Sequence analysis of E2 glycoprotein genes of classical swine fever viruses: identification of a novel genogroup in Thailand.

Thirty classical swine fever viruses (CSFV) isolated in Thailand between 1988 and 1996 were characterised by genetic sequence analysis of a part of their E2 coding regions, comparing the new data with that for representative reference viruses from other countries and continents. Thai isolates were divided into three distinct genogroups, indicating multiple origins for the outbreaks. Eighteen isolates from 1988-1995 form a new genogroup not previously described from any other geographical region. Eleven isolates from 1988-1995 are in the same genogroup as old US and European strains represented by reference strains Alfort 187 and Brescia. The viruses of this group seem to have died out in Europe but still persist in Thailand. One recent isolate from 1996 represents another previously described genogroup being closely related to Italian viruses isolated in the same year.

The Microphthalmia gene product interacts with the retinoblastoma protein in vitro and is a target for deregulation of melanocyte-specific transcription.

Little is known of the molecular mechanisms underlying the differentiation of the melanocyte from the melanoblast or the progression from the melanocyte to a malignant melanoma. Since the adenovirus E1A products have proved a useful tool for understanding control of differentiation in other systems, we explored the possibility of using E1A as a probe for factors controlling melanocyte-specific gene expression and differentiation. The results obtained show that the adenovirus E1A 13S, but not the 12S, product can transform the highly pigmented and TPA-dependent melanocyte cell line melan-a. Transformation is characterised by a morphological change, loss of TPA-dependence, the ability to grow in soft agar and strikingly, loss of pigmentation which correlates with loss of expression of the melanocyte-specific TRP-1 and tyrosinase genes. Cotransfection assays demonstrated that repression of TRP-1 by E1A correlated with E1A binding to p105Rb and p300, with the target in the TRP-1 promoter being the M-box, and 11 bp basic-Helix-loop-Helix (bHLH) factor-binding motif conserved between melanocyte-specific promoters. Consistent with the M-box acting as a target for E1a-mediated transcription repression, we also show that the basic-helix-loop-helix-leucine zipper (bHLH-LZ) protein (Mi) encoded by the microphthalmia gene (mi), which is required for pigment cell differentiation, is a positive acting transcription factor which can interact with the retinoblastoma product in vitro and activate the TRP-1 promoter. Moreover, expression of the mi gene was reduced around 50-fold in the non-pigmented E1a-transformed melan-a cells compared to the nontransformed melan-a cell line, with ectopic expression of Mi able to prevent repression of the tyrosinase and TRP-1 promoters in the presence of E1A. Mi therefore appears to play a crucial role in melanocyte-specific gene expression. The parallels between repression of myogenesis and muscle cell bHLH factors, and Mi and melanocyte differentiation are discussed.

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. Excessive exposure to UV, however, is the primary cause of malignant melanoma, an increasingly common and highly aggressive disease. As an initial approach to understanding the regulation of melanocyte differentiation and melanocyte-specific transcription, we have isolated the gene encoding TRP-1 and examined the cis- and trans-acting factors required for cell-type-specific expression. We find that the TRP-1 promoter comprises both positive and negative regulatory elements which confer efficient expression in a TRP-1-expressing, pigmented melanoma cell line but not in NIH 3T3 or JEG3 cells and that a minimal promoter extending between -44 and +107 is sufficient for cell-type-specific expression. Assays for DNA-protein interactions coupled with extensive mutagenesis identified three factors, whose binding correlated with the function of two positive and one negative regulatory element. One of these factors, termed M-box-binding factor 1, binds to an 11-bp motif, the M box, which acts as a positive regulatory element both in TRP-1-expressing and -nonexpressing cell lines, despite being entirely conserved between the melanocyte-specific tyrosinase and TRP-1 promoters. The possible mechanisms underlying melanocyte-specific gene expression are discussed.

Identification of new actin-associated polypeptides that are modified by viral transformation and changes in cell shape.

By using a monoclonal antibody we have identified a new polypeptide doublet (C4h and C4l) of Mr approximately 21 kD and pI 8 and 7, respectively, that is associated with and (at the immunofluorescence level) uniformly distributed on actin filament bundles in rat, mouse, and other vertebrate species. C4 is absent in neurones, erythrocytes, and skeletal muscle but the epitope is evolutionarily conserved as it is present in invertebrates such as molluscs and crustaceans. C4h is not found in cells such as lymphocytes and oncogenically transformed mesenchymal cells where actin stress fiber bundles are reduced in number or absent. C4l, on the other hand, is always present. C4h expression can also be blocked by switching normal nontransformed mesenchymal cells from adherent to suspension culture. Reexpression of C4h occurs 24 h after these cells are returned to normal adherent culture conditions, but can be blocked by either actinomycin D or cycloheximide, suggesting that the expression of this epitope is regulated at the transcriptional level.