A mutation within exon 14 of the TGFBI (BIGH3) gene on chromosome 5q31 causes an asymmetric, late-onset form of lattice corneal dystrophy.

PURPOSE: Two forms of autosomal-dominant lattice corneal dystrophy (LCD), types I and IIIA, have previously been shown to be caused by different mutations within the transforming growth factor, beta-induced (TGFBI) gene. A clinical and molecular analysis of three unrelated kindreds with a clinically distinct late-onset LCD was undertaken to determine whether this phenotype is also caused by mutations within the TGFBI gene. DESIGN: Experimental study. PARTICIPANTS: Thirty-two members of three kindreds with corneal dystrophy. DNA from 100 normal control subjects was used as a control population. METHODS: Members of three kindreds with LCD were examined clinically, and blood samples were taken for DNA analysis. Mutation analysis was undertaken on all individuals for the coding region of the TGFBI gene by means of polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism/heteroduplex analysis, subcloning, and sequencing. MAIN OUTCOME MEASURES: Detection of mutations within the TGFBI gene. RESULTS: Clinical examination revealed a form of LCD that was bilateral in all but one case, with onset around the fourth to fifth decade. The majority of cases showed significant asymmetry, and in one case there was evidence of onset directly after minor superficial corneal trauma. Molecular analysis in all families demonstrated sequence changes within exon 14 of the TGFBI gene on chromosome 5q31, at codon 622 in family 3, and at codon 626 in families 1 and 2, which are presumed to be responsible for the disease. CONCLUSIONS: Previously, a late-onset form of LCD, termed IIIA, was shown to be caused by a P501T mutation in exon 11 of TGFBI. The authors present the first description of mutations in exon 14 of TGFBI causing an LCD, also of late onset. Although the condition presented is morphologically and histopathologically typical of an isolated lattice dystrophy, the age of onset and clinical course is not typical of type I, III, or IIIA lattice dystrophy. This, in conjunction with recent developments in our understanding of the molecular genetics of these disorders, calls into question the usefulness and validity of the current classification of the isolated lattice dystrophies.

Reticuloendotheliosis virus long terminal repeat elements are efficient promoters in cells of various species and tissue origin, including human lymphoid cells.

Promiscuous transcriptional activity of the reticuloendotheliosis virus (REV) long terminal repeat (LTR) was detected in transient expression assays using LTR-chloramphenicol acetyltransferase-encoding gene chimeras, and cells of diverse species and tissue type; levels of expression from two different REV LTRs correlate with reports of pathogenicity of the respective viruses in vivo. REVs do not encode a transactivator targeted to the viral LTR, and cells infected with Marek's disease virus, a herpesvirus with an overlapping host range, do not express factors that preferentially enhance expression from REV or avian sarcoma/leukemia virus LTRs. REV LTRs work efficiently in human lymphoid cells, and are viable alternatives to promoters commonly used for expression of cloned genes. They may also prove useful in the identification of new, ubiquitous cellular transcription factors.

Transient expression analysis of the reticuloendotheliosis virus long terminal repeat element.

A region of the Reticuloendotheliosis virus (REV) long terminal repeat (LTR) harbouring single or duplicated copies of 46-bp and 26-bp sequence elements is implicated in enhancer activity. Sequences residing upstream from the proviral 3' LTR did not contribute to activity of the intact LTR. Gene expression regulated by a combination of REV enhancer and SV40 early region promoter was 50-fold less than from the analogous construct containing the chicken syncytial virus promoter. Deletion of LTR sequences immediately downstream of the CAP site, which include a region capable of forming a stable hairpin in the mRNA, decreased expression by 70%. Expression assays and S1 nuclease mapping showed that a second transcriptional start site, identified by transcription in vitro using HeLa cell lysates and purified DNA templates, was not used in vivo in the cell lines examined.

