Masaru Nonaka

Molecular cloning of mouse β2-glycoprotein I and mapping of the gene to chromosome 11

beta 2-Glycoprotein I (beta 2 GPI), a plasma protein that binds to anionic phospholipids, is composed of five repeating units called a short consensus repeat (SCR), which is found mostly in the regulatory proteins of the complement system. Recently the human beta 2 GPI gene has been assigned to chromosome 17, not to chromosome 1 where most of the genes of the SCR-containing proteins are clustered. In this report, we have isolated a full-length cDNA clone of mouse beta 2 GPI and determined the chromosomal localization of the gene. The amino acid sequence deduced from the nucleotide sequence of mouse beta 2 GPI revealed 76.1% identity with that of human beta 2 GPI. A genetic mapping by in situ hybridization and linkage analysis using 50 backcross mice has shown that the mouse beta 2 GPI gene (designated B2gp1) is located on the terminal portion of the D region of chromosome 11, closely linked to Gfap, and is 18 cM distal to Acrb, extending a conserved linkage group between mouse chromosome 11 and human chromosome 17. On the basis of these results, the evolutionary relationships among the SCR-containing proteins are discussed.

Three extra copies of a C4-related gene in H-2w7 mice are C4/Slp hybrid genes generated by multiple recombinational events

Mice bearing the H-2w7 haplotype have five C4-related genes and constitutively express the Slp antigen. To understand the structure and evolution of the five C4-related genes of the C3H.W7 mouse, we have determined nucleotide sequences of the 5′ end region of these genes. A C4/Slp hybrid nature was confirmed for three of five C4-related genes as predicted previously by restriction enzyme analysis. The nucleotide sequences of the 5′ flanking regions of these three hybrid genes showed close similarity to that of the C4 gene, while the 3′ side of the ninth exon of the three hybrid genes showed close similarity to that of the Slp gene. In contrast, the regions between the first exon and the middle of the ninth exon of the three hybrid genes showed a mosaic structure of C4-like and Slp-like sequences. Moreover, the boundaries of the C4-like and Slp-like sequences were quite different among the three hybrid genes. The pattern of nucleotide sequence diversity in this region among the five C4-related sequences could be mainly explained not by point mutations but by gene conversions or unequal crossovers. These results suggest that multiple genetic recombinational events between two homologous sequences played an important role in the generation and diversification of the extra copies of the C4/Slp gene in the H-2w7 mouse.

Post-transcriptional regulation of complement C4 in low C4-producing strain of mouse

The expression of the fourth component of complement (C4) of the mouse can differ 20-fold and is determined byC4-high (C4h) orC4-low (C4l) alleles. To investigate the molecular mechanisms underlying the differences in C4 expression, we compared the transcriptional activity of theC4 genes between high and low C4-producer strains of mice (B10 and FM vs B10.BR) using nuclear transcriptional and chloramphenicol acetyltransferase (CAT) assays. We also compared the level of C4-specific RNA in total and nuclear RNA of the liver. The results revealed no significant difference in transcriptional activity betweenC4h andC4l genes. However, the steady-state levels of C4 mRNA are ten times lower inC4l strains than inC4h strains, suggesting that the major regulation of C4 plasma levels occurs at the post-transcriptional level.

Tissue-specific RNA processing for the complement C4 gene transcript in the H-2 k mouse strain

Biosynthesis of mouse C4 has been investigated intensively because of the recognized genetic variation in serum C4 levels among different inbred strains characterized by C4-high (C4h) and C4-1ow (C41) alleles (Shreffier 1976). Indeed, the S region of the mouse major histocompatibility complex (MHC, H-2) was originally defined as the locus that controls the quantitative difference of serological substance (Shreffler and Owen 1963) that was subsequently identified as mouse homologue of the complement component C4 (Shreffier 1982). Thus, mice carrying the H-2 k haplotype have only 5-10% of the serum C4 levels of other common inbred strains with non-H-2k haplotypes. Two main sources of mouse C4 are liver and peritoneal macrophages, although other organs or cell types produce C4 at much lower rates (Colten 1976; Cox and Robins 1988). Studies of C4 production in cultures of mouse hepatocytes and peritoneal macrophages indicated that C4 biosynthesis is regulated differently in macrophages and liver: the amounts of C4 produced by hepatocytes from different inbred strains reflect the observed differences in plasma C4 levels among the strains, whereas resident peritoneal macrophages isolated from strains with C4 h and C41 alleles produce similar amounts of C4 in culture (Rosa and Shreffler 1983; Sackstein and Colten 1984). Assays for C4 mRNA [5.4 kilobase (kb)] in liver and macrophages coincided with the studies at the protein level (Sackstein and Colten 1984; Cox and Robins 1988). Furthermore, C4 genes of C4h and C41 alleles showed a similar level of transcriptional activity indicating the gene regulation at the post-transcriptional level (Nakayama et al. 1990). Recently, we identified abnormally processed C4 mRNA in the liver of mice with the C41 allele but not in mice with C4 h alleles (Pattanakitsakul et al. 1992). The abnormal C4 mRNA contained a 200 base

Differential expression of the five C4-related genes of H-2w7mice

Mice bearing the H-2w7 haplotype have five C4-related genes, one C4, one Slp, and three C4/Slp hybrid genes. The expression of these five genes in the liver of their respective mRNA. We have amplified by the polymerase chain reaction (PCR) three regions of the Ca/Slp mRNA where some of these five genes show nucleotides substitution. A relative amount of each gene product was estimated by single-strand conformation polymorphism (SSCP) analysis or by direct counting of the number of respective clones after subcloning into a plasmid vector. A steady state level of the C4 mRNA was most abundant among C4-related gene transcripts. The hybrid 1 and 3 genes were expressed at a similar level which is about 1/2-1/3 of the C4 level. The hybrid 2 gene was expressed at about 1/5 of the hybrid 1 or 3 level. Neither male nor female H-2w7 mice expressed the Slp gene. These results showed that the expression of the five C4-related genes of H-2w7 mice is differentially regulated in spite of the close similarity in the nucleotide sequences in both the 5′ flanking and coding regions of these genes.

Evolution and Genetic Regulation of Complement C3 and C4 Genes

Evidence has been accumulated that the complement system predated the immune system as the body defence mechanism in the vertebrates. Although the occurrence of immunoglobulines inducible by antigenic stimulation in the most primitive of the extant vertebrates has been widely accepted as a solid fact since the classical work of Marchalonis and Edelman (1968), more recent works conducted utilizing the techniques of molecular biology pointed to the other conclusion(Ishiguro et al. 1992).