Fig. 5. TD formation correlates with the length of homologous ß-globin gene sequence. (A) Plasmid maps showing the design of the ß plasmids. Boxes represent promoters and exons, black arrows indicate transcriptional start sites, and `pA' indicates polyadenylation sites. The plasmids were based on the pUC18 backbone (not shown) and contained the HIV-LTR promoter upstream of the plasmid gene sequences. ß-globin sequences are shown in green while 2-globin sequences are shown in red. (B) Plasmid pHIV-ß was transiently transfected into HeLa cells with each of the ß plasmids, and the TDs were labelled by RNA in situ hybridisation using ß-globin and 2-globin intronic probes. The degree of colocalisation between the TDs was calculated using the overlap coefficient which ranges from 0 to 1, with 1 indicating perfect colocalisation. The blue curve indicates the length of ß-globin gene sequence in the co-transfected plasmids. The highest overlap coefficients were observed when pHIV-ß was transfected alone and the nascent transcripts were double-labelled with two different probes to the ß-globin first intron (bar 1). Co-transfections of pHIV-ß and plasmid pHIV- (which contains no ß-globin gene sequence) gave the lowest overlap coefficients (bar 2). When pHIV-ß was co-transfected with plasmids containing varying lengths of ß-globin gene sequence (bars 3-7) the value of the overlap coefficient correlated with the length of homologous ß-globin gene sequence. (C) Homologous 2-globin gene sequences can also mediate TD formation. Plasmid pHIV- was co-transfected with each of the ß plasmids shown in Fig. 3A. The TDs were labelled by RNA in situ hybridisation using ß-globin and 2-globin intronic probes. The degree of colocalisation between the transcription signals was measured using the overlap coefficient. The blue curve indicates the length of 2-globin sequence in the co-transfected plasmids. The highest overlap coefficients were observed when pHIV- was transfected alone and the nascent transcripts were double-labelled using two different 2-globin first intron probes (bar 1). Co-transfections of pHIV- and plasmid pHIV-ß gave the lowest overlap coefficients (bar 2). When pHIV- was co-transfected with plasmids containing varying lengths of 2-globin gene sequence (bars 3-7) the value of the overlap coefficient correlated with the length of homologous 2-globin gene sequence.