Homozygous Strains Research Articles

Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice

Irradiated MHC-heterozygous mice often reject bone marrow cells transplanted from one of the homozygous parental strains, a phenomenon ('hybrid resistance') that appears to violate the laws of transplantation. Rejection of parental and allogeneic marrow cells also differs from conventional T cell-mediated rejection mechanisms as it is effected by NK1.1+ cells. To account for the unusual specificity of bone marrow rejection, it has been proposed that NK1.1+ cells destroy marrow cells that fail to express the full complement of self MHC class I (MHC-I) molecules. We show here that NK1.1+ cells in normal mice reject haemopoietic transplants from mice that are deficient for normal cell-surface MHC-I expression because of a targeted mutation in the beta 2-microglobulin gene. These findings demonstrate that deficient expression of MHC-I molecules renders marrow cells susceptible to rejection.

Gynogenesis in common carp ( Cyprinus carpio L.) : II. The production of homozygous gynogenetic clones and F 1 hybrids

Homozygous gynogenetic fry of common carp ( Cyprinus carpio L.) were produced by heat-shocking eggs, activated with UV-irradiated sperm (1 : 3 diluted, 10 ml, at 2200 J m −2 min −1) during metaphase of the first mitosis. Consistent yields of 5–15% viable, gynogenetic fry were obtained when eggs were shocked at 40°C for 2 min, 28–30 min after fertilization. The homozygous nature of the gynogenetic fry was demonstrated by the mendelian segregation patterns of three recessive mutant pigment genes. Homozygous inbred strains were produced by heterozygous gynogenetic reproduction (second polar body retention) of homozygous gynogenetic females, while F 1 hybrids were produced by crossing these females with homozygous gynogenetic male siblings. The clonal nature of these strains was unequivocally demonstrated by the acceptance of reciprocally exchanged skin allografts.

Structure of miniature swine class II DRB genes: conservation of hypervariable amino acid residues between distantly related mammalian species.

As part of our studies of the class II genes of miniature swine, we have isolated and characterized cDNA clones corresponding to DRB genes from two major histocompatibility complex homozygous strains. Comparison of the sequences of these clones to those of human DRB genes revealed a striking amino acid homology between the hypervariable residues of SLA-DRBc and the human DRB1-0101 allele. The percentage of differences in these residues between the pig DRBc allele and the human DRB1-0101 allele was significantly lower (29%) than that between the DRB1-0101 allele and all other human alleles (average, 66.2%). This similarity was not seen in a comparison of the number of silent substitutions, by which the swine DRBc and the human DRB-0101 differed. Since phenotypic selection operates at the level of protein products rather than nucleotide sequences, these data suggest the existence of selective mechanisms that have resulted in similar hypervariable regions in certain alleles even in these widely disparate species. Consistent with this hypothesis, an examination of available murine and bovine class II sequences revealed a homology in hypervariable residues between the human DRB1-1401 allele and the mouse E beta s allele as well as a cow DRB allele. Consideration of these data along with intraspecies allelic sequence comparisons suggests that at least some of the interspecies similarities have emerged as the result of convergent evolution, possibly as the result of a need to react to common pathogens.

Novel sterile mutants of the fission yeast Schizosaccharomyces pombe which are defective in their response to starvation

The mating reaction in the fission yeast, Schizosaccharomyces pombe, is induced by nitrogen deprivation. In order to understand the genetic control of cellular responses to starvation, sterile mutants defective in the mating reaction were isolated and analyzed. Tetrad analysis and complementation tests of ethyl methanesulfonate-induced Ste- mutants revealed three novel ste loci, designated ste11, ste12 and ste13. These genes were mapped on chromosome II. ste11 was allelic to stex; ste12 was tightly linked to ste2 (0.8 cM). Homozygous diploid strains harboring either of these ste alleles were defective in meiosis. Vegetative cells of the ste11 mutant showed the normal resting state in stationary phase. In contrast, the ste12 and ste13 mutant cells rapidly lost viability after nutrient deprivation and the latter were sensitive to heat shock in stationary phase. The transcript level of the mat1-Pi and mei2 genes was greatly reduced in these mutants. These results indicate that the ste12 and ste13 genes are responsible not only for entry into sexual reproduction but also for entry into the G0 phase.

Tolerance to 1-pentene-3-ol and to 1-pentene-3-one in relation to alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities inDrosophila melanogaster

The detoxification of 1-pentene-3-ol (pentenol) and 1-pentene-3-one (pentenone) by Drosophila melanogaster adult flies has been studied in two homozygous lines for the AdhF and AdhS alleles (LRC lines), in their respective lines selected for tolerance to ethanol (LRSe lines) and in a homozygous strain for the Adhn4 null allele. For each line, the genotype and sex LDs50 of both compounds were estimated. Then, in order to explain the differences in LD50, both alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities were assayed. In addition, the effects of pentenone on AKR activity were also studied. Our results show that ADH-positive flies exhibit a much higher sensitivity to pentenol than ADH-null flies. However, both ADH-positive and ADH-null flies show a similar tolerance to pentenone. Our results show that flies selected for improving tolerance to ethanol also have increased tolerance to pentenol (FF and SS flies) and pentenone (SS flies). However, this improved ability to tolerate pentenol and/or pentenone cannot be explained by changes in ADH or AKR activities. On the other hand, we have observed a beneficial effect of pentenol, but not of pentenone, in n4 flies. We also show that AKR activity is not modified by the administration of pentenone. These results suggest that, in the absence of ADH activity, pentenol may be transformed into a compound that is less toxic than pentenone and that pentenone itself might also be transformed into a less toxic compound.

A new class of S sequences defined by a pollen recessive self-incompatibility allele ofBrassica oleracea

Self-incompatibility in Brassica oleracea is controlled by a single genetic locus (the S locus) with nearly 50 different alleles. In this paper, we report the characterization of the S2 allele, a pollen recessive self-incompatibility allele that exhibits weak DNA homology to the other previously sequenced S locus glycoprotein genes (SLG-6, -13, -14, -22 from alleles S6, S13, S14 and S22, respectively). Stigma cDNA clones with sequence homology to SLG-13 were isolated from two different S2 homozygous strains belonging to two different B. oleracea cultivars, var. alboglabra (Chinese kale) and var. italica (broccoli). The two S2 cDNA sequences are 90% homologous to each other, but only 70% homologous to SLG-13. Using the Chinese kale S2 genetic background, we demonstrate that the isolated alboglabra cDNA sequence is a transcript from a gene, designated SLG-2A that resides at the S locus, and propose that it is a putative determinant of S2 allelic specificity. Among the estimated 10-15 genomic copies of SLG-related genes detected in the S2 genome, we cloned and characterized the SLG-2A gene and another closely related and genetically linked gene copy, SLG-2B. A complete open reading frame that is 94% homologous to SLG-2A is located within SLG-2B. The existence of this intact duplicated S gene raises the possibility that SLG-2B may also be involved in the functioning of self-incompatibility in Brassica oleracea.

Centromeric regions control autonomous segregation tendencies in single-division meiosis of Saccharomyces cerevisiae.

We have previously shown that yeast cdc5 or cdc14 homozygotes can be led through a single-division meiosis in which some of the chromosomes segregate reductionally whereas others, within the same cell, segregate equationally. Chromosomes XI tend to segregate reductionally, whereas chromosomes IV tend to segregate equationally. In this report we present experiments with cdc5 homozygous strains, in which the centromeres of one or both chromosomes XI was replaced by the centromeric region from chromosome IV. Analysis of the products of single-division meioses in these strains demonstrates that the choice between reductional or equational segregation is directed by sequences in the vicinity of the centromeres. Although the choice is made separately for each individual chromosome, the analysis also reveals the existence of a system responsible for coordinated segregation of the two chromosomes of a given pair.

Cerebellar phenotype of two alleles of the ‘reeler’ mutation on similar backgrounds

Using congenic strains, the two alleles (‘Ed’ and ‘Orl’) of the reeler mutation in mice have been compared on two genetic backgrounds (B6 and C). The phenotypic differences in cerebellar organization between the 4 homozygous reeler mutant strains appeared related to the genetic background rather than to allelic differences.

SKIN GRAFTING IN GYNOGENETIC COMMON CARP (Cyprinus carpio, L.)

The fate of skin allografts exchanged among heterozygous and homozygous gynogenetic common carp siblings, and among newly developed inbred strains and F1 hybrids, is described. Heterozygous gynogenetic offspring were produced by fertilizing eggs with UV-irradiated sperm and by treating the resulting zygote with a cold shock (0 degree C, 45 min). The temperature shock causes retention of the second polar body, which allows the eggs to develop into normal diploid fry. Homozygous gynogenetic offspring were similarly produced by using a heat shock (40 degrees C, 2 min), which suppresses the first mitotic division. Skin allografts exchanged among heterozygous gynogenetic carp exhibited prolonged survival, with some allografts (21.8%) surviving for over 28 days. Furthermore, a strong histocompatibility locus was seen to segregate in this group. In contrast, skin allografts exchanged among homozygous gynogenetic siblings were all rejected within 14 days (MST 9.4 days). New homozygous inbred strains, designated JJ and MM, were produced by gynogenetic reproduction of homozygous female carps, while F1 hybrids were produced by crossing of these homozygous females with homozygous male siblings. All grafts exchanged among members of the same strain were permanently accepted. Likewise grafts from homozygous strain members were accepted by fish from the related F1-hybrids, while the reverse grafts were rejected. These results provide evidence for the idea that in the carp histocompatibility genes exist at least one major locus and multiple minor loci, which are codominantly expressed.

Purification and properties of an esterase from organophosphate-resistant strain of the mosquito Culex quinquefasciatus

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.

Relative fitness of a malathion-resistant strain of Cryptolestes ferrugineus (Coleoptera: Cucujidae) when development and oviposition occur in malathion-treated and untreated wheat kernels

Malathion resistance in an English strain of Cryptolestes ferrugineus (Stephens) was triphenyl phosphate suppressible, autosomal, monofactorial, and semi-dominant. The resistant strain (RR) was crossed with a susceptible strain (SS) and heterozygotes were backcrossed to the SS strain for 6 generations then reselected on malathion-treated feed to produce another homozygous resistant strain (BC 6RR) with most of the genome derived from the SS strain. Intrinsic rates of increase on whole-kernel wheat at 30°C, 75% r.h. were similar for the SS strain, the F 1 reciprocal hybrids and the BC 6RR strain but 22–28% lower for the original RR strain. On wheat kernels treated with 4, 8 and 16 ppm malathion, the intrinsic rates of increase of the BC 6RR strain and the heterozygous strains were equal to or greater than the intrinsic rates of increase of the RR strain. Therefore, lower fitness of the RR strain relative to the SS strain could not be attributed to the R gene itself. The tendency of larvae to wander from the germ of the wheat kernel (treated or untreated) in which they had emerged from the egg was greatly increased in the presence of the R allele but only when it was homozygous.

Chemical communication of emigration behavior of Drosophila melanogaster. I. Strain difference.

Experiments were conducted with six homozygous strains of D. melanogaster belonging to a population from Matsuyama, Ehime Prefecture, in order to examine whether or not a substance(s) secreted by one strain affects the emigration behavior of another strain. From the results of experiments in which flies of one strain in a central tube were discarded and those of another strain were newly introducted, it appeared that in some combinations emigration behavior of the latter strain was influenced by a substance(s) secreted by the former strain. The result suggests that the behavior should be under the genetic control, because the differential responses to the substance secreted by fruit flies were observed among homozygous strains. Bioassay experiments were performed to detect the biologically active substance extracted from adult flies using two strains of the population. The results of bioassay with materials obtained from several steps of separation suggested that the substance may be a kind of fatty acid and different among strains.

The influence of the swine major histocompatibility genes (SLA) on variation in serum immunoglobulin (Ig) concentration

Variation in serum IgG and IgM concentration was determined in three homozygous SLA-defined strains of miniature swine (SLA a, SLA c and SLA d) and one recombinant strain SLA g (AB cD d) as part of a study of SLA and other genetic effects on immune response. Data were obtained from 119 8-week-old piglets from 29 litters by 12 sires and analyzed using a SAS linear model for the effects of SLA haplotype, sire, dam, litter, sex, season of birth and sow parity. SLA haplotype ( P < 0.10) and other genetic effects due to sire ( P ≤ 0.001) and dam ( P ≤ 0.002) contributed to the variation in serum IgG concentrations. Season of birth and sow parity also affected IgG concentration as did litter effects. Least squares mean comparisons indicated that pigs of the dd, dg and gg haplotypes had significantly higher serum IgG than did pigs of the other haplotypes. Heritability estimates for IgG, calculated by paternal half-sib correlation, ranged from 0.31 to 0.27, indicating that selection for increased serum IgG concentrations would be possible. For serum IgM concentrations, only the effect of litter was significant at P ≤ 0.001 and P ≤ 0.009 by the radial immunodiffusion test read at 24 or 48 h. Since sire variance components estimates were negative, heritabilities were not calculated for IgM and are assumed to be zero.

Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae.

A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.

The Brassica self-incompatibility multigene family

The pollen–stigma interaction of self-incompatibility in Brassica species is controlled by a single genetic locus, the S locus. This locus encodes the S locus specific glycoproteins of the stigma. Hybridization of restriction enzyme digested genomic DNA isolated from homozygous strains of Brassica with cDNA probes encoding these glycoproteins yields a pattern of multiple fragments of varying size and intensity. The presence of S sequences as multiple related copies in the Brassica oleracea genome has been verified by the cloning of several different genomic regions containing S homology. Probes capable of distinguishing between S gene copies have been obtained and have demonstrated that two such copies are expressed. Characterization of additional probes specific for S gene copies will permit the study of the expression of the other copies, and the analysis of the genomic organization of the self-incompatibility multigene family.Key words: self-incompatibility, Brassica, cDNA probes.

DNA rearrangements associated with the H3 surface antigen gene of Tetrahymena thermophila that occur during macronuclear development.