Dibutyryl cAMP inhibits expression of transformation-related properties in Kirsten murine sarcoma virus transformed Balb/c-3T3 cells despite continued presence of p21v-Ki-ras.

We studied the effects of simultaneous treatment with 0.1 mM N6, O2'-dibutyryl cAMP (dbcAMP) and 1 mM theophylline on several transformation-specific properties and on levels of the Kirsten murine sarcoma virus (Ki-MSV) transforming gene product p21v-Ki-ras, in a Ki-MSV-transformed mouse cell line (Balb/c-3T3, clone A31; KA31). The rate of logarithmic growth, cell motility, and final saturation density were reduced in dbcAMP-treated KA31 cultures. Capabilities for anchorage-independent growth were reduced in treated cells, to levels similar to those observed for the untransformed parental A31 cell line. Treatment with dbcAMP had no observable effect on the binding of 125I-labeled epidermal growth factor and did not alter fluorescence staining patterns for actin microfilaments and fibronectin which, although characteristic of normal cells, were also present in KA31 cells. Changes induced by dbcAMP were readily reversible, except for loss of anchorage-independent growth. However, this property was also reversible, provided removal of dbcAMP occurred 48 h prior to inoculation into soft agar medium. Immunoprecipitation with a monoclonal antibody directed against the protein p21v-Ki-ras (Y13-259) revealed the continued presence of this protein in dbcAMP-treated KA31 cells. We, therefore, conclude that cAMP mediates the inhibition of growth-related transformation-specific properties either by acting at steps subsequent to the expression of p21v-Ki-ras or on a pathway independent of p21ras function.

In vitro transcription analysis of the viral promoter involved in c-myc activation in chicken B lymphomas: detection and mapping of two RNA initiation sites within the reticuloendotheliosis virus long terminal repeat.

Chicken syncytial virus, a member of the reticuloendotheliosis virus family, induces B-cell lymphomas in chickens that arise by transcriptional activation of the chicken c-myc gene. In vitro transcription studies on cloned tumor DNA containing a deleted chicken syncytial virus provirus integrated upstream from, and in the same transcriptional orientation as, the chicken c-myc coding region were utilized to map possible transcriptional promoters and initiation sites. In vitro transcripts extending into c-myc sequences were initiated at two sites within the downstream long terminal repeat (LTR) closest to c-myc coding sequences. Both initiation sites have been precisely mapped by S1 nuclease and DNA sequencing methods. One site (I1) lies at the U3-R junction of the LTR, and the other site (I2) lies approximately 160 nucleotides upstream. Transcriptional control signals, including TATA- and CAAT-like sequences are present at appropriate distances upstream from the initiation sites. Both initiation sites are utilized to a similar extent. The upstream chicken syncytial virus LTR was also shown to be transcriptionally active in vitro. Two strong transcriptional initiation sites were also found in the LTR of spleen necrosis virus, a related member of the reticuloendotheliosis virus family; therefore, it seems likely that the existence of two transcriptional initiation sites is a common feature of the reticuloendotheliosis virus LTR, in contrast to other previously studied retroviral LTRs that exhibit one such site. The possible implications of these findings are discussed.

Restriction of the rat-specific RNS sequences of Ki-MSV to the nucleus of dibutyryl cyclic AMP-treated K-A31 cells.

The treatment of oncogenically transformed cells in culture, with dibutyryl cyclic AMP (cAMP) has, in many cases, resulted in a general phenotypic change towards the normal state. A virus-specific gene product(s) is responsible for the transformation of cells by sarcoma viruses and it has been suggested that the src gene product may act through the alteration of cAMP levels. With these premises we have studied the effects of dibutyryl cAMP on cell growth and virus genome expression in a Kirsten sarcoma virus-transformed mouse cell line. Our results suggest that certain virus-specific RNA sequences are restricted to the nucleus of these cells after several days of growth in medium containing dibutyryl cAMP and that these sequences appear to be those coding for the sarcoma information.