H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

The rates of emergence of H-2^b– and H-2^d– variants from an H-2^b+/ H-2^d+ heterozygous mouse leukemia cell line were determined by fluctuation analyses. The results show that the wild type cells throw off the H-2^b– variants at about 10^–5 per cell per generation and the H-2^d– variants at 10^–6 per cell per generation. The reason for this tenfold difference in the rates for the two symmetrical variants is not clear. The fluctuation analyses also indicate that the variants exist in the wild type population prior to exposure to the selective agents.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An (H-2k/H-2d)F1 sarcoma cell line was subjected to immunoselection using ascites fluid from a mouse growing a hybridoma secreting an anti H-2Kk antibody.One hundred random clones were picked from the surviving population and screened by direct cytolysis using the hybridoma antibody or alloantisera against H-2Kk and H-2Dk. Fifty-nine clones were resistant to all three antisera, indicating that they no longer expressed the entire H-2k haplotype. Thirty-two were resistant to the ascites and to the anti H-2Kk alloantiserum, but sensitive to the anti H-2Dk serum, indicating that they had lost H-2Kk antigen, but retained H-2Dk. Nine clones were sensitive to the alloantisera, but resistant to the hybridoma, indicating that, though they retained the product(s) recognized by the alloantiserum against H-2Kk, they had lost the site(s) that bound the hybridoma antibody. Quantitative absorption assays using lymph-node cells from young BALB.K (H-2Kk) mice as targets show that one representative clone from the last group absorbs the anti H-2Kk activity in the alloantiserum. This implies that the sensitivity of the variant clone to the alloantiserum is not due to contaminating anti C-type virus antibodies in the serum. The possible implications of these data are discussed.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

In previous studies on the emergence of H-2 antigen loss variants from an H-2^b/H-2^d heterozygous mouse leukemia cell line, it has been shown that stable variants could be obtained after a single-step selection procedure with antibody and complement. Selection against the K-end alleles revealed an asymmetry in the types of variant obtained. This paper deals with the results of selection against theD-end alleles. Once again, an asymmetry is noticed. Selection against the H-2D^d gene product results in the isolation of stable variants that have lost H-2D^d, either alone or in combination with the linked H-2K^d antigens. Selection against H-2D^b, on the other hand, has not, so far, resulted in the isolation of any stable variants. These experiments have not demonstrated unequivocally the mechanism by which these H-2 antigen loss variants are generated. However, certain preliminary considerations suggest the possibility that these variants may not, in fact, be true genetic mutants. The alternative -that these variants arose through some nongenetic, but stable mechanism — must be considered seriously.     

H-2 Antigen variants in a cultured heterozygous mouse leukemia cell line

H-2 antigen variants, derived from a heterozygous mouse Friend leukemia cell line by selection with anti-H-2 antisera and complement, were tested for susceptibility to cell-mediated cytolysis, using T-lymphocytes directed against individual H-2 antigens. The cytotoxic cells were generated in the BALB and B10 backgrounds by a combination of in vivo and in vitro immunizations. The phenotypes of the variants inferred from the patterns of resistance or susceptibility to CML were consistent with those presumed from earlier assays using antisera. The one exception was the variant cell line which was H-2D(d-) in assays using antisera, but was H-2D(d+) by CML.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An H-2 antigen variant, referred to as ?4 + 31 clone 1, was selected by its resistance to an anti-H-2D^d antiserum (BALB.G anti-BALB/c.H-2 ^g antiH-2 ^d ). When tested by cell-mediated cytolysis, this variant was found to be sensitive to cytolytic T lymphocytes raised in the same donor-host combination as that used in raising the antiserum. Further CML characterization of this variant, reported here, indicates that the cell line is in fact resistant to anti-H-2D^d killer cells raised in a more restricted immunization, viz. BALB.G anti-BALB/cH-2 ^db ,H-2 ^g anti-H-2 ^db . It is, however, sensitive to cytolytic cells raised in (BALB.B xBALB/c-H-2db) F¹ H-2 ^b /H-2 ^db ) against the BALB/c strain. These results suggest that the variant does not express H-2D^d itself, but probably expresses CML target antigens that are missing in theH-2 ^db mutation. This in vitro-isolated variant might thus be the complementary mutation to the in vivoobtainedH-2 ^db mutation.     

Rate and mechanism of generation of beta 2-microglobulin mutants from a heterozygous murine cell line

Mutants that fail to express the B2mb allele can be readily isolated from a B2ma/b heterozygous cell line called R8 by a single step immunoselection protocol using a mAb against beta 2 microglobulin protein in the presence of complement. We present data that demonstrate that the mutants preexist the selection and arise at a rate of about 10(-6)/cell per generation. Southern blot analyses suggest that the mutants have lost the B2mb gene and the linked allele at the Emv-15 proviral integration site telomeric to the B2m locus. Surprisingly, the deletion of the first exon of the B2mb gene that is observed in other cell lines is not found in R8. Possible reasons for this observation are discussed.     

H-2 antigen variants in a cultured mouse leukemia cell line

We have investigated the effect of immune selection against a single gene product on a cultured mouse Friend leukemia cell line. The clonal cell line used is heterozygous at theH-2 complex and expresses theH-2 ^d andH-2 ^b haplotypes. The genes selected against were theH-2K locus alleles. Variants were obtained after a single-step selection using either antiH-2K^b or anti-H-2K^d serum. The phenotypes of the variants obtained showed an interesting asymmetry between the two haplotypes. Selection against theH 2K ^b allele resulted in the isolation of the two expected types of variant-those that had lost only H-2K^b and those that had lost both H-2K^b and the linked H-2D^b. Selection against H-2K^d yielded, exclusively, variants that had lost both the selected antigen and the linked H-2D^d. None of the variants showed an alteration in expression of antigens intrans configuration. Karyotypic analyses of the variants revealed that all the cells had retained both copies of chromosome 17 present in the wild-type cells. The results suggest that the variants did not emerge through chromosome loss.     

Serological analysis of H-2 mutants

Serological absorption experiments showed that mutant B6.M505 (H-2 ^bd ) lacks an H-2K end specificity present in the parental B6 strain. It has a molecular weight of 45,000 dalton, indicating it is an H-2 antigen. It is a private specificity ofK ^b strains, and has been designated H-2.62.     

Segregation of proteinase-negative mutants from heterozygous Candida albicans

The extracellular acidic proteinase (EC 3.4.23.6) produced by Candida albicans has been reported to be a virulence factor. In studying the role of this proteinase in human disease, we determined the optimum conditions for stimulating proteinase production in order to isolate proteinase-negative (Prt-) mutants. We found that in liquid medium containing bovine serum albumin (BSA) as the sole nitrogen source, at pH 4 and 27 degrees C, the sensitivity of proteinase detection was considerably greater than when assayed on BSA agar at 37 degrees C. This observation is due, in part, to temperature sensitivity of proteinase induction. Nitrogen starvation did not induce proteinase. Proteinase production on agar was increased by adding 0.01% yeast extract (YE) to BSA medium. Using BSA + YE agar to isolate mutants, it was discovered that C. albicans ATCC 28366 was heterozygous for a Prt- mutation. Spontaneous Prt- mutants occurred at a frequency of 2 x 10(-3). Ultraviolet light increased the mitotic segregation of Prt- cells to a frequency of 1 x 10(-2). The Prt- phenotype showed a large inoculum effect, Prt- segregants reverted with a high frequency, and the revertants were unstable.     

Spontaneous loss and subsequent stimulation ofH-2 expression in clones of a heterozygous lymphoma cell line

The expression of H-2K^k antigens in a (C3H × DBA/2)F₁ lymphoma cell line growing in vitro was investigated with monoclonal antibodies specific for a public antigen of theH-2K ^k region (H-2.m3) in fluorescence analysis and microcytotoxicity assays and in cell-mediated cytotoxicity with allogeneically stimulated effector cells. Estimates of relative levels of H-2K^k-antigen expression obtained by the different methods were highly correlated. The uncloned, unselected population gradually lost H-2K^k surface antigen expression under culture conditions. This was due to the appearance of H-2K^k negative variants. Fifteen cloned sublines of a population enriched for cells expressing antigen H-2.m3 in the fluorescence activated cell sorter contained either two distinct populations, one consisting of H-2.m3 negative and one of H-2.m3 positive cells, or consisted of H-2.m3 negative cells only. The expression of the H-2.m3 determinant of H-2K^k paralleled that of other serological H-2K^k determinants and of H-2K^k target determinants for cell-mediated cytotoxicity. In nearly all clones where two populations could be detected, the proportion of H-2.m3 negative cells increased with time in culture. The amounts of H-2K^k antigen expressed by the clones appeared not to be correlated to the amounts of H-2D^k antigens on the cell surface as judged by cell-mediated cytotoxicity.In at least one clone and in the uncloned population, H-2K^k-antigen expression detectable by fluorescence analysis could be stimulated by growing the cells in the peritoneal cavities of (C3H × DBA/2)F₁ mice or by adding mouse interferon preparations to the cell cultures. The increase in susceptibility to cell-mediated lympholysis of cells grown in vivo paralleled the increase inH-2 expression detected by fluorescence. In contrast, cells growing in the presence of interferon in vitro showed reduced sensitivity to lysis by alloreactive lymphocytes, although H-2 antigens were strongly expressed as measured by fluorescence.     

Intra-H-2 recombination in the mouse. III. I-Region characterization of haplotypes H-2at2, H-2at3 and H-2a2

Serological characterization of three K-S interval recombinant strains, TBR2 (H-2at2), TBR3 (H-2at3) and AIR 1 (H-2a2) was performed using anti-H-2, Ia, Ss and Slp antisera. The data presented here reveal that the crossover events in both TBR2 and TBR3 occurred between the I-A and I-E subregions. In both cases, the H-2K and I-A subregions were derived fron the H-2t1 of chromosome, while the I-E, S and H-2D regions were derived from the H-2b chromosome (KsAkEbSbDb). The H-2a2 chromosome resulted from a crossover event between the H-2a1 and H-2i9 chromosomes. Ia and Ss typing of AIR 1 suggested that the K to I-E regions originated from H-2a1 and the S and D regions originated from H-2i9 (KkAkEkSbDd).     

Generation and characterization of a conditionally immortalized lung clara cell line from the H-2Kb-tsA58 transgenic mouse

The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.     

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5′ and 3′ deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the β-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9×10⁻⁴, whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8×10⁻⁴. The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.     

The role of H-2 in T cell recognition of Mls

The role of H-2 was evaluated in T cell recognition of Mls-encoded antigens during primary mixed lymphocyte responses (MLR). Mlsc was used as a stimulating determinant in MLR and its recognition by T cells was assessed by linear regression analysis under culture conditions in which (A x B)F1 responder cell number was the factor limiting total response. Results of such experiments indicated the presence of distinct (A x B)F1 responder T cell subpopulations capable of differentially recognizing the foreign Mls antigen in association with one or the other parental H-2 haplotype. These findings demonstrate that T cells do not recognize Mlsc products in isolation, but rather are restricted to recognition of Mlsc in the context of "self" H-2 determinants.     

Segregation of recessive phenotypes in somatic cell hybrids: role of mitotic recombination, gene inactivation, and chromosome nondisjunction.

Somatic cell hybrids heterozygous at the emetine resistance locus (emtr/emt+) or the chromate resistance locus (chrr/chr+) are known to segregate the recessive drug resistance phenotype at high frequency. We have examined mechanisms of segregation in Chinese hamster cell hybrids heterozygous at these two loci, both of which map to the long arm of Chinese hamster chromosome 2. To follow the fate of chromosomal arms through the segregation process, our hybrids were also heterozygous at the mtx (methotrexate resistance) locus on the short arm of chromosome 2 and carried cytogenetically marked chromosomes with either a short-arm deletion (2p-) or a long-arm addition (2q+). Karyotype and phenotype analysis of emetine- or chromate-resistant segregants from such hybrids allowed us to distinguish four potential segregation mechanisms: (i) loss of the emt+- or chr+-bearing chromosome; (ii) mitotic recombination between the centromere and the emt or chr loci, giving rise to homozygous resistant segregants; (iii) inactivation of the emt+ or chr+ alleles; and (iv) loss of the emt+- or chr+-bearing chromosome with duplication of the homologous chromosome carrying the emtr or chrr allele. Of 48 independent segregants examined, only 9 (20%) arose by simple chromosome loss. Two segregants (4%) were consistent with a gene inactivation mechanism, but because of their rarity, other mechanisms such as mutation or submicroscopic deletion could not be excluded. Twenty-one segregants (44%) arose by either mitotic recombination or chromosome loss and duplication; the two mechanisms were not distinguishable in that experiment. Finally, in hybrids allowing these two mechanisms to be distinguished, 15 segregants (31%) arose by chromosome loss and duplication, and none arose by mitotic recombination.     

Genetic demonstration of mitotic recombination in cultured Chinese hamster cell hybrids

Chinese hamster ovary cell hybrids were constructed that are heterozygous for two markers, leuS and emtB, linked to the long arm of chromosome 2. In addition, the chromosome 2 carrying the wild-type leuS and emtB alleles contains, on its short arm, a homogeneously staining region (hsr) in which the gene encoding dihydrofolate reductase (dhfr) is amplified approximately 50-fold. This provides a convenient cytogenetic and biochemical means to distinguish the chromosome 2s from the different parents. Analysis of emetine-resistant segregants isolated from such hybrids identified three distinct classes of segregants. One rare class of segregants loses the wild-type leuS and emtB gene functions on the long arm of the hsr chromosome 2 (H-2) but retains the amplified dhfr genes on the opposite arm. Detailed genetic analysis of two such segregants that did not arise by chromosome loss or deletion revealed that new gene linkage relationships had been established on the H-2 chromosome in each, demonstrating that the segregation events in these cell lines involved mitotic recombination.     

Ionic coupling and mitotic synchrony of siblings in a Drosophila cell line

Following mitosis in many cell lines, siblings remain adjoined in dyads until further cell division. We report here a series of experiments designed to ascertain the nature of this apposition in the embryonic Kc cell line of Drosophila melanogaster. We have found that (1) cell division in siblings is highly synchronized when compared to that in nonsiblings: (2) siblings in dyads are dye coupled with respect to Lucifer Yellow, but intercellular diffusion of larger molecules (FITC-dextran at 6 and 24 kDa) is retarded: (3) siblings are electrically coupled by an ungated low-resistance intercellular connection which is resistant to treatment with octanol and CO2, both known to close gap junction channels: and (4) members of a dyad are joined by a cytoplasmic bridge. Structures resembling septate junctions are also found between siblings and between cells in aggregates. The evidence accumulated here suggests that cytokinesis in Kc dyads is incomplete, resulting in an intercellular pathway that may provide for the passage of a molecular or electrical signal that regulates subsequent mitosis.