Mapping of a major surface-exposed site in herpes simplex virus protein Vmw65 to a region of direct interaction in a transcription complex assembly.

The cellular factor Oct-1 is selectively recruited, together with at least one other cellular protein (CFF), into a multicomponent transcription complex whose assembly is directed by the herpes simplex virus regulatory protein Vmw65 (VP16). The acidic carboxy terminus of Vmw65 is not involved in assembly of the complex but is absolutely required for subsequent transcriptional activation. Elucidation of the mechanism of action of Vmw65 is important for an understanding not only of combinatorial control of gene expression by POU- and homeodomain proteins but also of the interaction(s) between activation domains of regulatory proteins and components of the basal transcriptional apparatus.

Immunocytochemical localization of c-erbB-2 protein in transitional cell carcinoma of the urinary bladder.

The level of expression and cellular localization of the c-erbB-2 gene product in transitional cell carcinoma of the urinary tract is controversial. Analysis of the c-erbB-2 gene structure and comparison of its expression in the same cells by Southern, Northern and immunoblotting, and by immunocytochemistry minimize the errors of interpretation inherent in one technique. Such a 'correlative study' has been performed on tumours from 82 patients.

Mutation of H-ras is infrequent in bladder cancer: confirmation by single-strand conformation polymorphism analysis, designed restriction fragment length polymorphisms, and direct sequencing.

A series of 152 human bladder tumors, 14 bladder tumor cell lines, and 1 immortal urothelial cell line were examined by single-strand conformation polymorphism (SSCP) and designed restriction fragment.length polymorphism analyses for mutations in exons 1 and 2 of the H-ras gene. Nine tumors (6%) contained mutations.

Single amino acid substitutions alter helix-loop-helix protein specificity for bases flanking the core CANNTG motif.

While all basic region/helix-loop-helix (bHLH) proteins bind the consensus CANNTG motif, other factors must be involved in determining regulatory specificity. In this report we show that bases outside this core 6 bp are involved in determining the specificity of binding. Thus, binding of the yeast bHLH protein PHO4, but not CPF-1, is inhibited by the presence of a T residue immediately 5' to their common CACGTG recognition sequence.

An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DR alpha promoter and activation by SV40 T-antigen.

Constitutive expression of major histocompatibility complex class II (MHC II) antigens normally occurs in B-lymphocytes and antigen presenting cells of the monocyte/macrophage lineage. However, many malignant tumours and transformed cells express these proteins aberrantly. We demonstrate here that the MHC II DR alpha promoter is constitutively active both in the SV40 large T antigen transformed cell line, COS, and in CV1 cells from which they are derived.

A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization.

The p53 gene product has been implicated in both human and animal tumorigenesis. p53 forms heterologous complexes with the transforming proteins encoded by several different DNA tumor viruses. p53 also assembles into stable homo-oligomers.

Positive and negative elements regulate a melanocyte-specific promoter.

Melanocytes are specialized cells residing in the hair follicles, the eye, and the basal layer of the human epidermis whose primary function is the production of the pigment melanin, giving rise to skin, hair, and eye color. Melanogenesis, a process unique to melanocytes that involves the processing of tyrosine by a number of melanocyte-specific enzymes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), occurs only after differentiation from the melanocyte precursor, the melanoblast. In humans, melanogenesis is inducible by UV irradiation, with melanin being transferred from the melanocyte in the epidermis to the surrounding keratinocytes as protection from UV-induced damage.

Localization of cis-acting sequence requirements in the promoter of the latency-associated transcript of herpes simplex virus type 1 required for cell-type-specific activity.

We have previously demonstrated (A. H. Batchelor and P.

Structural studies of the acidic transactivation domain of the Vmw65 protein of herpes simplex virus using 1H NMR.

We have overproduced and purified the carboxy-terminal transactivation domain of Vmw65 (VP16) of herpes simplex virus, and studied potential folding of the domain by 1H NMR. Two species of the acidic domain were obtained from the bacterial expression system, and we demonstrate that one of these represents read-through of the natural amber termination codon of the Vmw65 reading frame producing a larger polypeptide. Additional residues in the read-through product were identified by total amino acid analysis and by NMR.

