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To identify the promoter sequence(s) of the CG beta gene, genomic fragments derived from a cosmid containing the CG beta gene family were transfected into mouse Y1 adrenal cortical cells. Using this system, we showed that the CG beta genes 5, 3, and 8 have functional promoters, the basal element of which in the case of CG beta 5, was within 78 base pairs 5‘ ward from the CAP site. The size of the CG beta transcripts and identity of the transcription start site was the same for CG beta mRNA synthesized in Y1 cells as in first trimester placenta. The promoter region identified by this system was also capable of driving the chloramphenicol acetyltransferase gene when transfected stably into choriocarcinoma cells. Chloramphenicol acetyltransferase constructs bearing variable length of 5‘-flanking sequences from CG beta 5 and transfected into trophoblast cells suggest the presence of regulatory sequences within 700 base pairs from the CAP site. The information obtained here provide a foundation for studies of analyzing trans-acting placental and pituitary proteins to the defined CG beta promoter region.

Two functionally distinct but homologous sites in complement receptor type 1 (CR1) (CD35) were further characterized by homologous substitution mutagenesis of two CR1 derivatives, each containing one site. In both sites, reducing negative and/or increasing positive charge augmented interaction with iC3/C3b and C4b, supporting a role of ionic forces in the binding reaction. In one case, substitution of Asp at the end of complement control protein repeat (CCP) 2 with an Asn transformed the protein, with negligible cofactor activity and iC3 binding, into a mutant with activities similar to native CR1. Consequently, this protein, one-fourth the size of CR1, is a therapeutic candidate for a complement inhibitor. Another important observation is that the residues between two CCPs contribute to activity, probably because they influence positioning of one CCP relative to the next. The initial characterization of the third CCP of an active site led to identification of three peptides necessary for binding. In line with earlier findings for the first two CCPs, interactions with iC3/C3b are similar but not identical to those with C4b, implying overlapping but distinct binding domains. Moreover, changes in cofactor activity usually, but not always, parallel alterations in binding, indicating that these two activities are separable. We also mapped epitopes for a blocking and a function enhancing monoclonal antibody. Their effects can be explained by epitope location. The first antibody binds near functionally important residues. The second may shield inhibitory (negatively charged) residues. These results represent a comprehensive analysis of the active sites of CR1, which is built of modules found in more than 50 mammalian proteins.

To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus. Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed.