GENOMICS 7,644-646 (1990)

BRIEF REPORT

Physical Linkage of the Human Genes Coding for Complement Factor H and Coagulation Factor XIII B Subunit

JAVIER REY-CAMPOS, DOMINGO BAEZA-SANZ, AND SANTIAGO RODRIGUEZ DE CORDOBA

Unidad de Inmunologia, Centro de lnvestigaciones Biologicas (CSIC), Velazquez 144, 28006-Madrid, Spain

Received December 11, 1989

The B subunit of coagulation factor XIII (F13B) be- longs to the so-called super-family of C3b/C4b-binding proteins. These proteins are characterized by a struc- tural organization based on the presence of internal repeat units of 60 amino acids that share a framework of highly conserved residues (Reid et al., 1986). F13B is composed of 10 of these repeats (Ichinose et aZ., 1986). Among the C3b/C4b-binding proteins F13B is genet- ically related to MCP (membrane cofactor protein), CR1 (C3b/C4b receptor), CR2 (C3dg receptor), DAF (decay accelerating factor), C4BP (C4b-binding pro- tein), and factor H. All these proteins are encoded by linked genes that organize the regulator of complement activation (RCA) gene cluster located on the long arm of chromosome 1 (Rodriguez de Cordoba et aZ., 1985; Rey-Campos et al., 1987; Weiss et al., 1987; Rey-Cam- pos et al., 1988; Carroll et uZ., 1988; Bora et al., 1989; Rodriguez de Cordoba and Rubinstein, 1987). Studies to determine the genetic organization of the RCA gene cluster have shown that the MCP, CRl, CR2, DAF, and C4BP genes are located, in this order, in a segment of 800 kb of DNA (Rey-Campos et al., 1988; Carroll et

al., 1988, Bora et aZ., 1989). This group of genes is, however, some 7 CM (N 7 Mb) apart from that encoding factor H (HF) (Rodriguez de Cordoba and Rubinstein, 1987). Using formal genetic analysis we have recently shown that the human gene encoding F13B (Fl3B) is linked to the RCA gene cluster and suggested that it is probably located in close vicinity to the HF gene (Rodriguez de Cordoba et uZ., 1988). To gain insight into the organization of the RCA gene cluster we have elaborated a physical map of the region including the F13B and HF genes and sought to establish its location in relation to other (RCA or non-RCA) genes in that chromosomal region.

To elaborate a physical map of the chromosomal re- gion including the HF and F13B genes we have per- formed pulsed-field gel electrophoresis (PFGE) anal- ysis to separate large fragments of human genomic

DNA generated by digestion with infrequently cutting restriction enzymes and sequentially probed Southern blots of these gels with specific cDNA probes. PFGE was performed essentially as described in Rey-Campos et al. (1988), using a 120“ angle alternating homoge- neous electrical field apparatus constructed according to Chu et al. (1986) and a Pulsewave 760 electrophoretic pulse switcher (Bio-Rad, Richmond, CA). The source of genomic DNA was an EBV-transformed human B cell line. Several DNA probes were used in these ex- periments. The human CR1 (CRl-l), factor H (NZ- 2B12), and LCA/CD45 (pXHCL-1) probes have been previously described (Wong et al., 1985; Kristensen et

al., 1986; Ralph et al., 1987) and were generous gifts of Dr. D. T. Fearon (Johns Hopkins School of Medicine, Baltimore, MD), Dr. B. F. Tack (Scripps Clinic Re- search Foundation, La Jolla, CA), and Dr. M. L. Thomas (Washington University School of Medicine, St. Louis, MO), respectively. The human F13B probe was isolated in our laboratory from a human cDNA library using oligonucleotide probes derived from the published human F13B cDNA sequence (Ichinose et

al., 1986). The human C4BP probe was also isolated in our laboratory and it is described in Rey-Campos et

al. (1988). The human renin probe (pHRnX3.6) (Ho- bart et al., 1984) was obtained from the American Type Culture Collection (Rockville, MD).

