Heterozygous Mutant Cells Research Articles

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Flexible Frogs (Natural variation in embryo mechanics: gastrulation in Xenopus laevis is highly robust to variation in tissue stiffness, by Michelangelo von Dassow, Lance A. Davidson, Dev Dyn 238:2–18) This study opens up a discussion rarely heard in developmental biology circles, how does tissue stiffness contribute to morphogenesis? To address this question, the authors used a microaspirator, placing a gastrulating Xenopus embryo against a channel that bridged low and high pressure reservoir. Apparent stiffness was gauged by measuring the amount of tissue displacement upon application of suction at a specified pressure. This approach revealed that tissue rigidity is surprisingly variable. From the onset of gastrulation to blastopore closure, there was an increase of apparent stiffness. What's more, when comparing embryo-to-embryo or clutch-to-clutch, tissue stiffness varied as much as twofold. Variability is likely innate, and not a product of environment, because changes in pH and salt concentration of surrounding embryonic medium had no affect on stiffness. These data lead the authors to conclude that their fellow morphogenesis researchers should be more flexible—mechanisms that allow normal development to proceed in the face of variable tissue rigidity are arguably just as important as changes in cell shape and cell movement. Size Matters (Many ribosomal protein mutations are associated with growth impairment and tumor predisposition in zebrafish, by Kevin Lai, Adam Amsterdam, Sarah Farrington, Roderick T. Bronson, Nancy Hopkins, Jacqueline A. Lees, Dev Dyn 238:76–85) It is often the little things which matter most. Here, the authors follow up on their previous finding that heterozygous zebrafish lines with retroviral insertions in ribosomal protein (rp) genes are susceptible to developing tumors. They report that 60% of 28 rp mutant lines examined are prone to acquiring malignant tumors, predominantly peripheral nerve sheath tumors which occur in only 1 in 2,000 non-rp mutant fish. Strikingly, the most tumor-prone lines are also growth impaired, but the correlation between the two is not absolute. Nevertheless, the data suggest that the same cellular defect may underlie both phenotypes. To determine the nature of the defect, donor heterozygous mutant cells were injected into wild-type hosts and vice versa. In both cases mutant cells were out-competed by wild-type cells in chimeric fish, as determined by real time polymerase chain reaction, suggesting the defect manifests itself at the cellular level. Precisely what the defect is remains to be determined. The distinction between rp mutations that lead to slower growth and tumorigenesis, and those that do not, does not correlate with the level of knockdown of a given rp gene. Also translation is not globally impaired in slow growing mutants since the proportion of ribosomes bound in monosomes or polysomes in rp heterozygotes appears the same as in wild-types. It will be important to determine what these little fish are telling us about mechanisms of tumorigenesis. Expect the Unexpected (Muscle degeneration and leukocyte infiltration caused by mutation of zebrafish fad24, by Kevin B. Walters, M. Ernest Dodd, Jonathan R. Mathias, Andrea J. Gallagher, David A. Bennin, Jennifer Rhodes, John P. Kanki, A. Thomas Look, Yevgenya Grinblat, Anna Huttenlocher, Dev Dyn 238:86–99). Leave it to a screen to yield unexpected answers to the question asked. In search of genes that regulate inflammation, Huttenlocher's screen identified zebrafish with increased neutrophil distribution in the trunk. The cause was unanticipated—a near or total loss of function of factor for adipocyte differentiation 24 (fad24), a gene they show regulates lipid metabolism like its human counterpart. In addition to decreased lipid concentrations in multiple tissues, embryos also had small eyes, heart folding defects, disorganized skeletal muscle, and other developmental abnormalities. These phenotypes are similar to those found in embryos that are nutritionally deprived, and those in which yolk lipids fail to be transported throughout the body. Further characterizing the inflammation phenotype, they found that skeletal muscle, which continued to degenerate over time, was populated by neutrophils. Application of pan-caspase inhibitors reduced observed apoptosis in skeletal muscle, as well as neutrophil infiltration. These data are evidence that apoptosis is involved in recruiting neutrophils to the trunk—a revealing finding because signals that regulate neutrophil recruitment are largely unknown. The screen has thrust the authors into relatively unexplored territory: mechanisms that connect lipid metabolism to muscle degeneration and neutrophil infiltration.

