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Herpes simplex virus type 1 ICP22-/U(S)1.5- mutants initiate viral gene expression in all cells; however, in most cell types, the replication process stalls due to an inability to express gamma2 late proteins. Although the function of ICP22/U(S)1.5 has not been established, it has been suggested that these proteins activate, induce, or repress the activity of cellular proteins during infection. In this study, we hypothesized that cell cycle-associated proteins are targets of ICP22/U(S)1.5. For this purpose, we first isolated and characterized an ICP22-/U(S)1.5- mutant virus, 22/n199. Like other ICP22-/U(S)1.5- mutants, 22/n199 replicates in a cell-type-specific manner and fails to induce efficient gamma2 late gene expression in restrictive cells. Although synchronization of restrictive human embryonic lung cells in each phase of the cell cycle did not overcome the growth restrictions of 22/n199, synchronization of permissive Vero cells in S phase rendered them less able to support 22/n199 plaque formation and replication. Consistent with this finding, expression of cellular S-phase cyclins was altered in an ICP22/U(S)1.5-dependent manner specifically when S-phase Vero cells were infected. Collectively, these observations support the notion that ICP22/U(S)1.5 deregulates the cell cycle upon infection of S-phase permissive cells by altering expression of key cell cycle regulatory proteins either directly or indirectly.

The genomic structure and apparently complete coding sequence of the myotonic dystrophy protein kinase gene have been determined. The gene contains 15 exons distributed over about 13 kb of genomic DNA. It codes for a protein of 624 amino acids with an N-terminal domain highly homologous to cAMP-dependent serine-threonine protein kinases, an intermediate domain with a high alpha-helical content and weak similarity to various filamentous proteins, and a hydrophobic C-terminal segment. Located in close proximity is a second gene, coding for a transcript of about 3 kb, that is homologous to the gene DMR-N9 in the corresponding mouse locus, but has no homologies to other known genes or proteins. Strong expression of the latter gene in brain suggests that it may have a role in the development of mental symptoms in severe cases of the disease.

Myotonic dystrophy (dystrophia myotonica), the commonest and most variable of the muscular dystrophies of adult life, has long been known to be associated with cataract, while slit-lamp examination for specific lens opacities has been one of the principal methods of presymptomatic detection of gene carriers. The recent discovery that the myotonic dystrophy mutation is an unstable DNA sequence, composed of varying numbers of CTG triplet repeats, now allows a specific molecular test for this disorder, as well as explaining the phenomenon of anticipation. A series of case reports is presented to illustrate the important practical applications of this development in relation to ophthalmic aspects of the disorder. Reassessment of the specificity of the ophthalmic changes may be required and it will be important for molecular analysis to be used alongside ophthalmic studies, when determining whether family members carry the mutation for myotonic dystrophy.

A clinical and molecular analysis of 439 individuals affected with myotonic dystrophy, from 101 kindreds, has shown that the size of the unstable CTG repeat detected in nearly all cases of myotonic dystrophy is related both to age at onset of the disorder and to the severity of the phenotype. The largest repeat sizes (1.5-6.0 kb) are seen in patients with congenital myotonic dystrophy, while the minimally affected patients have repeat sizes of < 0.5 kb. Comparison of parent-child pairs has shown that most offspring have an earlier age at onset and a larger repeat size than their parents, with only 4 of 182 showing a definite decrease in repeat size, accompanied by a later age at onset or less severe phenotype. Increase in repeat size from parent to child is similar for both paternal and maternal transmissions when the increase is expressed as a proportion of the parental repeat size. Analysis of congenitally affected cases shows not only that they have, on average, the largest repeat sizes but also that their mothers have larger mean repeat sizes, supporting previous suggestions that a maternal effect is involved in the pathogenesis of this form of the disorder.

We have examined the hypothesis that the severe congenital form of myotonic dystrophy is caused by genomic imprinting at the level of differential DNA methylation of maternal and paternal alleles. Probes encompassing the 5', central, and 3' regions of the myotonic dystrophy protein kinase gene were used on blots of blood DNA from congenital and adult onset patients, digested with combinations of methylation sensitive and insensitive restriction enzymes. We observed similar patterns of methylation in each of the different classes of patient, and found no methylation differences between paternally and maternally derived alleles. Within the limitations of the experiment, our results provide no evidence for a role for genomic imprinting in congenital myotonic dystrophy and suggest that the explanation for this form of the disease will be found elsewhere.