Figure 1 shows the hybridization of the factor H and F13B probes with Sfi I, SacII, iVru1, and NurI digests. Both probes hybridized with the same 1050- and 800- kb Sfi I fragments. Further digestion of these fragments with NruI, although incomplete, generates a 700-kb DNA fragment which retains the hybridization with both probes. Similarly, the double-digestion with NruI and Sac11 produces a fragment of 650 kb which is also recognized by the two probes. Finally, these probes hy- bridize with different NurI fragments. Taken together these data are compatible only with the HF and F13B genes being included within the same S/i I, NruI, and

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BRIEF REPORT 645

Sac11 fragments, therefore establishing the physical linkage of these two human genes. Both enzymes NruI and Sac11 generate fragments containing the HF and F13B genes which are too large to be resolved under the conditions used in the experiment shown in Fig. 1. In a different experiment the NruI fragment was per- fectly resolved and its size estimated to be around 2500 kb. The two Sfi I fragments of 1050 and 800 kb hy- bridizing with both factor H and F13B probes are no- tably reproducible and most likely the consequence of an Sfi I polymorphic restriction site in this genomic region. This interpretation is supported by double- digestion experiments, not shown here, demonstrating that both Sfi I fragments of 1050 and 800 kb are further digested with MluI, resulting in two Sfi I/MU frag- ments of 500 and 700 kb which hybridize with the factor H probe and a single Sfi I/MluI fragment of 450 kb hybridizing with F13B probe.

Figure 2, a summary of all experiments performed, provides a restriction map of the human RCA gene cluster region containing the HF and F13B genes. The smallest DNA segment found to hybridize with both probes is a 650-kb-long fragment generated by the double digestion with NruI and SacII. Our results sug- gest that these two genes are not contiguous. However, the length of the DNA segment that separates the HF and the F13B genes remains to be determined.

In an attempt to precisely locate the DNA segment including the factor H and F13B genes in relation to other RCA and non-RCA genes within this chromo- somal region, the same nylon membranes used in the experiments described above were hybridized with CRl-, C4BP-, LCA-, and renin-specific cDNA probes.

- 1250 Kb -- 1050 Kb

Hf FN B

FIG. 1. Pulsed-field gel electrophoresis analysis of human ge- nomic DNA using factor H- and F13B-specific cDNA probes. Ex- perimental conditions for this gel were as follows: 1.5% agarose in 0.25X TBE, run at 180 V (voltage gradient of approx 6 V/cm) for 44 h with pulse lengths of 120 s for the first 22 h and 50 s during the last 22 h. Molecular sizes of selected yeast chromosomes are indicated on the right. The restriction enzymes are abbreviated as follows: Sf, Sfi I; SC, SacII; Nu, NruI; and Nr, NurI.

too Kb

FIG. 2. Restriction map of the chromosomal region containing the HF and F13B genes. Asterisks indicate probable polymorphic restriction sites. The regions to which the factor H and F13B probes hybridized are indicated by hatched bars. The restriction enzymes are abbreviated as follows: S, SalI; Sf, Sfi I; SC, Sⅈ M, MU; N, NotI; Nu, NruI; Nr, NurI; and Na, NaeI.

These probes, however, did not hybridize with any of the fragments recognized by the factor H or F13B probes and, thus, the physical linkage to these genes could not be revealed.

The RCA gene cluster is thought to have originated as a result of the continuous duplication of genes in this chromosomal region. The reasons, if any, for the manifest evolutionary stability of the RCA gene cluster, where the “new genes” appear to have remained tightly linked to those from which they originate, are currently unknown. However, the physical distances between the RCA genes are, most likely, an indication of their evo- lutionary distances. In this respect, our data support the concept that complement factor H and the B sub- unit of coagulation factor XIII are evolutionarily closer to each other than to the other known members of the RCA gene cluster.

ACKNOWLEDGMENT

This work was partially supported by the Spanish Government DGICYT Grant PM%-0002.

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