Margriet Snoek

The recombinant congenic strains—a novel genetic tool applied to the study of colon tumor development in the mouse

The development of tumors in mice is under multigenic control, but, in spite of considerable efforts, the identification of the genes involved has so far been unsuccessful, because of the insufficient resolution power of the available genetic tools. Therefore, a novel genetic tool, the RC (Recombinant Congenic) strains system, was designed. In this system, a series of RC strains is produced from two inbred strains, a “background” strain and a “donor” strain. Each RC strain contains a different small subset of genes from the donor strain and the majority of genes from the background strain. As a consequence, the individual genes of the donor strain which are involved in the genetic control of a multigenic trait, become separated into different RC strains, where they can be identified and studied individually. One of the RC strains series which we produced is made from the parental strains BALB/cHeA (background strain) and STS/A (donor strain). We describe the genetic composition of this BALB/cHeA-C-STS/A (CcS/Dem) series and show, using 45 genetic autosomal markers, that it does not deviate from the theoretical expectation. We studied the usefulness of the CcS/Dem RC strains for analysis of the genetics of colon tumor development. The two parental strains, BALB/cHeA and STS/A, are relatively resistant and highly susceptible, respectively, to the induction of colon tumors by 1,2-dimethylhydrazine (DMH). The individual RC strains differ widely in colon tumor development after DMH treatment; some are highly susceptible, while others are very resistant. This indicates that a limited number of genes with a major effect are responsible for the high susceptibility of the STS strain. Consequently, these genes can be mapped by further analysis of the susceptible RC strains. The differences between the RC strains were not limited to the number of tumors, but the RC strains differed also in size of the tumors and the relative susceptibility of the two sexes. Our data indicate that the number of tumors and the size of tumors are not controlled by the same genes. The genetics of these different aspects of colon tumorigenesis can also be studied by the RC strains. The DMH-treated mice of the parental strains and the RC strains also developed anal tumors and haemangiomas in varying numbers. The strain distribution pattern (SDP) of susceptibility for each of the three types of tumors induced by DMH is different, indicating that development of these tumors is under control of different, largely non-overlapping, sets of genes. Thus, with a single series of RC strains, genes involved in tumorigenesis in various organs and tissues can be studied separately. These results indicate that the novel genetic tool, the RC strain system, offers new possibilities for analysis of the multigenic control of tumor development.

A susceptibility gene for alveolar lung tumors in the mouse maps between Hsp70.3 and G7 within the H2 complex

Lung tumor susceptibility (LTS) in the mouse is influenced by multiple loci within the H2 complex. We compared the LTS of two H2 congenic strains with intra H2 recombinations, B10.A(1R) and B10.A(2R), whose genetic difference has been reduced to a region of approximately 50 kilobases within the C4-H2D interval, between Hsp70.3 and G7. After transplacental induction with N-ethyl-N-nitrosourea the load of alveolar lung tumors in strain B10.A(2R) is significantly higher than in strain B10.A(1R) (P <0.001). For papillary tumors no significant differences were observed. We conclude that the alveolar lung tumor load is influenced by an LTS gene located within the Hsp70.3-G7 interval.

A new H-2.1-PositiveD region allele,D dx , controlling two molecules, H-2Ddx and H-2Ldx

The inbred strains GRS/A and LIS/A carry the haplotypeH-2dx, which had earlier been shown to have theKd,If,Sf, andGf alleles and a previously unknownD region allele,Ddx. We show here that theDdx allele determines a new private specificity, H-2.63, is H-2.28 negative, and determines at least one public specificity of the H-2.1 family. It is thus a second example (afterDk) of a H-2.1-positive H-2.28-negativeD region allele. Capping experiments show that the Ddx product comprises two molecules: H-2Ddx bearing the private specificity H-2.63, and H-2Ldx, which is H-2.63-negative but reacts with sera against the H-2.1 family of specificities. SDS gel electrophoresis of detergent-solubilized immunoprecipitated Ddx products shows that the H-2Ldx antigen has a molecular weight of approximately 45,000 daltons and is associated with a smaller polypeptide (mol. wt. 12,000).

Fine mapping of the crossover-sites in the C4-H-2D region of H-2 recombinant mouse strains

The class I and class II regions of the major histocompatibility complex (MHC) encode polymorphic molecules that present antigen to the T-cell receptor. The class HI region which lies between the class I and class II region contains genes for complement components (C4, Bf, and C2) and steroid 21 hydroxylase (21-OHA and B). This stretch of DNA of the class 11I region encompasses approximately 1100 kilobases (kb) in human and mouse (Dunham et al. 1987; Carrol et al. 1987; Miiller et al. 1987a). This region appears to be conserved between several species, as the genes for tumor necrosis factor (Tnf) and heat shock protein (Hsp70) have been mapped to this region and found to be located in the same order in several mammals (Miiller et al. 1987b; Dunham et al. 1987; Sargent et al. 1989a; Cameron et al. 1990; Wurst et al. 1989; Gaskins et al. 1990). Recently, 15 transcribed genes have been identified in the HLA complex in the interval corresponding to the mouse C4-H-2D segment (G 1-11 and BAT 1-9, respectively; Sargent et al. 1989b; Spies et al. 1989). A recent paper comparing human and mouse for the BAT1 gene confirms the existence of interspecific conservation by showing that its mouse homologue maps at a comparable distance to Tnf, although the distance to the nearest class I locus appears to be larger in humans (Wroblewski et al. 1990). Numerous recombinant mouse haplotypes have arisen by a crossover in the C4-H-2D interval. However this region has not yet been analyzed by genetic fine mapping. Within the mouse MHC recombination is not random but occurs more often at specific s i tes -hot spots. Such hot spots have been mapped to the H-2K-I interval and the 1-1-21 region (Steiumetz et al. 1986; Lafuse and David 1986). Whether the recombination within the class III region is also nonrandom is as yet not sufficiently studied. However, the presence of a possible recombination hot

Coding sequences and levels of expression of Hsc70t are identical in mice with different Orch-1 alleles

Experimental allergic orchitis (EAO) is an autoimmune disease of the testis that is controlled by multple genes. The use of recombinant mouse strains has defined the map position of the H-2-associated locus controlling disease susceptibility, Orch-1, within the H-2S/H-2D interval. Over the last few years the definition of the structural organization of the C4-H-2D segment and identification of the recombination sites of the various intra-H-2 recombinations has reduced the map position of Orch-1 to the Hsp70.1-G7 interval. Three Hsp70 genes, Hsp70.1, Hsp70.3, and Hsc70t, and the genes G7b and G7a are located in this segment of DNA. In order to investigate whether Hsc70t is a suitable candidate for Orch-1 we have compared the sequence of the gene from a susceptible and a resistant haplotype.

Mapping of a Hot Spot in the Major Recombination Area of the Mouse H-2 Complex

Analysis of the class III region of the mouse, using human probes, showed that all human class III genes tested thusfar seem to have homologous counterparts in the mouse genome. Furthermore, the relative gene order of these genes appears to be conserved also. The fine mapping of the C4-H-2D segment shows that the recombination in this interval is not random, but that recombination occurs preferentially in the Hsp70-Bat-5 segment, indicating the existence of a “hot spot”. The determination of crossovers at the molecular level will be helpful in the identification of functionally defined genes, since our structural data gives more information of their localisation of genes controlling disease susceptibility.