The third component of human complement (C3) is a key molecule in the activation of the complement cascade. C3 cDNA fragments were used to identify seven cosmid clones that covered all but 1 kilobase pair (kb) of the C3 gene. The remainder of the gene was cloned by using the polymerase chain reaction. These clones were used to identify the intron/exon boundaries and to map the gene. The C3 gene is 42 kb in length and comprises 41 exons ranging in size from 52 to 213 base pairs (bp). The transcription start site was identified by primer extension, and approximately 1 kb of DNA upstream of this site was sequenced. Putative TATA and CAAT boxes were identified along with a number of regions that shared homology with known regulatory sequences. These include responsive elements for interferon-gamma, interleukin-6, nuclear factor kappa B, estrogen, glucocorticoids and thyroid hormone. Several of these agents have been shown to affect C3 synthesis and mRNA levels. The sizes of the exons in C3 were compared to those of C4 and alpha 2-macroglobulin (alpha 2M). Thirty-nine of 41 exons in C4 were found to be of similar size to the analogous ones in C3, and two-thirds of those in alpha 2M were also similarly sized, supporting the hypothesis that these genes arose from a common ancestor.

Using an interspecific cross, gene linkage relationships among members of the murine complement receptor-related genes, C4bp, Cfh, Mcry, and Mcr2, were analyzed by segregation of RFLP in 200 mice. The human homologues of these genes are tightly linked, composing the RCA locus, which maps to human chromosome (Chr.)1q32, within a large linkage group conserved between human Chr.1q21-32 and mouse Chr.1. RFLP associated with C4bp and Cfh map within this conserved linkage group; Cfh is located 9 cM telomeric to C4bp, which is consistent with linkage data for their human homologues. Mcry and Mcr2, while tightly linked, are located outside the conserved group, 40 cM telomeric to C4bp. These data suggest that a translocation or inversion occurred within the RCA family during the evolution of the mouse, defining a breakpoint of this large conserved linkage group.

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Factor H is a regulatory protein of the alternative pathway of complement activation that is synthesized mainly in the liver. The authors used the +/+ Li murine liver cell line as a model for examining its regulation. When +/+ Li cells were incubated with IFN-gamma, the levels of factor H mRNA increased in a dose-dependent manner, achieving a maximal response at a concentration of 50-100 units/ml. The increase in factor H mRNA levels was paralleled by an increase in factor H secretion. The kinetics of induction of factor H mRNA were slow, with the response reaching near maximal levels at 24 h. The increase in factor H mRNA by IFN-gamma was dependent on protein synthesis, as cycloheximide abolished the response. The presence of IFN-gamma was required for the entire incubation period in order to produce a maximal response. The luciferase system was used in an attempt to identify an interferon-responsive element. Luciferase constructs containing from 807 to 236 bp of upstream sequence responded to IFN-gamma with a twofold induction of luciferase activity, whereas a construct containing 83 bp of 5' sequence did not. Thus, IFN-gamma stimulates factor H mRNA transcription through a protein intermediary that interacts with the promoter between positions -83 and -236.

Williams SA, Vik DP. Characterization of the 5 0 Flanking Region of the Human Complement Factor H Gene. Scand J Immunol 1997;45:7-15 The promoter region of the human factor H gene was cloned and a 3 kb Eco RI fragment was sequenced. Primer extension and S1 nuclease analysis were used to determine the transcription start site, which was found to be 10-11 nucleotides upstream of the published cDNA sequence. No canonical TATA or CCAAT boxes were found in conjunction with this site. The sequence from the human H promoter region was compared to that from the mouse gene. There was a region of 800 bp that was 62.5% identical between the two sequences. The sequences of the two promoter regions were compared to a database of transcription factor binding sites. Five elements were identified that matched the consensus sequence 100% and were identical in the two promoter sequences. Promoter assays using the luciferase reporter gene demonstrated that this region contained a functional transcription start site and putative enhancer elements. U118-MG astroglioma cells and Hep3b hepatoma cells were incubated with various cytokines to measure effects on their factor H mRNA levels. Interferon-(IFN-), but not interleukin-1 (IL-1), tumour necrosis factor (TNF-) or IL-6, was able to increase the level of H mRNA in both cell lines.

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Murine complement protein H is encoded by a 100-kb gene on chromosome 1. A 3.2-kb fragment of the 5' flanking region of the H gene was sequenced, and two transcription start sites for this gene were identified by RNase protection and S1 nuclease analyses, each of which had upstream TATA and CAAT boxes. This region shares sequence homology with known regulatory elements, including the SV40 enhancer consensus, the Sp1 binding site, and two glucocorticoid-responsive core elements (GRE). Tissue and cell-line specificity has been examined by Northern analysis, and the 4.4-kb full-length H messenger RNA was identified in liver, kidney, spleen, thymus, liver cell line 1469, and L cells. IFN-gamma did not induce H mRNA expression in the macrophage cell line P388D.1 but had a positive effect on both the mRNA and protein levels of H in L cells. PMA, LPS, and vitamin D did not increase H mRNA levels in L cells. Pursuant to the discovery of two GRE in the 5' regulatory region of the H gene, we examined the effects of glucocorticoids on H mRNA expression. Dexamethasone (10(-7) M) was found to increase markedly the levels of H mRNA and protein after 24 h of incubation, and the effect on the mRNA was detectable by 30 min. The fact that H is a down-regulator of complement activation is consistent with the known immunosuppressive role of glucocorticoids. To our knowledge, this is the first time that dexamethasone has been shown to increase the levels of a complement protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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