The surfaces of Tetrahymena thermophila cells grown between 20 and 35 degrees C are covered by one or more variants of H antigens. A cDNA clone, pC6, has previously been identified that hybridizes to a unique polyA+ RNA that appears to code for the SerH3 variant of the H antigens. pC6 and a subclone of it, pGpC6.295, were used to analyze the genomic organization of the corresponding gene(s) in both the macronucleus and the micronucleus. It was determined that pC6 hybridizes to a small family of sequences in the macronucleus, only one of which also hybridizes to pGpC6.295. The latter is a strong candidate for the gene encoding the SerH3 antigen. Sequences homologous to pC6 - but not to pGpC6.295 - are present in strains carrying the other SerH alleles. Shifts in antigen switching during vegetative growth do not result in any detectable DNA rearrangements in the vicinity of the pC6-hybridizing sequence family. Analysis of micronuclear DNA from a homozygous SerH3 strain revealed that it also contains a family of sequences that are homologous to pC6; but, in contrast to the macronuclear DNA, two members of this micronuclear sequence family hybridize to pGpC6.295. Comparison of micro- and macronuclear DNA indicate that some members of the pC6-positive sequence family rearrange during macronuclear development. These rearrangements fall into two classes: those which occur reproducibly, and those which show variability. The gene homologous to pGp6.295 falls into the former category.

Production and characterization of radiation-sensitive meiotic mutants of Coprinus cinereus.

We have isolated four gamma-ray-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1; rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the few viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants.

Activation properties of T cell receptor-gamma delta hybridomas expressing diversity in both gamma- and delta-chains.

To elucidate the structure, diversity, and activation properties of the murine T3-associated gamma delta-receptor, examination was made of the gamma delta and T3 components, T cell receptor (TCR) gene transcription, activation properties, and lymphokine production in a panel of four cloned T cell hybridomas expressing a TCR-gamma delta. Biochemical analysis of the gamma and delta proteins expressed on these hybridomas reveals new gamma and delta species not observed in whole populations of dLy-1 Lyt-2-L3T4-thymocytes from which these hybridomas were derived. Thus, analysis of expression of the TCR-gamma delta complex at the clonal level indicates that both gamma and delta appear to be expressed as multiple distinct gene products within a homozygous inbred mouse strain. Northern blot analysis reveals that, whereas all gamma delta hybridomas had mature 1.5-kb TCR-alpha-chain and mature TCR-gamma-chain transcripts, none had mature 1.3-kb TCR-beta-chain transcripts, thus indicating that the type of TCR heterodimer expressed reflects of the state of TCR gene transcription in these hybridomas. Our results also reveal striking similarities between TCR-gamma delta and TCR-alpha beta cells with respect to their activation properties. First, all five of the T3 components associated with the gamma delta-complex are of the same size and have the same glycosylation patterns as those associated with alpha beta-heterodimers. Second, induction of function in these gamma delta cells (assayed by lymphokine production) can be achieved with a variety of stimuli known to elicit activation signals in alpha beta cells as well: direct receptor-engagement (i.e., through anti-Thy-1), and phorbol 12-myristate 13-acetate-plus-ionomycin-mediated. Collectively, these findings suggest that gamma delta T cells express a receptor of at least limited diversity and use T3-mediated activation pathways very similar to those employed by TCR-alpha beta-bearing T cells.

Low molecular weight thiol content in glutathione synthetase-deficient human fibroblasts

The activity and the kinetic properties of glutathione synthetase and the concentrations of non-protein bound thiols of the γ-glutamyl cycle were measured in 11 human fibroblast cell strains. Six of these strains were derived from patients suffering from 5-oxoprolinuria, a recessive genetic disease characterized by a deficiency in glutathione synthetase; the other cell strains were derived from healthy heterozygous or homozygous relatives of the patients. The glutathione synthetase activities of homozygous deficient strains were 1/3 of control values while those of heterozygous strains were 2/3 of control values. The total thiol concentration was lower in only 3 of the 6 deficient homozygotes and that of glutathione (GSH) was lower in only 4 of the 6 deficient homozygotes. This lower GSH level was at least partly offset by an accumulation of γ-glutamylcysteine, a precursor of GSH, which is almost completely absent from control cells. The total quantities of thiols and GSH in plateau phase cells were about 50% and 30% respectively of the levels in growth phase cells. Approximately 80% of the GSH was in the reduced form in both quiescent and growing cells.

Genetic Analysis of Antheridiogen Sensitivity in Ceratopteris richardii

The pheromone antheridiogen promoted the production of males in gametophyte populations of the fern Ceratopteris richardii. Two homozygous strains of this species exhibited markedly different sensitivities to the pheromone, resulting in populations with either high or low frequencies of males. The two strains were characterized by dose responses to antheridiogen and by gametophyte population density studies to investigate the effect of antheridiogen produced within cultures. Analyses of Fl hybrids and an F2 generation indicated that differences in the antheridiogen responses of the two parental strains are associated with either one or two nuclear genes.