Effects of the Bgs locus on mouse β-galactosidase

The Bgs locus determines tissue levels of β-galactosidase in the mouse, so that enzyme levels are twice as high in mice carrying the Bgs ^hallele as in mice carrying the Bgs ^dallele (Felton et al., 1974). By immunotitration with antiserum to purified β-galactosidase, we have found that the Bgs locus influences the amount of enzyme protein present in the tissues. We have utilized recombinant inbred lines derived from a cross between C57BL/6J and DBA/2J mice to confirm the location of the Bgs locus on chromosome 9. The inhibition of mouse β-galactosidase by the active-site-directed reagent N-bromoacetyl-β-d-galactosylamine has been investigated. β-Galactosidase from the high and low Bgs strains has identical affinity for this inhibitor.     

Genetic analysis of anH-2 mutant,B6.C-H-2 ba,using cell-mediated lympholysis: T- and B-cell dictionaries for histocompatibility determinants are different

B6.C-H-2 ^ba [H (z1)] is a mutant derived from C57BL/6. The two strains mutually reject their skingrafts and are incompatible in the mixed leucocyte reaction (MLR) and in cell-mediated lympholysis (CML) assays. They are serologically indistinguishable. This report shows that H(z1) carries a new, privateK end CML specificity clearly distinguishable from that of B6 by a third party strain, HTG. Antisera directed against the private H-2K specificity of B6 present on H(z1) cells) can block CML between the two strains in either direction. The new CML specificities of H(z1) cross-react with (public) CML specificities controlled by bothK andD regions of other unrelated haplotypes. The results suggest that H(z1) carries a mutation in theH-2K locus itself or in a closely linked gene, the product of which is also physically associated with the H-2K molecule corresponding to the cis-configuration of the alleles in both loci. These findings indicate that T- and B-cell dictionaries for histocompatibility determinants are different.     

Esterase-16 (Es-16): Characterization,polymorphism, and linkage to chromosome 3 of a kidney esterase locus of the house mouse

A polymorphism for an isozyme of a presumed arylesterase, esterase-16 (EC 3.1.1.2), has been detected in kidney, heart, and spleen of the house mouse, Mus musculus, by means of isoelectric focusing and by disc electrophoresis. Three phenotypes can be distinguished: the ES-16A phenotype (IEP 5.9) was found in C57BL/10Sn and many other laboratory inbred strains; the ES-16B phenotype (IEP 6.1) was found in M. m. molossinus; and the ES-16C phenotype (IEP 5.9; very weak activity) was found in Peru-Coppock. Esterase-16 is strongly inhibited by 10^?3 m p-chloromercuribenzoate, but not by 2·10^?4 m bis-p-nitrophenyl phosphate or by 10^?3 m Diamox. It stains well with indoxyl acetate and other indigogenic substrates but only weakly with α-naphthyl acetate. Esterase-16 is completely insoluble in water. It is apparently governed by a structural gene locus, Es-16, with three alleles, Es-16 ^a , Es-16^b, and Es-16 ^c, respectively. Es-16 is closely linked to Car-1 and Car-2 on chromosome 3. Typing of 94 animals of the backcross (C57BL/10Sn × M. m. mol.) F₁ × M. m. mol. revealed a recombination frequency of 8.51±2.9%.     

Monoclonal antibody reveals H-2-linked quantitative and qualitative variation in the expression of a Qa-2 region determinant

We have produced a monoclonal antibody, Y-7, that reacts with a Qa-2 region-controlled determinant. Cellular and strain distribution analyses, coupled with quantitative variation in the amount of Y-7 antigen expressed among strains, provide overwhelming evidence that Y-7 reacts with the Qa-2^a determinant. The determinant detected by Y-7 is differentially expressed in T and B lymphocytes in a strain specific manner. Y-7 reacts with the majority of T lymphocytes (> 95%) and approximately one-half of B lymphocytes in certain strains (++ strains), and with the majority of T lymphocytes (> 95%) and no B lymphocytes in other strains (+ strains). T lymphocytes in + strains express approximately three fold less of the Y-7 determinant than T lymphocytes from ++ strains. In addition, we show that the Y-7 determinant is expressed in approximately one-third to one-half of Lyb-3^?, 5^? B lymphocytes. Possible mechanisms determining quantitative and qualitative variation in the expression of the Y-7 determinant in T and B lymphocytes are discussed.     

A new locus regulating the expression of the Ldh-2 gene in mouse liver

Patterns of lactate dehydrogenase (LDH) isozymes of tissues from various mouse strains were examined. An interstrain polymorphism for LDH isozymes of liver was established. One phenotype (CBA/Lac and AKR/J mice) yielded a five-banded LDH pattern, another phenotype (DBA/1J, DBA/2J, C57BL/6J and C3H/He) showed a three-banded one. Immunochemical evidence was obtained indicating that differences in the LDH pattern are mainly due to different contents of the B subunit of LDH. Linkage tests indicated that the locus Ldr-2 determining the amounts of the LDH B subunit in mouse liver tissue is located in chromosome 6, 19 ± 4.1 cm away from the earlier described Ldr-1 locus. The effect of locus Ldr-2 is strictly tissue-specific; it is manifest only on days 6–8 after birth.     

