The mayor part of the work reported in this thesis, aimed at the generation and isolation of DNA probes complementary to the messenger RNA for the complement components C3 and C4. Indeed for the complement component C3 the isolation of a complementary DNA probe has been successful and therefore it has become possible to perform detailed studies on the gene(s) and the RNA transcripts for this component.Recent results revealed that among the clones harboring recombinant plasmids (310, see chapter 3) eleven (3%) contained C3-specific sequences. Nine of these contained different plasmids with partially overlapping C3-cDNA inserts, covering in total 4700 nucleotides (90/1) of the C3-mMA.The C3-cDNA probes are now used for a variety of experiments in different lines of investigation. a) Further characterization of C3-mMA. In chapter 3 the C3-mRNA was estimated to contain 7500 nucleotides. This value was obtained by electrophoresis of mRNA in presence of the denaturing agent methyl mercury hydroxide. In chapter 4, using glyoxal for the denaturation of the RNA, C3- mRNA was found to contain only 5300 nucleotides. several independent experiments i.e. primer extended reverse transcription and digestion of C3- mRNA with RNase H after hybridization to C3-cDNA fragments, indicate that the value of 5300 probably is correct. Also the length of the mRNA for vitellogenin, which has a similar size as C3-mRNA, has been overestimated, using the methyl mercury hydroxide method (Dr. G. Ryffel, personal communication). b) Gene expression. Several aspects of the regulation of the expression of the gene for the complement component C3 have been studied in chapter 4. Sofar no convenient model system for the modulation of the expression of the C3-gene has been found, in which it will be possible to study the mechanism of control of gene expression. The continuous presence of complement component C3 might be so essential that its gene is expressed constitutively in the producer tissues and that major regulation never takes place. In any case, if in future regulatory stimuli will be found which have a strong effect on the expression of the C3-gene, tools for a detailed study of the mechanism are available. c) Gene structure. The mouse and human C3-genes have been analysed by digestion with several restriction enzymes, followed by size fractionation and detection of the C3-specific fragments by hybridization to radioactively labeled C3-cDNA. Fragments of the C3-gene, comprising at least 90% of the sequences coding for C3-mRNA, have been isolated from a mouse gene library. Results from several experiments are to be expected soon : the determination of the number of C3-genes in the mouse and human genome; the location of the human gene(s) on a chromosome(s), with the use of human-mouse hybrids cells; sequence analysis of parts of the C3-cDNA, which will possibly reveal the amino acid sequence of the C3a polypeptide and of the portion of C3b which contains the binding site for cells and particles (see 1.3.4 and 1.3.5).All these examples show that a new approach has become available to answer questions about the expression, genetic aspects and structural aspects of the gene for the complement component C3.For the complement component C4 the isolation of a complementary DNA probe has not been successful so far. The serum level of C4-protein is approximately three fold lower than for C3-protein. This might reflect a lower abundancy of the C4-mRNA in the liver. Indeed, after invitro translation of liver mRNA and immunoprecipitations using anti-C3 and anti- C4 sera, much less (at least ten fold) pro-C4 was found than pro-C3 (chapter 3). But low efficiency of the immunoprecipitation with anti-C4 could also explain these results. However, if macrophage mRNA is used for invitro translation, the immunoprecipitations with anti-C4 and anti-C3 are equally efficient. Therefore it is likely that part of the polypeptides, obtained after invitro translation of liver mRNA, which have a molecular weight of 190'000 is not related to pro-C4 and that indeed C4-mRNA appears approximately ten fold less abundant than C3-mRNA in liver. This in turn could explain why no recombinant plasmids were found which contained cDNA inserts for C4-mRNA. A larger collection of clones containing recombinant plasmids should be made in order to increase the chance of isolating a C4-cDNA probe. A C4-DNA probe would provide an entrance into the major histocompatibility complex and could help to elucidate whether there is a relation between this complex and the complement system, other than the location of some of the complement genes within the major histocompatibility complex.It is conceivable that in the future, apart from the complement components C3 (and C4?), for the other components of the complement system cDNA clones will be isolated. However, serious problems might be encountered due to low abundancies of their mRNA. If these isolations will be succesfull, the coherance in the regulation of the biosynthesis of the complement components might become understood.
|Qualification||Doctor of Philosophy|
|Award date||25 Jun 1981|
|Place of Publication||Wageningen|
|Publication status||Published - 1981|