Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene.

Myotonic dystrophy, or dystrophia myotonica (DM), is a highly variable multisystem disease in which the classic adult-onset form displays progressive muscle wasting, cataracts, heart block, gonadal atrophy, insulin resistance and neuropsychiatric impairment. Its genetic basis is an expansion of CTG trinucleotide repeats in the DMPK protein kinase gene. Among the triplet repeat expansion disorders, DM is distinguished by the extended length of the repeat tract (5-13 kb in postmortem tissue) and its location in the 3' untranslated region of the gene that contains it. The pathophysiological mechanism for multisystem degeneration in DM is not understood. In contrast to the profound muscle wasting that characterizes advanced DM, only minor histopathological abnormalities have occurred in DMPK knockout mice or in mice that overexpress a human DMPK transgene, making it unlikely that changes in DMPK activity provide a unitary explanation for the disease. A DNAse hypersensitive site that maps 0.7 kb downstream (centromeric) from the CTG repeats is eliminated on DM chromosomes. This finding indicates that the repeat expansion may alter the adjacent chromatin structure and raises the possibility that it may also affect the expression of flanking genes. An interesting candidate flanking gene is DMAHP, a recently discovered homeodomain-encoding gene. We show here that DMAHP expression in myoblasts, muscle and myocardium is reduced by the DM mutation is cis, and the magnitude of this effect depends on the extent of CTG repeat expansion. These observations support the hypothesis that DMAHP participates in the pathophysiology of DM.

Acute and delayed cerebral infarction after wasp sting anaphylaxis.

A 52-year-old man developed a severe anaphylactic reaction after a wasp sting. Slurred speech and left hemiparesis were noted a few hours later. Three-and-one-half weeks later, he became acutely obtunded and quadriparetic. Angiographic studies demonstrated complete and near-complete occlusions of the right and left internal carotid arteries, respectively. A mechanism is suggested for delayed ischemic stroke after wasp sting anaphylaxis that involves cerebrovascular sympathetic innervation.

Expression of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for virus growth and neoplastic transformation.

The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) has protein kinase (PK) activity and properties similar to those of growth factor receptor kinases which can be activated to transforming potential. DNA sequences that encode the PK domain cause neoplastic transformation of immortalized cells. The studies described in this report used a spontaneous mutant (ts5-152) temperature-sensitive for the synthesis of ICP10 and the previously described ICP10 expression vectors to study the role of ICP10 expression in HSV-2 growth and neoplastic potential. The titres of the ts5-152 mutant are 1000-fold lower at 39 degrees C compared to 34 degrees C after 12 h post-infection. The efficiency of plaquing is 0.003. The growth defect at 39 degrees C correlates with decreased ICP10 synthesis. Sequence analysis of the PK domain of the ts5-152 ICP10 gene identified a pair of frameshift mutations resulting in a 19 amino acid residue substitution at positions 275 to 293 and a downstream single base pair mutation causing a substitution at position 309. Cloning of the mutant ICP10 gene from ts5-152 into a wild-type HSV-2 isolate resulted in a recombinant (859/152) with growth properties and rates of ICP10 synthesis at 39 degrees C similar to those of ts5-152. Cells transformed with u.v.-inactivated ts5-152, or the recombinant 859/152, have significantly decreased cloning efficiency in agarose at 39 degrees C, but only during the first 250 post-transfer population doublings. Anchorage-independent growth was observed in cells transfected with expression vectors pJW17 or pJW32 that express ICP10 or its PK domain, respectively. Cells transfected with the frameshift mutant pJW21 or the ICP10 carboxy-terminal vector pJW31 did not form clones in agarose.

Immediate early and functional AP-1 cis-response elements are involved in the transcriptional regulation of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

Expression from the promoter of the herpes simplex virus type 2 (HSV-2) large subunit of ribonucleotide reductase (ICP10) is stimulated by co-transfection with DNA that encodes the virion protein Vmw65 previously shown to activate in trans the transcription of all IE genes (Wymer et al., 1989). Specific cis response elements involved in ICP10 transcriptional regulation were studied by chloramphenicol acetyltransferase analysis with hybrid ICP10 promoter/CAT structural gene constructions containing wild type or site-directed mutations of the promoter sequences. The data indicate that Vmw65 activation requires an intact TAAT-GARAT motif while complex formation requires an intact Oct-1 element, and the AP-1 consensus elements in the ICP10 promoter are functional in vitro. Thus, expression from the wild type and GA-rich mutant constructions was enhanced 10-20-fold by co-transfection with DNA encoding Vmw65. The GARAT and POU homeobox (PHB) binding motifs were required for Vmw65 mediated activation but the mutant in the POU specific box (PSB) binding motif was activated at higher concentrations of Vmw65 DNA (1.0-3.0 micrograms). The PHB and PSB binding motifs were necessary for complex formation as determined by gel retardation analysis with in vitro synthesized OTF-1 and Vmw65 proteins. The GARAT and GA-rich elements were not required. CAT expression from pICP10-cat was enhanced by co-transfection with jun and fos encoding DNA, and the ICP10 promoter complexed with in vitro synthesized jun protein.

Genomic sequences homologous to the protein kinase region of the bifunctional herpes simplex virus type 2 protein ICP10.

