Restriction analysis shows that wild Scandinavian mice belonging to the species Mus mtuculus contain the mitochondrial DNA of a neighboring species, M. domesticus. This demonstration results from comparisons of Scandinavian mice with authentic M. dwometicus and M. muscdus from other parts of Europe. Electrophoretic and immunological analysis of eight diagnostic proteins confirms that mice from north of the hybrid zone in Denmark are M. musculus in regard to their nuclear genes. In contrast, the mice tested from this region and a nearby part of Sweden have exclusively M. domesticus types of mitochondrial DNA. Phylogenetic analysis of the restriction maps suggests that the mitochondrial DNAs found in Scandinavian M. musculus could stem from a single M. domesticus female.The growing use of mtDNA as a tool for genetic research on animal populations (1, 2) makes it important to compare the ability of nuclear and mitochondrial genomes to move between populations. [mtDNA differs conspicuously from nuclear DNA not only by being outside the nucleus but also by existing in thousands of copies per cell, being inherited maternally, and evolving quickly (3,4).] Such a comparison can be made by examining the distribution of genes across a hybrid zone-i.e., a geographic zone where two species meet and interbreed but where there is limited flow of nuclear genes (5).Of all the hybrid zones examined by both organismal and molecular biologists, that between two species of mice in Denmark is the best known (6-8). The comprehensive study by Hunt and Selander (7) of proteins encoded by the nuclei of 2,696 mice caught at 44 Danish localities delineated the hybrid zone as regards nuclear genes. In addition, the protein evidence agrees with anatomical evidence as to the geographic location of this hybrid zone (6-8).Further protein work has shown how these Danish mice are related to other commensal mice (9, 10). Commensal mice are those species that live in close association with buildings used by humans. They contrast with aboriginal mice (in Europe: Mus spretus, M. hortulanus, and M. abbotti), which live predominantly independent of human dwellings and, in nature, do not interbreed with commensal mice (10-12). According to a phylogenetic analysis of the protein data, there are two commensal mouse species in Europe. One, known as M. domesticus, lives in southern Denmark, in most of the rest of western Europe, and around the Mediterranean Sea (11, 12) (see Fig. 1). The second, M. musculus, lives in northern Denmark, the rest of Scandinavia, and eastern Europe (11, 12). The hybrid zone defined by Ursin (6) and Selander and co-workers (7, 8) is the meeting place of M. domesticus and M. musculus in Denmark (see Fig. 1). These two types of mice are sometimes considered as semispecies. Our decision to refer to them as separate species is based not only on extensive morphological and biochemical evidence (10-12) but also on the observation that there is a high incidence of sterility in the male offspring of crosses between M. musc...

Human esterase D (carboxylesterase; carboxylic-ester hydrolase, EC 3.1.1.1), a genetic marker of retinoblastoma, was purified to biochemical homogeneity from erythrocytes. The purification scheme including carboxymethylcellulose, phenyl-Sepharose, chromatofocusing, and hydroxylapatite chromatographies resulted in a 10,000-fold purification of the enzyme with 15% recovery of total activity. The Km of esterase D was estimated to be 10 x 10-6 M using 4-methylumbelliferyl acetate as substrate. (8,9). In addition, the defective gene of Wilson disease was found to be linked to the esterase D gene (10). Thus, it is important to investigate the nature of the esterase D protein and its gene so as to understand these inherited disorders.Esterase D has been found in most tissues, but not much is known about its structure and function. A protocol for partial purification of this enzyme has been described (11).However, it remains difficult to obtain a sufficient amount of the homogeneous enzyme and to generate specific antibodies. In this communication, we describe the purification and characterization of this enzyme as well as the preparation of esterase D-specific polyclonal and monoclonal antibodies. Furthermore, we observed that this enzyme was mainly distributed in liver and kidney and could be induced by treatment with phenobarbital but not with phorbol ester. MATERIALS AND METHODSEsterase D Enzymatic Assays. (i) Quantitative assay. Esterase D activity was determined essentially as described (11). The reaction was started by adding the enzyme into 1 ml of reaction buffer containing 50 mM potassium phosphate, 1 mM EDTA (pH 6.0), with 0.1 mM 4-methylumbelliferyl acetate as substrate. The increase in absorbance at 340 nm was recorded. A unit of activity was defined as the amount of enzyme that hydrolyzed 1 /.kmol of substrate per min at 230C using 7.27 mM-1 cm-1 as the coefficient of absorption.(ii) Spot test. A rapid semiquantitative test was developed by us for assaying enzyme activity in column fractions.Briefly, 5 jul of 4-methylumbelliferyl acetate (20 Ag/ml) in sodium acetate (pH 5.2) was spotted onto a piece of SaranWrap over a UV transilluminator together with 5 A.l of enzyme samples from each column fraction. The intensity of fluorescence emitted corresponded with the enzyme activity.(iii) Qualitative assay. Electrophoresis in 1% agarose gels was performed as described (12) with slight modifications. The agarose gel buffer was a 1:5 dilution ofrunning buffer that contained 62 mM Tris, 15.5 mM citric acid, 18 mM boric acid, and 1.65 mM lithium hydroxide (pH 7.5). Electrophoresis was performed at 40C for 90 min at 20 V/cm. The gel was stained in 50 ml of 50 mM sodium acetate (pH 5.2) containing 1.0 ml of 4-methylumbelliferyl acetate (1 mg/ml in acetone) for 5 min and then photographed under UV light.Purification of Human Esterase D.Step 1. Outdated human erythrocytes were washed once in phosphate-buffered saline (PBS) with 1 mM EDTA. A precooled (-20°C) mixture of chloroform and butanol was added to the pa...

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Ighp.

We have generated four xenogeneic rat antimouse IgG3 monoclonal antibodies recognizing at least three different antigenic determinants (epitopes) on BALB/c IgG3 molecules. These antibodies were used in solid-phase blocking radioimmunoassays for detection of the epitopes in sera of 40 inbred strains and 134 wild mice. These antibodies detect genetic polymorphism of IgG3 isotype among wild mice even though there is no polymorphism found among 40 inbred strains tested (except X-chromosome-linked immunodeficient CBA/N strain which lacks IgG3 molecules). An IgG3 variant was also isolated from hybridomas derived from Mus spretus.

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1a/Igh-4a heavy chain molecules. The Igh-1a determinants were maintained, but the Igh-4a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.