A clinical and molecular study is reported of 83 patients considered to be minimally affected with myotonic dystrophy (DM). These had been identified in three ways: 60 subjects were identified on clinical grounds and were divided into those with and those without neuromuscular involvement (groups I and II); nine subjects were at high risk of carrying the DM gene but had a normal phenotype (group III); and 14 were parents of definitely affected patients where neither parent showed clinical abnormalities (group IV). PCR analysis of the CTG repeat in the DM gene showed a range of 70 to 230 repeats for the younger at risk patients in group III, while the asymptomatic gene carriers in group IV had 53 to 60 repeats. The sensitivity of diagnosis by EMG was found to be 39%. For ophthalmic signs this was 97.5%. This suggests that assignment on the basis of minimal clinical features carries a significant error. Molecular analysis, in conjunction with established clinical investigations, should prove valuable in the identification and exclusion of minimal myotonic dystrophy.

The myotonic dystrophy (DM) gene maps to the long arm of human chromosome 19 and is flanked by markers ERCC1 and D19S51. Also mapping to this region is the polio virus receptor gene (PVS). To produce more markers for this interval, we have constructed radiation-reduced hybrids by selecting for the retention of ERCC1 and for the loss of PVS. One of the cell lines produced has been characterized extensively and contains about 2 Mb of human DNA derived exclusively from chromosome 19, and includes ERCC1 and D19S51. Phage libraries constructed from DNA of this cell line have been screened and several new markers identified, including two for which cDNAs have been isolated. These represent candidate genes for DM. The new markers have also been used to extend the long-range restriction map of this region.

A variable DNA sequence has been detected in patients with myotonic dystrophy. We set out to determine whether identification of this specific molecular defect would improve clinical management of patients and families with myotonic dystrophy. 127 affected patients who were studied had an expanded DNA fragment not seen in 73 normal controls. The increase in length of the fragment correlated broadly with disease severity, and we noted expansion of the sequence in successive generations of the same family. Progressive expansion of the affected gene provides a molecular explanation for an apparently earlier onset in successive generations (anticipation) in myotonic dystrophy and supports the role of an unstable repeat sequence as the basis of the defect. The specificity of this finding will assist in accurate diagnosis of myotonic dystrophy and genetic counselling of affected families.

Myotonic dystrophy is the commonest adult form of muscular dystrophy, with an estimated incidence of 1 per 7,500, although this is likely to be an underestimate because of the difficulty of detecting minimally affected individuals. It is a multisystem autosomal dominant disorder of unknown biochemical basis. No case of new mutation has been proven. We have isolated a human genomic clone that detects novel restriction fragments specific to individuals with myotonic dystrophy. A two-allele EcoRI polymorphism is seen in normal individuals, but in most affected individuals one of the normal alleles is replaced by a larger fragment, which varies in length both between unrelated affected individuals and within families. The unstable nature of this region may explain the characteristic variation in severity and age at onset of the disease. A second polymorphism at this locus is in almost complete linkage disequilibrium with myotonic dystrophy, strongly supporting our earlier results which indicated that most cases are descended from one original mutation.

The human selenocysteine tRNA gene (TRSP) has been localized on chromosome 19q13.2-->q13.3 by in situ hybridization and ordered with respect to other genes and anonymous DNA markers in this region by linkage analysis in the forty CEPH pedigrees. These loci span only 10 cM in males and about 30 cM in females. The order of the loci is cen ... D19S7-D19S9-D19S47-CYP2A-CYP2F1-APOC2++ +-(TRSP, CKM). CYP2B flanks the CYP2A and CYP2F1 loci, but it cannot be determined whether it is proximal or distal to the other two cytochrome P450 loci with respect to the centromere.

We have examined the linkage of two new polymorphic DNA markers (D19S62 and D19S63) and a previously unreported polymorphism with an existing DNA marker (ERCC1) to the myotonic dystrophy (DM) locus. In addition, we have used pulsed-field gel electrophoresis to obtain a fine-structure map of this region. The detection of linkage disequilibrium between DM and one of these markers (D19S63) is the first demonstration of this phenomenon in a heterogeneous DM population. The results suggest that at least 58% of DM patients in the British population, as well as those in a French-Canadian subpopulation, are descended from the same ancestral DM mutation. We discuss the implications of this finding in terms of strategies for cloning the DM gene, for a possible role in modification of risk for prenatal and presymptomatic testing, and we speculate on the origin and number of existing mutations which may result in a DM phenotype.

