Establishment of a congenic strain with complement regulatory protein of factor H allotype derived from Mus. musculus molossinus]

Factor H is a plasma glycoprotein with M. W of 160 KDa which serves as one of the regulatory proteins for C 3 convertases. We have previously reported three serologically defined mouse factor H allotypes by surveying many laboratory and wild mice. In the present work, we established a congenic strain with factor H allotype, H. 2. on BALB/c (H. 1 allotype) background and named this strain BALB-H.2. Alloantiserum against each allotype has been easily prepared using two congenic strains by immunization with a small amount of whole mouse serum. BALB-H.2 is valuable for the genetic studies on the genes in the vicinity of factor H gene (cfh) derived from Mus. m. molossinus.     

Rabbit secretory components: identification of a third allotype, t63

A third allotype of rabbit secretory component has been identified. The allotype previously referred to as t62 by our laboratory can now be subdivided into two allotypes, t62 and t63, with alloantisera capable of discriminating between the two. Results of family studies are consistent with a three allele system (t61, t62 and t63) at the t-locus. By SDS PAGE, electrophoretic mobilities of the multiple SC bands for each of the three allotypes are characteristic of the allotype; the apparent molecular sizes of the bands of the t62 allotype are 2 to 3 kDa lower than those for the t61 allotype. The banding patterns of the t61 and t63, although similar, are not identical to each other. Results of serologic cross-reaction studies and of tryptic peptide mapping studies suggest multiple structural differences between the allotypes as well as a closer relationship between t62 and t63 than between either of these allotypes and t61.     

Influence of the Ig H chain locus on autoantibody production in autoimmune mice.

Autoimmune disease is influenced by multiple genes. In this study, we investigated the role of one genetic locus, Ig H chain. IgG2a antichromatin, anti-ssDNA, and antihistone autoantibodies (autoAb) from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr), (Ighj/b); (C57BL/6-lpr/lpr x C57BL/6-lpr/lpr-Igha), (Ighb/a); and (MRL/Mp-lpr/lpr x MRL/Mp-lpr/lpr-Ighb), (Ighj/b) mice were determined using allotype-specific ELISA. Strikingly, antichromatin and antihistone antibodies (Ab) were comprised of significantly more b allotype than either a or j allotype in all cohorts of F1 mice examined. In mice that produced anti-Sm Ab, the b allotype was used preferentially for these autoAb as well. However, no allotype skewing was observed in IgG2a Ab directed against TNP or DNA, or for total IgG2a. An Igh recombinant locus was utilized to examine the genetic control of b allotype skewing in lpr mice and in chronic graft vs host disease. In both models, the VH region did not appear to be responsible for the preferential use of b allotype. These results indicate a contribution to autoimmunity by the Igh locus and raise the possibility that Ig allotype may influence autoimmune disease by its effect on the production of certain autoAb.     

Serum antigens of cattle

We have produced an alloimmune serum that detects a new cattle immunoglobulin light chain allotype. Genetic analysis involving more than 600 offspring showed that this allotype is controlled by an autosomal codominant allele [Ig(L)b ²], of theIg(L)b ¹ gene previously reported from this laboratory. This system also contains a recessive allele for which no product can now be detected. The frequencies of the three alleles in 5 dairy breeds are reported. The Ig(L)B1 and Ig(L)B2 allotypes are expressed as complexes of 2 or 3 precipitin bands in double diffusion gels containing polyethylene glycol-6000. The complexes are transmitted as genetic units.     

Allelic polymorphism of murine IgE controlled by the seventh immunoglobulin heavy chain allotype locus

Two alloantisera against hybridoma-derived IgE detected allotypic determinants expressed on the murine s chain. An antiserum raised in BALB/c mice against monoclonal IgE of C57BL/6 origin reacted exclusively with IgE of strains having Igh-1^b (IgG_2a) allotype. The second antiserum, C57BL/6 anti-BALB/c monoclonal IgE, reacted with IgE of strains having Igh-1^a, Igh-1^d, Igh-1^e and Igh-1^j allotypes. The genetic studies of (BALB/c x C57BL/6)F₁ and backcross F₂ animals indicated that the locus controlling the IgE allotype is linked to the Igh-1 locus. This was further confirmed by the possession of respective IgE allotypes by Igh-C congenic mice, BALB/c and BAB-14, C3H.SW/Hz and CWB/Hz. Thus, the allotype detected on the chain is controlled by the seventh murine immunoglobulin allotype locus, and should be designated as the Igh-7 allotype.Abbreviations used in this paper PCA passive cutaneous anaphylaxis - RID radioimmunodiffusion - i.p. intraperitoneally - EA egg albumin - Igh-C immunoglobulin heavy chain constant region locus - DNP 2,4-dinitrophenyl - PBS phosphate-buffered saline - NMS normal mouse serum - KLH keyhole limpet hemocyanin Visiting investigator supported by the Scientific and Humanistic Development Council from the Central University of Venezuela, currently at the following address: Consejo de Desarrollo Cientifico y Universidad Central de Venezuela, Av. Principal Urb. La Floresta Ota., Silenia Caracas, Venezuela.     