Analysis ofH-2 mutants: Evidence for multiple CML target specificities controlled by theH-2K b gene

C57BL/6 (H-2 ^b ) mice and two mutants derived from this strain, B6.C-H-2 ^ba (Hz1) andE6-H-2 ^bd (M505), were studied in a number of functional tests, in vitro and in vivo, that assay for differences at theH-2 complex. All three strains give rise to reciprocal mixed lymphocyte reactivity (MLR) and cell-mediated lympholysis (CML) in vitro as well as graft-host reactivity (GVHR) and skin graft rejection in vivo. Analysis for cross-reactivity between these strains in CML revealed that the gained antigens in each mutant do not cross-react, and that Hz1 has lost an antigen shared by C57BL/6 and M505 strains. In addition, spleen cells from B10.A(4R) mice, which differ from theH-2 ^b haplotype only at theK end of theH-2 complex, recognize a common antigen shared by all three strains tested. Provided that the mutations occurred in theH-2K ^b gene, these data indicate that a) there are at least three antigenic specificities coded for by theH-2K ^b gene(s) that serve as targets for receptors on thymus-derived (T) cells in CML; b) since C57BL/6 strain mice and the mutants are serologically indistinguishable on a qualitative basis, the antigens recognized by the receptors on T cells and by humoral H-2 antibody are nonidentical; and c) mutation in theH-2K ^b locus itself can give rise to allogeneic recognition phenomena such as MLR and GVHR.     

Evidence for the control of Eosinophilia by the major histocompatibility complex in mice

The genetic control of eosinophilia has been studied in congenic strains of mice. Eosinophilia was induced with cyclophosphamide followed by keyhole limpet hemocyanin in complete Freund's adjuvant. After this treatment, BALB/c mice developed a high eosinophil response, whereas CBA, C57BL and A/J mice developed a low one. The major histocompatibility complex (M-HQ was found to exert a control on eosinophilia, as B 10.D2 mice developed a higher eosinophil response than B10, B10.A, or B10.BR. BALB/c-H-2 ^k mice had a lower response than BALB/c, and A.TL mice had a higher response than A/J or A.TH. If a single gene within the MHC is responsible for these effects, the most likely position for it is in the vicinity of the Tla locus. Splenectomy reduced eosinophilia in BALB/c and A.TL mice, but not in A/J mice,indicating that the spleen is a significant site of eosinophil production in high responder strains.     

A new alloantigen,Ly-8, recognized by C3H anti-AKR serum

A new membrane alloantigen, designated Ly-8.2, is defined by a C3H anti-AKR serum. The locus,Ly-8, which controls this determinant is not linked toThy-1, Ly-4, Ly-6, H-2, albino (c), or brown (b). Ly-8.2 has a unique strain distribution, and appears to be present on both T and B lymphocytes.     

Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. 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-1 ^b, 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-1^a/Igh-4^a heavy chain molecules. The Igh-1^a determinants were maintained, but the Igh-4^a 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.     

Restriction fragment length polymorphism and chromosome mapping of a mouse homeo box gene,Hox-2.1

Restriction endonuclease fragment length polymorphisms (RFLPs) were found using the cDNA probe Hox-2.1 for the homeo box-2.1 gene in the mouse. Polymorphism was detected in restriction patterns generated by fragments fromHindIII digestion. The great majority of laboratory strains of mice carries theHox-2.1 ^a allele. Only two laboratory strains carry theHox-2.1 ^b allele. Among strains of wild origin, the European subspecies (Mus m. domesticus, M. m. brevirostris, andM. m. musculus) and some Asian subspecies (M. m. castaneus) carry theHox-2.1 ^a allele. The subspecies from Far Eastern countries (M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai) carry theHox-2.1 ^ballele. Using the RFLP, theHox-2.1 gene was mapped on chromosome 11. Three-point cross test data showed that the recombination frequency is 29.6% between theHba and theHox-2.1 genes and 23.5% between theHox-2.1 and theEs-3 genes. The gene order ofHba-Hox-2.1-Es-3 has been confirmed.     