We have constructed and analysed somatic cell hybrids from cell lines containing balanced reciprocal translocations involving chromosome 19 and providing two new breakpoints on 19q. These and other hybrids have been tested with a series of markers from 19q to enhance the existing map. Several new cloned DNA sequences that map to 19q13.3-19qter are reported; the locus D19Z1 has been analysed by CHEF gel electrophoresis.

The most useful markers for the prenatal diagnosis of myotonic dystrophy (DM) are APOC2 and CKM, both of which map proximal to DM. In order to produce other markers useful for DM, we have screened genomic DNA libraries constructed from cell line 20XP3542-1-4, which contains 20 to 30 Mb of human material including APOC2 and CKM. Of 51 human clones identified, seven map to chromosome 17, four to chromosome 8, and nine to chromosome 19, and the remaining 31 were excluded form chromosome 19 but not localised further. Four of the clones from chromosome 19 map distal to CKM and two of these clones (D19S62 and D19S63) are closely linked to DM. Analysis of a family in which a crossover between CKM and DM has occurred shows that neither D19S62 nor D19S63 and DM have recombined, suggesting that D19S62 and D19S63 are either closer to or flanking DM in relation to CKM. Pulsed field gel analysis showed that CKM, D19S62, and D19S63 map to a region of at least 1500 kb.

A 203-base-pair sequence 5' of the latency-associated transcripts (LATs) of herpes simplex virus type 1 contains a 7-base consensus sequence TGCGTCA that is identical to the cAMP-response element of the proenkephalin gene. This consensus sequence is at -38 relative to the putative 5' end of the LATs with a TATA box at the -24 position. In transient chloramphenicol acetyltransferase assays in rat pheochromocytoma (PC12) cells, this enhancer region stimulated gene expression up to 3-fold in the presence of dibutyryl cAMP, forskolin, nerve growth factor, or phorbol 12-myristate 13-acetate. Mutation of the cAMP-response element to TGCG-CAA resulted in a 4-fold reduction of basal activity and a complete loss of inducible stimulation. In DNA gel retardation assays, purified cAMP-response element-binding protein and a nuclear protein from PC12 cells were shown to bind specifically to this element. Furthermore, it was demonstrated that the reactivation of wild-type herpes simplex virus type 1 from dissociated latently infected murine trigeminal ganglia was significantly accelerated (P less than 0.005) by the addition of cAMP analogs or adenylate cyclase activators. However, these reagents did not accelerate reactivation of a deletion mutant that lacks the putative cAMP-response element-containing promoter region, transcriptional start site, and 1015 base pairs of the LATs. These studies demonstrate that the promoter region of the LATs contains a functional cAMP-response element and that expression of the LATs is likely controlled by second messenger signal transduction and imply a role for cAMP in triggering viral reactivation.

We have studied the genetic linkage of two markers, the apolipoprotein C1 (APOC1) gene and a cytochrome P450 (CYP2A) gene, in relation to the gene for myotonic dystrophy (DM). A peak lod score of 9.29 at 2 cM was observed for APOC1-DM, with a lod score of 8.55 at 4 cM for CYP2A-DM. These two markers also show close linkage to each other (theta max = 0.05, Zmax = 9.09). From examination of the genotypes of the recombinant individuals, CYP2A appears to map proximal to DM because in one recombinant individual CYP2A, APOC2 and CKMM had all recombined with DM. Evidence from another CYP2A-DM recombinant individual places CYP2A proximal to APOC2 and CKMM. Localisation of CYP2A on a panel of somatic cell hybrids also suggests that it is proximal to DM and APOC2/C1/E gene cluster.

Lead concentrations before, during and after external redecoration have been measured in seven school playgrounds. All playgrounds showed increases in lead concentration during redecoration but the extent of contamination and duration varied enormously. The geometric mean lead value per school playground, taken on the day of the highest concentration during redecoration, ranged from 370 to 53000 micrograms g-1. The two post-war schools had significantly lower lead in dust concentrations than the other older schools.

Measurements of the quantity of lead on the hands of schoolchildren and of the concentration of lead in dust from school playgrounds have been carried out; the hand-lead was collected by wiping with a moist tissue. When hand-lead and dust-lead values were averaged for each school, a statistically significant relationship between the two sets of means was obtained. Some limited measurements of size distribution of the dust particles on the children's wipes were also made; these indicated that most of the particles were less than 10 micron in diameter.