MSI and LOH in the development and prognosis of follicular cell-derived thyroid tumours

Microsatellite instability (MSI) and loss of heterozygosity (LOH) represent molecular disorders acquired by the cell during neoplastic transformation. Both are associated with genetic instability. Functional silencing of tumour suppressor genes may be the consequence of genomic instability, particularly of the globally occurring LOH phenomenon. Numerous studies have confirmed the role of MSI/LOH at both the early and the late stages of thyroid tumourigenesis. This paper reviews the available study results on MSI/LOH significance and prevalence in thyroid neoplasms. Additionally, it summarises the knowledge regarding the practical usage of the study findings on MSI/LOH in aspects of cancer risk assessment as well as the development of prognostic markers for thyroid neoplasms.     

Genetic control of the immune response to (T,G)-A--L in C3H in equilibrium C57 tetraparental mice

When 15 C3H ? C57 tetraparental (allophenic) mice were analyzed for coat color, hemoglobin, and immunoglobulin allotype, all but two were shown to be chimeric. These 15 tetraparental mice were immunized with the synthetic polypeptide (T,G)-A--L, and the origin of the (T,G)-A--L-specific antibody produced was determined by using genetic markers (allotypes) on the immunoglobulin heavy chain constant region. Five tetraparental mice were high responders to (T,G)-A--L and had significant amounts of a (low responder) allotype antibody in their total serum. Three of these mice had significant amounts of anti-(T,G)-A--L antibody of the a (low responder) allotype. The antigen binding capacities of the a allotype fractions of these three were 4–5 times higher than the antigen binding capacities of immunized C3H (low responder) control mice. These results are compatible with the hypothesis that the inability of low-responder mice to produce significant amounts of anti-(T,G)-A--L antibody is a function of Ir-1A gene expression at the level of T cells.     

Synthesis of Gm allotypes by human tonsillar lymphocytes in culture: concordance with serum phenotype.

Isolated human tonsillar lymphocytes were cultured with pokeweed mitogen, phytohemagglutinin, and without mitogen for 9 to 28 days. IgK, Gm(a) and Gm(f) were then quantitated in the cell suspensions. In cultures of cells derived from persons whose blood was heterozygous for IgGl allotype antigens Gm(a+f+), approximately equal amounts of Gm(a) and Gm(f) were found. In cultures of cells of Gma or Gmf homozygotes, there was complete concordance between the Gm allotype antigens produced by the cultures and the donor's serum phenotype-with no instance, either at zero time or at culture termination in which a Gm antigen was detected which was absent from the donor's serum. It was concluded that in vitro genetic allotype synthesis in tonsillar lymphocytes during short-term culture mirrored accurately in vivo Gm expression. IgK and Gm antigen synthesis was highest in the flasks containing pokeweed mitogen although both phytohemagglutinin and no-mitogen control flasks showed, in certain experiments, proliferation and an increase in the Ig per viable cell. It was observed that no-mitogen flasks contained twice as much allotype antigen as did phytohemagglutinin flasks suggesting an inhibition of Ig synthesis associated with the mitogen. The tonsillar lymphocytes, under the experimental conditions employed, were shown by a radio-incorporation and immunoprecipitation technique to be synthesizing polyclonal Ig de novo, at the termination of the cultures.     

Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones

Many plant species and/or genotypes are highly recalcitrant to Agrobacterium-mediated genetic transformation, and yet little is known about this phenomenon. Using several Arabidopsis: genotypes/ecotypes, the results of this study indicated that phytohormone pretreatment could overcome this recalcitrance by increasing the transformation rate in the known recalcitrant genotypes. Transient expression of a T-DNA encoded ss-glucuronidase (GUS) gene and stable kanamycin resistance were obtained for the ten ARABIDOPSIS: genotypes tested as well as for the mutant uvh1 (up to 69% of petioles with blue spots and up to 42% resistant calli). Cultivation of Arabidopsis: tissues on phytohormones for 2-8 d before co-cultivation with Agrobacterium tumefaciens significantly increased transient GUS gene expression by 2-11-fold and stable T-DNA integration with petiole explants. Different Arabidopsis ecotypes revealed differences in their susceptibility to Agrobacterium-mediated transformation and in their type of reaction to pre-cultivation (three types of reactions were defined by gathering ecotypes into three groups). The Arabidopsis uvh1 mutant described as defective in a DNA repair system showed slightly lower competence to transformation than did its progenitor Colombia. This reduced transformation competence, however, could be overcome by 4-d pre-culture with phytohormones. The importance of pre-cultivation with phytohormones for genetic transformation is discussed.     

A new cross-reactive idiotype-defined family in the phthalate humoral immune response of mice. I. Linkage of VH-Xmp to IgCH allotype locus and mapping with respect to other known VH genes

A cross-reactive idiotype family was previously identified from a very large library of phthalate-specific hybridoma clones. The prototype of this idiotype family is the hybridoma, 2E9, secreting an IgM antibody with phthalate specificity. A portion of both primary and secondary anti-phthalate antibodies elicited in all BALB/c mice tested expresses the 2E9 cross-reactive idiotype. This idiotype has now been found in the anti-phthalate antibodies of several other inbred strains of mice (A/HeHa, DBA/2, and C3Hf/HeHa) tested but not in C57BL/6 mice. Anti-phthalate antibodies elicited from congenic mice BC.8, which express the same IgCH allotype as BALB/c mice but possess C57BL/6 genetic background, contain the 2E9 cross-reactive idiotype, whereas this idiotype is not expressed on the anti-phthalate antibodies derived from another congenic mouse CB.20, which expresses a C57BL/6 IgCH allotype and a genetic background of the BALB/c strain. These results indicate that the gene controlling the 2E9 idiotype is closely linked to the IgCH allotype locus. The 2E9 cross-reactive idiotype was also found in all of the F1 mice (BALB/c X C57BL/6) tested, and the level of expression of this idiotype in the F1 mice was quantitatively equivalent to the allotype/idiotype homozygous mice. The expression of the 2E9 idiotype in the phthalate repertoire has been followed in 12 different wild mouse populations. As expected, the 2E9 idiotype was observed in a large proportion of the wild mouse strains. Surprisingly, several examples of nonconcordance in the expression of idiotype and allotype were observed in these mice. One likely explanation for the linkage breakdown is a crossing over of the heavy chain constant and variable region gene complexes. In the SM/J inbred strain of mice, where such a crossover has occurred, nonconcordance between allotype and 2E9 idiotype expression was demonstrated. By using the recombinant inbred BXD strains of mice, the VH gene encoding the 2E9 idiotype has been mapped with respect to other known VH gene families. Relative to other VH genes the VH-Xmp is situated very close to the IgCH gene region.     

Immunogenetics of immunoglobulins of the American mink. VI. Deviation from Mendelian segregation for Cgamma-allotypes H2, H3 and H4

The non-expression of parental allotype among mink F1 offsprings of monohybrid analyzing crosses was established for C gamma-allotypes. A phenotype ratio 0:1, instead of the expected Mendelian 1:1, was observed in some families. The non-expression of allotype may occur in the progeny of mink with normal allotypic specificity. Deviations of allotypic expression had both qualitative and quantitative nature and did not depend on the direction of crossings. Sometimes, the appearance of allotype in a mink could not be expected on the basis of its pedigree. Instability of expression and inheritance of H2, H3 and H4 in many families may have masked the allelic or linkage relationships of these genetic markers.     

Genetic Variability of Flight Metabolism in DROSOPHILA MELANOGASTER . III. Effects of Gpdh Allozymes and Environmental Temperature on Power Output