Human papillomavirus sequences are not detectable by Southern blotting or general primer-mediated polymerase chain reaction in transitional cell tumours of the bladder.

A large series of transitional cell tumours has been screened for the presence of human papillomavirus (HPV) sequences using Southern blotting and general primer-mediated polymerase chain reaction (GP-PCR). The latter technique allows the detection of a broad spectrum of both sequenced and unsequenced HPV types using two primer pairs located in the highly conserved L1 and E1 regions of the HPV genome. No evidence for HPV infection was found in 100 transitional cell tumours, 6 cases of carcinoma in situ, 2 adenocarcinomas and a squamous carcinoma of the bladder and 3 cases of cystitis.

Loss of heterozygosity at the RB locus is frequent and correlates with muscle invasion in bladder carcinoma.

Studies of second, non-ocular tumours in surviving retinoblastoma patients and their families have reported a higher than expected incidence and lower age at diagnosis of bladder tumours. This suggests that RB mutations may predispose to bladder cancer. To determine whether this gene is involved in the development of sporadic bladder tumours we have examined 162 bladder tumours for evidence of structural alterations to the RB gene.

Sequence, function, and regulation of the Vmw65 gene of herpes simplex virus type 2.

We determined the sequence of the gene for the virion transactivator protein Vmw65 of herpes simplex virus type 2 (HSV-2), strain 333. An analysis of the coding sequence revealed an overall high degree of primary sequence conservation (86%) relative to the HSV-1 protein, although the carboxy-terminal region which encompasses the powerful acidic transactivation domain of the HSV-1 protein was slightly less well conserved (70%). One important change in this region was the presence of a proline residue in a region of the HSV-2 protein which is thought to form an amphipathic alpha-helix in the HSV-1 homolog.

Transcriptional activation by heterodimers of the achaete-scute and daughterless gene products of Drosophila.

The achaete-scute complex (AS-C) and the daughterless (da) genes encode helix-loop-helix proteins which have been shown to interact in vivo and to be required for neurogenesis. We show in vitro that heterodimers of three AS-C products with DA bind DNA strongly, whereas DA homodimers bind weakly and homo or heterocombinations of AS-C products not at all. Proteins unable to dimerize did not bind DNA.

C-myc and the yeast transcription factor PHO4 share a common CACGTG-binding motif.

The basic-helix-loop-helix (b-HLH) motif is common to a number of proteins involved in transcriptional regulation and cell-type determination. The b-HLH motif is also present in the S. cerevisiae transcription factor PHO4 which positively regulates the acid phosphatase gene PHO5.

Amplification at chromosome 11q13 in transitional cell tumours of the bladder.

Amplification of several markers which map to chromosome 11q13 was detected by Southern blotting in transitional cell tumours of the urinary bladder. The oncogenes INT2 and HST and the BCL1 locus were co-amplified in 20/97 (20.6%) tumours and the locus-specific minisatellite probe pMS51 (D11S97) detected amplification in 17/97 (17.

Amplification and over-expression of c-erbB-2 in transitional cell carcinoma of the urinary bladder.

The structure and expression of the proto-oncogene c-erbB-2 was studied in 86 patients with transitional cell carcinoma. Initial tissue samples comprised 37 grade 1, 32 grade 2 and 13 grade 3 tumours and four cases of carcinoma in situ. At the time of this first tumour sample, amplification of the c-erbB-2 gene was demonstrated by Southern blotting in 1/37 grade 1, 5/32 grade 2 and 6/13 grade 3 tumours (0.

Loss of a tumor suppressor function during neoplastic progression of epithelial cells in vitro.

In vitro transformation of rat urothelial cells is a multi-step process. We have used cell fusion to analyse the role of recessive events during in vitro progression of an immortal urothelial cell line. Somatic cell hybrids were made between the transformed cell line RM2T and a series of immortal urothelial cell lines, including the progenitor line RM2AD, from which RM2T was isolated.

