonist activity of C-terminal cholecystokinin peptides at the cloned human CCK-A receptor expressed in Chinese hamster ovary cells.

The agonist activities of the C-terminal cholecystokinin peptides sulfated cholecystokinin octapeptide (CCK-8S), non-sulfated cholecystokinin octapeptide (CCK-8NS), pentagastrin and CCK-4 at the cloned human CCK-A receptor expressed in Chinese hamster ovary cells were evaluated in two functional assays of receptor activation. [125I]-CCK-8S displacement studies employing membranes derived from these cells revealed the expected rank order of affinity for a number of CCK receptor ligands. CCK-8S was a potent agonist in (i) stimulating the mobilization of intracellular free Ca2+, measured with the Ca2+ sensitive fluorescent indicator FURA-2, and (ii) stimulating increases in extracellular acidification rates, measured by microphysiometry. Consistent with their lower affinities for CCK-A receptors, CCK-8NS, pentagastrin and CCK-4 were weaker agonists in both functional assays. In addition, these peptides exhibited partial agonist activity relative to the maximum response observed with CCK-8S in both assays. These results demonstrate that CCK-8S represents the minimum ligand requirement for both high affinity and full agonist activity at the human CCK-A receptor subtype.

Animals↗

PubMed1997

Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors.

We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.

Cell Nucleus↗