The large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) consists of two functional domains. The amino (N)-terminal domain at residues 1-411 has serine/threonine-specific kinase activity (PK domain) and is encoded by a DNA fragment with transforming potential (15,17). The remaining region is required for ribonucleotide reductase activity (RR domain) (14, 15). Computer-assisted comparison of the ICP10 sequence to the EMBL database 21 has revealed sequences within the RR domain that are common to all RR1 proteins. Motifs homologous to the catalytic domains of all PKs were identified in the PK region (15). However, based on this database all other sequences were unique. Secondary structure analysis of the PK and RR junction region of ICP10 identified twist angle variations with helical periodicity characteristic of enhancer elements. Sequences homologous to a segment of the PK domain were amplified and cloned from human DNA using the polymerase chain reaction (PCR), suggesting that the PK domain may have originated from a cellular gene.

Leucine repeats in the large subunit of herpes simplex virus type 2 ribonucleotide reductase (RR; ICP10) are involved in RR activity and subunit complex formation.

Computer-assisted comparison of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase large subunit (RR1) sequence with the known primary structures of other RR1 proteins revealed a motif consisting of five leucines occurring at every seventh residue between positions 409 to 437. This motif is specific to HSV RR1 proteins. A synthetic oligopeptide (LA-4) corresponding to 15 residues in the internal portion of the motif inhibited HSV-2 RR activity. In immunoprecipitation experiments, LA-4 disrupted a complex consisting of RR1, the small RR subunit and a previously uncharacterized 180K protein, apparently of cellular origin. We deduce that the LA-4 sequence represents a critical RR1 site involved in RR complex formation and enzymic activity.

Myristylation and polylysine-mediated activation of the protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) was previously shown to possess protein kinase (PK) activity that localizes to the cytosolic, cytoskeletal, and plasma membrane fractions. Further studies of the PK domain using computer-assisted sequence analysis have identified a single transmembrane segment and fatty acid incorporation findings indicate that ICP10 is myristylated. Myristylation does not depend on a viral enzyme, since myristic acid is incorporated into ICP10 precipitated from cells transfected with an ICP10 expression vector. It is also incorporated into the 57-kDa protein expressed by the amino-terminal PK expression vector. The myristyl moiety is linked through an amide bond. The basic protein polylysine stimulates the kinase activity and alters its divalent cation requirements resulting in 20- to 40-fold stimulation in the presence of 0.1 mM Mn2+. The PK activity is inhibited by antibody to synthetic peptides corresponding to residues 355-369 and 13-26, respectively, located within, and amino-terminal to, the predicted PK catalytic domain.

Papillomavirus trans-activator protein E2 activates expression from the promoter for the ribonucleotide reductase large subunit from herpes simplex virus type 2.

Activation of the herpes simplex virus type 2 (HSV-2) large subunit of the ribonucleotide reductase (ICP10) gene by papillomavirus DNA encoding the E2 or E7 proteins was studied directly by immunofluorescence or by chloramphenicol acetyltransferase (CAT) analysis with hybrid ICP10 or IE175 and 38K promoter constructions. Cotransfection with bovine papillomavirus type 1 or human papillomavirus type 16 (HPV-16) E2 DNA enhanced CAT expression from constructions in which CAT is regulated by the ICP10 but not by other HSV promoters. Expression was not enhanced by cotransfection with HPV-16 E7 DNA. Sequence analysis of the ICP10 promoter identified a consensus E2-binding motif. Activation was significantly reduced by site-directed mutagenesis of the consensus motif.

Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The large subunit of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR1) is demonstrated to possess serine/threonine-specific kinase activity. Computer-assisted sequence analysis identified regions within the amino terminus of ICP10 that are homologous to the catalytic domain of known protein kinases (PKs). An in vitro kinase assay confirmed the ability of ICP10, immunoprecipitated from either HSV-2-infected cells or from cells transfected with an ICP10 expression vector, to autophosphorylate and transphosphorylate exogenous substrates in the presence of ATP and Mg2+. The HSV-1 RR1 was shown to be negative for PK activity under these conditions. PK activity was localized to a 57-kDa amino-terminal region within ICP10 that lacked RR activity.

Identification of immediate-early-type cis-response elements in the promoter for the ribonucleotide reductase large subunit from herpes simplex virus type 2.

Regulation of the expression of the herpes simplex virus (HSV) type 2 large subunit of ribonucleotide reductase (ICP10) gene was studied directly by immunofluorescence or by chloramphenicol acetyltransferase analysis with hybrid ICP10 promoter constructions. In Vero cells, cotransfection with DNA encoding HSV IE110 or Vmw65 proteins or HCMV IE2 enhanced expression at least 10-fold. In contrast, expression was minimally enhanced by DNA encoding IE175 at low doses and slightly reduced at high doses. IE110-mediated trans-activation was minimal in primary astrocytes and cells from line 293. However, Vmw65 enhanced expression 20-fold in all cell types. cis-Response elements in the ICP10 promoter include a TAATGARAT-like element and other sequences associated with regulation of IE gene expression and potential SP-1, consensus AP-1, and octamer transcription factor 1 binding elements. Factors that bind to the ICP10 promoter were identified in mock and HSV-infected cell extracts. DNA-protein complex formation, presumably involving Vmw65, was demonstrated by gel retardation analysis with mixtures of uninfected cell nuclear extracts and virion lysates. The octamer transcription factor 1 motif (ATGCAAAT) was necessary for optimal Vmw65 binding to the ICP10 promoter as evidenced by competition experiments with oligonucleotides overlapping the consensus IE110 promoter virion response element. The data suggest that ICP10 can be regulated as an immediate-early gene.