The eighth component of human complement (C8): Characterization of the gene encoding the C8[alpha] subunit and analysis of its linkage to C[sup [beta]]
Thesis/Dissertation
·
OSTI ID:7273126
Human C8[alpha] genomic phage clones were isolated from two human lymphocyte genomic DNA libraries. Exon-containing restriction fragments were isolated, sonicated and shotgun subcloned into M13. Exon-containing subclones were isolated and sequenced. The sequence was compared to the published C8[alpha] cDNA sequence to establish exon boundaries. Results showed that the C8[alpha] gene is composed of eleven exons with span [approximately]70 Kb. It is organized similar to the human C8[beta] and C9 genes, further supporting the evolutionary relationship among these proteins. Primer extension, SI nuclease and anchored PCR analysis were used to identify the transcription initiation site. Subsequent sequence analysis of the promoter region revealed two liver specific (HNF1 and hAFP2) and two IL-6 promoter elements. Earlier genetic studies of C8 polymorphisms showed that the C8[alpha] and C8[beta] loci were closely linked on chromosome 1. Analysis of genomic blots using end-specific but relatively long C8[alpha] and C8[beta] cDNA probes initially indicated that the two genes were linked by <2.5 Kb and in a 5[prime] C8[alpha]-3[prime] C8[beta] orientation. In the present study, PCR was used in an attempt to isolate the intergenic fragment. Repeated attempts were unsuccessful, therefore a re-analysis of genomic blots was performed using exon-specific probes. Probes specific for the last exon in C8[alpha] and the first exon in C8[beta] revealed that the fragments thought to be common to the respective cDNA probes were actually fortuitously co-migrating fragments. Thus, the two loci are not as closely linked as originally believed. Finally, efforts were made to express C8[alpha] in insect cells using a baculovirus expression system. Cells were infected with recombinant baculovirus that contained a copy of the C8[alpha] cDNA. Extracellular and intracellular samples were analyzed by Western blots for expression of C8[alpha].
- Research Organization:
- South Carolina Univ., Columbia, SC (United States)
- OSTI ID:
- 7273126
- Country of Publication:
- United States
- Language:
- English
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