The effect of allozyme variation at the sn-glycerol-3-phosphate dehydrogenase (Gpdh) locus on variation in the mechanical power output of the flight muscles of Drosophila melanogaster was investigated. The influence of different rearing and flight temperatures and of their interactions with the Gpdh allozymic genotypes (allotypes) on flight ability also were analyzed. Populations from three continents were used, and Gpdh allotypes were generated from crosses between randomly paired isofemale lines made autozygous for each of the two alleles by inbreeding. Measurements made during tethered flight, together with wing morphology, were used to estimate power output using both Weis-Fogh's and Ellington's formulas. Analyses of variance (ANOVA) indicated significant main effects for both environmental components (rearing and flight temperatures) but for only one of the three genetic components (genetic backgrounds within continent); Gpdh allotypes and populations (continent of origin) were not significant. The interaction between rearing and flight temperature was highly significant, indicating some physiological adaptation. The effect of Gpdh allozymes depended on both rearing and flight temperature and was either significant or marginally so, depending on which set of formulas was used. In either case, the S/S allotype showed a 2-4% greater power output than the F/F allotype at low temperature for both interactions. In addition, the S/S allotype showed significantly greater power output than the F/F allotype among flies raised at 15 degrees and flown at 15 degrees, whereas the reverse was true for flies raised at 30 degrees and flown at 30 degrees. Significant differences among the three allotypes for GPDH activity level were found in general, with S/S having the highest, F/S intermediate and F/F the lowest activity, and an inverse relationship existed between rearing temperature and activity. The temperature effects on power output are consistent with the geographical and seasonal variation observed at the Gpdh locus in nature. In general, the results show that Gpdh can be considered a minor polygene affecting quantitative variation in the power output during flight and that genotype-by-environment interaction is an important component of that effect.     

The chemical characterization of the gene: vicissitudes of evidential assessment

The chemical characterization of the substance responsible for the phenomenon of "transformation" of pneumococci was presented in the now famous 1944 paper by Avery, MacLeod, and McCarty. Reception of this work was mixed. Although interpreting their results as evidence that deoxyribonucleic acid (DNA) is the molecule responsible for genetic changes was, at the time, controversial, this paper has been retrospectively celebrated as providing such evidence. The mixed and changing assessment of the evidence presented in the paper was due to the work's interpretive flexibility--the evidence was interpreted in various ways, and such interpretations were justified given the neophytic state of molecular biology and methodological limitations of Avery's transformation studies. I argue that the changing context in which the evidence presented by Avery's group was interpreted partly explains the vicissitudes of the assessments of the evidence. Two less compelling explanations of the reception are a myth-making account and an appeal to the wartime historical context of its publication.     

Genetic transformation and cell morphology of Bacillus subtilis grown in Mg+(+)-limited chemostat culture

The rate and frequency of genetic transformation of Bacillus subtilis grown in Mg+(+)-limited chemostat culture are dependent on the dilution rate (D) of the system and achieved maximum values at D = 0.23 h-1. Mg+(+)-limitation induced a morphological change in the cells from their normal rod shape to extended helices. Although this change in shape was a transient phenomenon, under some conditions it persisted for several days and resulted in an apparent increase in the transformation frequency.     

Quantitative analysis of idiotypic mimicry and allelic exclusion in mice with a mu Ig transgene

Analysis of C57BL/6 mice (IgM allotype, Igh-6b or mu b) that carry an Ig H chain transgene of a different allotype (mu a) shows that IgM molecules of mixed allotype (mu a mu b) are present among serum antibodies. The finding was extended to hybridomas prepared from nonimmune transgenic mice, many of which also failed to exhibit allelic exclusion. The proportions of mu a and mu b secreted by individual hybridomas varied markedly, and the product of an individual hybridoma was found to be heterogeneous with respect to the allotype content of individual molecules. The ratio of mu a:mu b chains secreted by individual hybridomas was found to correlate with the number of transgene copies remaining in each hybridoma, and several hybridomas that secrete only mu b-positive molecules had apparently lost all but one copy of the transgene. An idiotype characteristic of the transgene was found to be present only in association with the transgenic (mu a) allotype, and indirect evidence strongly suggests that the idiotype was present only on mu a polypeptide chains. Thus, there is no evidence in this system for the induction of idiotypically cross-reactive endogenous molecules.     

Rapid stimulation of large specific antibody responses with conjugates of antigen and anti-IgD antibody