Characterization of a cellular factor which interacts functionally with Oct-1 in the assembly of a multicomponent transcription complex.

Induction of transcription of the immediate-early (IE) genes of herpes simplex virus involves the assembly of a DNA-binding complex containing the viral protein Vmw65 and the cellular transcription factor Oct-1. We show that Oct-1 is not sufficient for complex formation and that another cellular factor(s) which is absolutely required for complex formation can be separated from Oct-1 under native conditions. We have purified this factor by approximately 100-fold using DNA-cellulose, ion-exchange and size-exclusion chromatographies.

Simultaneous isolation of DNA, RNA, and antigenic protein exhibiting kinase activity from small tumor samples using guanidine isothiocyanate.

Correlative studies of genes and their expression in human tumors are often hampered by the small sample size and the need to use differing and incompatible techniques to obtain DNA, RNA, and protein. We describe an extension of the established guanidine isothiocyanate method for isolation of DNA and RNA which allows the simultaneous isolation of total cellular protein. The protein obtained by this method (from solid tumors and cell lines) was comparable to protein extracted by a standard detergent solubilization method.

Regulation and cell-type-specific activity of a promoter located upstream of the latency-associated transcript of herpes simplex virus type 1.

To identify promoter regions which control expression of the latency-associated transcript (LAT) of herpes simplex virus type 1 (HSV-1), we constructed a series of recombinant vectors in which various sequences upstream of LAT were linked to the chloramphenicol acetyltransferase gene and tested for expression efficiency by transfection into tissue culture cells. In HeLa cells no activity was observed from the region (-250 to +201) immediately surrounding the nominal 5' end of LAT, but high levels of activity were observed by using different fragments within the region -1267 to -594. This promoter activity was largely contained within the 140-base-pair region from -797 to -658 and was 20- to 50-fold stronger than typical HSV delayed-early promoters and at least as strong as the activity from the simian virus 40 (SV40) enhancer-promoter region or the HSV immediate-early 110,000-Mr (IE110K) promoter region.

Structural requirements in the herpes simplex virus type 1 transactivator Vmw65 for interaction with the cellular octamer-binding protein and target TAATGARAT sequences.

Herpes simplex virus type 1 virion protein Vmw65 forms a complex (TRF.C) with TAATGARAT sequences and the cellular transcription factor oct-1, which has been implicated as an intermediate in the activation of gene expression by Vmw65. To examine structural requirements within Vmw65 for this interaction, we analyzed extracts of transfected cells that express mutant Vmw65 proteins by gel retardation assay and identified two regions in the primary sequence of Vmw65 which are necessary for in vitro assembly of TRF.

p53 interacts with p34cdc2 in mammalian cells: implications for cell cycle control and oncogenesis.

The p53 gene product has been implicated in both human and animal tumorigenesis. p53 complexes with the transforming proteins encoded by several different DNA tumour viruses. We demonstrate that human p53 is phosphorylated by the mammalian p34cdc2 kinase in vitro and coprecipitates with p34cdc2 in vivo.

Interference with the assembly of a virus-host transcription complex by peptide competition.

Induction of transcription of the immediate-early (IE) genes of herpes simplex virus (HSV) involves the assembly of a DNA-binding complex containing the cellular transcription factor Oct-1 and the virus regulatory protein Vmw65 (VP16). Complex assembly can be observed using deletion variants of Vmw65 which lack the acidic C-terminal activation domain and are therefore defective for IE transactivation. Similar variants of Vmw65 interfere with IE activation by the normal protein, and with HSV replication.

Repopulation of guinea-pig skin by melanocytes during wound healing: a morphometric study.

Epidermal keratinocytes and melanocytes have a close functional interrelationship. In order to study this relationship we used computer-assisted three-dimensional morphometry (CAM) to investigate the shape and size changes of the cutaneous melanocyte in healing guinea-pig skin. The combination of CAM with osmium iodide staining and resin embedding of tissue gave excellent results and allowed qualitative and quantitative morphometric assessment of melanocytes in vertical epidermal sections.