Genetic aspects of tuberous sclerosis in the west of Scotland.

Complete ascertainment of tuberous sclerosis was attempted in the west of Scotland (population 2,763,000). A total of 101 patients was identified, giving an overall minimum prevalence of 1 in 27,000, but for children under 10 years of age the minimum prevalence was 1 in 12,000. Both parents of 84 of the ascertained cases were assessed for signs of tuberous sclerosis. In 51 pairs of parents no evidence of the condition was seen, indicating that up to 60% of the cases were new mutations. The mutation rate was estimated at 2.5 X 10(-5) mutations per gene per generation. Analysis of parental ages for the new mutations did not show a significant age effect. Thirty-five patients occurred in 13 families containing other affected subjects. The pattern of inheritance was consistent with an autosomal dominant trait in these families. In one sibship, non-penetrance or gonadal mosaicism resulted in affected sibs with normal parents. Of two further sibships where non-penetrance was suspected, one was shown to represent a single new mutation in monozygotic twins and the other to involve non-paternity.

Purification of recombinant glycosylated human gamma interferon expressed in transformed Chinese hamster ovary cells.

Human IFN-gamma was produced in cultures of a Chinese hamster ovary (CHO) cell line transformed with a combination of plasmids encoding HuIFN-gamma cDNA and mouse DHFR cDNA and subsequently selected for growth in the presence of methotrexate. Confluent monolayers of these cells constitutively secrete HuIFN-gamma into the medium reaching a concentration of 2-5 micrograms/ml; the supernatant of the monolayer could be harvested daily for a period of more than 10 days. IFN-gamma was purified by passing the filtered CHO cell culture medium directly through a phosphocellulose column followed by elution and adsorption on a Con A-Sepharose column. Further concentration on an AMICON PM 10 filter and removal of high mw contaminating proteins with DEAE-Sephacel resulted in a IFN-gamma preparation of more than 99% purity (specific activity of about 10(8) International units per mg of protein). Each liter of CHO conditioned culture medium yielded 1-2 mg pure HuIFN-gamma. Its molecular weight, as determined by gel filtration, is about 50 kD and corresponds to a dimer structure. SDS-polyacrylamide gel electrophoresis indicated the presence of a 21 kD and a 25 kD polypeptide as compared with 17 kD for unglycosylated, bacterially made HuIFN-gamma and consistent with the two glycosylated forms of HuIFN-gamma produced in mitogen-stimulated human lymphocyte cultures.

Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells.

Cotransformation with two plasmids, one [pSV2-IFN-gamma] encoding human immune interferon (Hu IFN-gamma) and the other [pAdD26SV(A)-3] encoding mouse dihydrofolate reductase, has been used to establish Chinese hamster ovary (CHO) cell lines that secrete high levels of Hu IFN-gamma. Hu IFN-gamma production by the transformed CHO cell lines E-10B and E-10C reached approximately 50,000 units/ml of culture medium, which compares favorably with that of stimulated lymphocytes. Furthermore, as the Hu IFN-gamma cDNA gene used in these studies is under the transcriptional control of the simian virus 40 early promoter, Hu IFN-gamma production is constitutive and thus does not require induction. CHO-produced Hu IFN-gamma migrates as two bands corresponding to molecular weights of 25,000 and 21,000 on NaDodSO4/polyacrylamide gels. These two species are shown to be the products of a single gene. As the molecular weight of native Hu IFN-gamma is around 55,000, it is likely to be a dimer. We have shown that the subunits of such a dimer cannot be linked by a disulfide bridge(s). Hu IFN-gamma from CHO cells is likely to be glycosylated and this should now permit comparison of the biological activities of glycosylated and nonglycosylated (bacterially produced) Hu IFN-gamma in animal studies.