Injection of mice with goat anti-mouse IgD antibody stimulates a large IgG1 anti-goat IgG antibody response, as well as polyclonal IgG1 production. To determine if this phenomenon could be used to induce large antibody responses to other Ag, covalent conjugates were produced between BSA or other Ag and H delta a/1, a mAb specific for IgD of the a allotype, and between BSA and AF3.33, a mAb specific for IgD of the b allotype. Injection of H delta a/1-BSA into BALB/c mice, which express Ig of the a allotype, or into (BALB/c x CB20)F1 mice (a x b allotype heterozygotes) induced IgG1 anti-BSA antibody responses that peaked 8 to 9 days after injection, and were more than 1000 times larger than those induced by injection of BSA alone, and 100 times larger than those induced by injecting unconjugated BSA plus H delta a/1. H delta a/1-BSA was no more immunogenic than unconjugated BSA when injected into CB20 mice, which express Ig of the b allotype, while AF3.33-BSA greatly enhanced anti-BSA antibody production in CB20, but not in BALB/c mice. Mice serially immunized with three different Ag conjugated to H delta a/1 made large antibody responses to all three Ag, provided that the mouse strain used did not recognize allotypic determinants on H delta a/1 as foreign and produce a neutralizing antibody response. Intravenous and s.c. routes of inoculation produced responses of similar magnitude and relatively low variability; responses to footpad or intramuscular inoculation were more variable, and i.p. inoculation induced smaller responses. Injection of BALB/c mice i.v. with 100 micrograms of H delta a/1-BSA induced an IgG1 anti-BSA response of 5.6 mg/ml, which was approximately 70% of the total IgG1 response. Anti-BSA responses to 30 micrograms of conjugate or less were much smaller, but could be considerably enhanced by adding unconjugated H delta a/1 to the inoculum. This system will be useful for the rapid stimulation of large antibody responses to biologically important Ag, and for investigating mechanisms of Ag processing and B and T cell activation.     

Immunogenetic study on the polymorphism of serum α2-lipoproteins in mink. IV. Diallelism at the Ld locus of low-density lipoprotein

Antibodies against a new allotype, Ld2, of mink low-density lipoprotein (LDL) were obtained by alloimmunization with a preparation of this lipoprotein. The two known allotypes of LDL, designated Ld1 and Ld2, are coded for by codominant alleles of the autosomal Ld locus. This locus is probably involved in the genetic control of the whole serum pool of LDL molecules. In Ld1/Ld2 heterozygotes, LDL is represented by two homozygous types of molecules, Ld1 and Ld2; it has no hybrid molecules bearing both allotypic specificities together. The results suggest that the Ld locus has, presumably, only two alleles in the mink populations studies. Mink LDL having allotypes Ld1 and Ld2 was found to be homologous to human and pig LDLs. Antigenic specificity of Ld1 allotype was established in the sera of a wide phylogenetic range of mammals and in the human LDL. The parallelism between the phylogenetic antiquity of the Ld1 gene and its high frequency in mink and other species may be attributed to the selective value of this gene, which has been retained unaltered during macroevolution.     

Diversity of wild and domestic pig populations estimated by a set of serum allotypes

Variation of serum protein allotypes serving as genetic markers of the blood has been analyzed in 29 populations of the domestic pig and subspecies of the wild boar. The population biodiversity and genetic structure have been estimated by two methods: by the frequencies of allotype combinations and with the use of a map constructed in the space of two principal components. The results obtained are the basis for determining the characteristics of the microevolution of wild boars and formation of the breeds of domestic pigs.     

Diversity of wild and domestic pig populations estimated by a set of serum allotypes

Variation of serum protein allotypes serving as genetic markers of the blood has been analyzed in 29 populations of the domestic pig and subspecies of the wild boar. The population biodiversity and genetic structure have been estimated by two methods: by the frequencies of allotype combinations and with the use of a map constructed in the space of two principal components. The results obtained are the basis for determining the characteristics of the microevolution of wild boars and formation of the breeds of domestic pigs.     

Genetic control of the immune response to staphylococcal nuclease. IX. Recombination between genes determining BALB/c antinuclease idiotypes and the heavy chain allotype locus.

The genetic linkage relationship of two antinuclease idiotypes produced by the BALB/c strain was investigated in the backcross (BALB/c x CB.20) X CB.20. These two idiotypes were detected by Lewis rat anti-idiotypic antisera prepared against affinity-purified A/J and SJL antinuclease antibodies, termed the A/J and SJL idiotypes, respectively. Both idiotypes were found to be linked to the IgCHa immunoglobulin heavy chain allotype locus. There was, however, a high frequency of recombination observed between both markers and the IgCHa locus, with eight of 83 backcross animals recombinant for the A/J idiotype and five of 83 recombinant for the SJL idiotype. All such recombinant animals were IgCHb/b homozygotes that had gained one or both idiotypes. These results are consistent with a genetic map of VHr region genes in the BALB/c strain in which genes determining the SJL idiotype are closer to the IgCHa allotype locus than are genes determining the A/J idiotype. This high frequency of recombination may indicate that the chromosome segment containing VH region genes is very large or that it has structural features that promote recombination.