Multigenic control of immune responses of inbred mice against the terpolymers poly(Glu57Lys38Ala5) and poly(Glu54Lys36Ala10) and linkage withH-2 haplotype

Results of immunizations of recombinant inbred and congenic strains of mice with the random polymers poly(glu⁵⁷ lys³⁸ala⁵) or GLA⁵ and poly(glu⁵⁴lys³⁶ala¹⁰) or GLA¹⁰ indicate that there is an association of the responsiveness with theH-2 haplotype. Although the C57BL/6J mice (H-2 ^b haplotype) are “non responders”, the C57BL/6By originally derived from mice of the same haplotype are responders. The immune response pattern of recombinant strains carrying haplotypes derived by crossing over within theH-2 complex indicate that the responsiveness is under control of anIr gene which maps to the left of theIB subregion. Studies with the backcross mice indicated multigenic control of the responsiveness, with one locus beingH-2 linked and another locus segregating independently ofH-2.     

Structural polymorphism of murine factor B controlled by a locus closely linked to the H-2 complex and demonstration of multiple alleles

New alleles of murine factor B (Bf) protein were demonstrated. When ethylenediaminetetraacetic acid (EDTA)-plasmas from inbred and wild mice were analyzed by isoelectro-focusing (IEF) and immunofixation, murine Bf proteins were visualized as distinct protein bands in all mice tested. Four variants of murine Bf could be demonstrated in a large number of tested mice: Bf 1 (isoelectro-focusing point (P.I.) range of 5.8–6.1) exemplified by B10 and B10.BR, Bf 2 (P.I. range of 5.8–6.0) exemplified by B10.MOL (OHM), Bf 3 (P.I. range of 5.6–5.9) exemplified by B10.MOL (TEN2) and Mus musculus (Mus m.) subspecies Chc, Bf4 (P.I. range of 6.0–6.3) exemplified by Mus m. subspecies Shh. The genetic linkage between S locus and Bf locus was studied with two backcross progenies — [B 10.BR × (B10.BR × Mus m. subspecies Chc)F₁] and [B 10.BR × (B10.BR × Mus m. subspecies Shh)F₁]. Totally, 256 backcross progenies were typed for Bf type and for Ss type (plasma level of the fourth complement protein regulated by S locus). The results indicated that murine Bf was controlled by a single codominant locus located close to the H-2 complex because no mouse showing recombination between Bf locus and S locus was found.     

H-2 restriction of cytolysis after immunization of minorH congenic pairs of mice

Mice of three congenic resistant lines differing from C57BL/10 at theH-3, H-13, H-7, andH-8 minor histocompatibility loci were used to immunize, and were immunized with, C57BL/10. Cytotoxic cells which were capable of causing rapid lysis of cells from the immunizing strain were generated at least one-way in all combinations tested. In order for a target to be susceptible to cytolysis, it had to carry both the sameH-2 ^b haplotype and the same differential minor histocompatibility alleles as the immunizing strain. That is, B10.C(47N) (H-2 ^b ,H-7 ^b ) anti-C57BL/10 (H-2 ^b ,H-7 ^a ) cytotoxic cells lysed C57BL/10 targets but not B10.BR (H-2 ^k ,H-7 ^a ) targets, nor BALB.B (H-2 ^b ,H-7 ^b ) targets. Crossreactions in the cytotoxic assay suggest that theH-3, H-13 congenic pair —B10.LP and C57BL/10 —may differ in at least two more minor histocompatibility loci which have not yet been defined. Although 6 x 10⁶6 C57BL/10 lymphoid cells primed B10.D2(57N) (H-8 ^b ) mice for a secondary in vitro cytotoxic response, a tenfold higher dose apparently made them tolerant. It is concluded that all minor histocompatibility differences can generate cytotoxic T cells which show specificity both for the minor and major histocompatibility alleles.     

Genetics of the anti-dextran B512 and the autoanti-idiotypic response: Codominant expression in F1 hybrids and dichotomy of response and allotype-linked idiotype

The IgM plaque-forming response to the alpha 1–6 epitope of dextran B512 is linked to the Ig-1 heavy chain allotypes j and b characteristic of CBA and C57BL strains, respectively, and the response typically induces the formation of autoanti-idiotypic antibodies that can distinguish between anti-dextran antibodies of CBA and C57BL origin. Nevertheless, some substrains of Balb/c mice (allotype a) and some Bailey recombinant stains give a PFC response although they do not possess allotypes j or b. The anti-dextran antibodies in these strains lack the idiotypes characteristic of either CBA and C57BL antibodies to dextran, but they possess their own particular idiotype. F₁ hybrids between two responder strains possessing different idiotypes on their antibodies against dextran, produce both idiotypes and two different autoanti-idiotypic antibodies. CBA(Ig-1^b) mice were high responders to dextran and possessed the idiotype of C57BL, whereas C57BL/6(Ig-1^a) mice were low responders. The V_H recombinant strains BAB.14 and CB-8KN that possess the Ig-1^b allotype of C57BL, but have some of theV _H genes from Balb/c and the rest from C57BL/6 were high responders to dextran, but did not possess the C57BL idiotype, suggesting that the genes determining the response against dextran and the idiotype may have different locations in the heavy chain locus.     

Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis

Strains of Azotobacter mediate in the nitrogen fixation process by reducing of N₂ to ammonia. In this study, 50 strains were isolated from different rhizospheric soil in central Iran, by using soil paste-plate method. These strains were biochemically identified and characterized on differential LG medium based on morphological and physiological properties. Results obtained showed that identified strains were belonging to three species, namely A. chroococcum, A. vinelandii and A. beijernckii. In order to molecular analysis, the 16S rRNA gene was amplified using 27f and 1495r primers and PCR products were subsequently restricted with RsaI, HpaII and HhaI. Cluster analysis based on amplified ribosomal DNA restriction analysis were revealed intraspecific polymorphism and differentiated strains into two mains clusters, clusters A and B. Cluster A strains were related to the A. vinelandii, whereas cluster B strains were related to the A. chroococcum and A. beijerinckii. The results show that amplified ribosomal DNA restriction analysis is a powerful and discriminatory tool for the identification of members of the genus Azotobacter.     

Immune response of mice to the Thy-1.1 antigen: Effect of theIr-5 alleles studied in 129/J and B10.129 (6M) mice

The primary immune response to the Thy-1.1 antigen was measured by a plaque assay that detected cells producing antibodies lytic for AKR thymocytes. B10.129(6M) mice carrying theH-2 complex of an intermediate responder (129) on a low-responder (B10) background, were low responders. Studies employing different F₁ hybrids and segregating generations of 129/J and 6M mice indicated that differences in responsiveness of those two strains depend on alleles at a single locus, loosely linked to theH-2 complex. These results lend further support to the previously advanced concept that the expression of theIr-Thy-1 allcles controlling the response to the Thy-1.1 antigen is influenced by the alleles at theIr-5 locus. In addition, studies employing F₁ hybrids produced through matings of 129/J, 6M, C3H.B10 and C57BL/6J mice to a panel of inbred strains suggested that in regard to the responsiveness to the Thy-1.1 antigen, 129/J and 6M mice are phenotypically, and presumably genotypically, similar to C3H.B10 and C57BL/6J mice, respectively.     

Cleft palate susceptibility linked to histocompatibility-2 (H-2) in the mouse

Data have been obtained indicating that cortisone-induced cleft palate in the mouse is linked to theH-2 ^a complex. Cortisone (2.5 mg) was administered to pregnant females on days 11 through 14 of pregnancy. On day 17 of pregnancy, the fetuses were inspected for cleft palates. Sham experiments were done by injecting sterile saline instead of cortisone. The inbred strains, A/J and C57BL/6, and the congenic strains C57BL/10ScSn and B10.A were tested for susceptibility to cleft palate. The clefting frequency was also observed in hybrids of the congenic strains. The A/J and B10.A strains showed a characteristic high susceptibility to cleft palate (i.e., 99% and 81% incidence of cleft palate, respectively) after teratogenic treatment. The C57BL/6 and C57BL/ 10ScSn demonstrated a significant resistance to the teratogen (i.e., 25% and 21 % incidence of clefting, respectively). The teratogenic treatment of congenic hybrids indicated that maternal influences significantly affected the incidence of cleft palate formation. The maternal influence appeared to depend upon the specificH-2 haplotype of the mother.     

Simiduia aestuariiviva sp. nov., a gammaproteobacterium isolated from a tidal flat sediment

A Gram-stain negative, aerobic, motile and rod-shaped bacterial strain, designated J-MY2^T, was isolated from a tidal flat sediment of the South Sea, South Korea. Strain J-MY2^T was found to grow optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain J-MY2^T forms a cluster with the type strains of Simiduia species. Strain J-MY2^T exhibited 16S rRNA gene sequence similarity values of 97.62–98.77 % to the type strains of four Simiduia species and of <92.95 % sequence similarity to the type strains of the other recognized species. Strain J-MY2^T was found to contain Q-8 as the predominant ubiquinone and summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_16:0, C_18:1 ω7c and C_17:1 ω8c as the major fatty acids. The major polar lipids of strain J-MY2^T were identified as phosphatidylethanolamine, phosphatidylglycerol, three unidentified glycolipids and one unidentified lipid. The DNA G+C content of strain J-MY2^T was determined to be 54.8 mol% and its mean DNA–DNA relatedness values with the type strains of the four Simiduia species were in the range 21–34 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain J-MY2^T is separated from other Simiduia species. On the basis of the data presented, strain J-MY2^T is considered to represent a novel species of the genus Simiduia, for which the name Simiduia aestuariiviva sp. nov. is proposed. The type strain is J-MY2^T ( = KCTC 42073^T = CECT 8571^T).     

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller “leaf-like” structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics’ analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu²⁺ stress. After 5 days of Cu²⁺ stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu²⁺-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu²⁺-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.