Most acute myeloid leukaemia patients with intermediate mutant FLT3/ITD levels do not have detectable bi‐allelic disease, indicating that heterozygous disease alone is associated with an adverse outcome

FLT3 internal tandem duplication mutant levels >50%, indicative of bi-allelic disease in some cells, are associated with a particularly poor prognosis in acute myeloid leukaemia; lower levels have an intermediate prognosis relative to wild-type FLT3. To examine whether a small population of homozygous mutant cells is responsible for the worse relapse risk rather than heterozygous disease per se, we determined the genetic composition of 34 intermediate mutant level (25-50%) samples. Only two had evidence of mutant homozygosity; only one had more homozygous than heterozygous mutant cells. Bi-allelic disease in intermediate mutant level cases is uncommon and heterozygous disease is sufficient for adverse outcome.

Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

Gene targeting by homologous recombination is a powerful and widely used technique for introduction of specific gene mutations (frequently a gene inactivation) in transgenic animals. The basic method detailed in this unit uses sequences homologous to the endogenous gene flanking the mutation. While methods using bacterial artificial chromosomes (BACs) and recombineering may be used, in most cases simpler bacterial plasmid clones with several kb of homology are sufficient. This protocol details the strategic factors in designing the constructs for selection and screening for homologous recombination.

Knock-in of Oncogenic Kras Does Not Transform Mouse Somatic Cells But Triggers a Transcriptional Response that Classifies Human Cancers

KRAS mutations are present at a high frequency in human cancers. The development of therapies targeting mutated KRAS requires cellular and animal preclinical models. We exploited adeno-associated virus-mediated homologous recombination to insert the Kras G12D allele in the genome of mouse somatic cells. Heterozygous mutant cells displayed a constitutively active Kras protein, marked morphologic changes, increased proliferation and motility but were not transformed. On the contrary, mouse cells in which we overexpressed the corresponding Kras cDNA were readily transformed. The levels of Kras activation in knock-in cells were comparable with those present in human cancer cells carrying the corresponding mutation. Kras-mutated cells were compared with their wild-type counterparts by gene expression profiling, leading to the definition of a "mutated Kras-KI signature" of 345 genes. This signature was capable of classifying mouse and human cancers according to their KRAS mutational status, with an accuracy similar to or better than published Ras signatures. The isogenic cells that we have developed recapitulate the oncogenic activation of KRAS occurring in cancer and represent new models for studying Kras-mediated transformation. Our results have implications for the identification of human tumors in which the oncogenic KRAS transcriptional response is activated and suggest new strategies to build mouse models of tumor progression.

Dominant-Negative but not Gain-of-Function Effects of a p53.R270H Mutation in Mouse Epithelium Tissue after DNA Damage

p53 alterations in human tumors often involve missense mutations that may confer dominant-negative or gain-of-function properties. Dominant-negative effects result in inactivation of wild-type p53 protein in heterozygous mutant cells and as such in a p53 null phenotype. Gain-of-function effects can directly promote tumor development or metastasis through antiapoptotic mechanisms or transcriptional activation of (onco)genes. Here, we show, using conditional mouse technology, that epithelium-specific heterozygous expression of mutant p53 (i.e., the p53.R270H mutation that is equivalent to the human hotspot R273H) results in an increased incidence of spontaneous and UVB-induced skin tumors. Expression of p53.R270H exerted dominant-negative effects on latency, multiplicity, and progression status of UVB-induced but not spontaneous tumors. Surprisingly, gain-of-function properties of p53.R270H were not detected in skin epithelium. Apparently, dominant-negative and gain-of-function effects of mutant p53 are highly tissue specific and become most manifest upon stabilization of p53 after DNA damage.

High Frequency of BRAFV600E Mutation in Acquired Nevi and Small Congenital Nevi, but Low Frequency of Mutation in Medium-Sized Congenital Nevi

To investigate whether the frequency of the BRAF(V600E) (V-raf murine sarcoma virus oncogene homolog B1) mutation in melanocytic nevi is associated with sun exposure patterns, we examined 120 acquired melanocytic nevi excised from various anatomic sites, including glabrous skin, as well as 62 congenital nevi. We used a new mutation detection system based on the shifted termination assay, called Mutector, which was able to detect only 5% of heterozygous mutant cells within the samples. We detected the mutation in 105/120 (87.5%) acquired nevi and 43/62 (69.4%) congenital nevi. Notably, we found the mutation in 35/43 (81.4%) acquired nevi excised from glabrous skin and genitalia. These results strongly suggest that UV light is not necessarily required for the acquisition of the BRAF(V600E) mutation, and suggest that non-mutagenic effects of UV light to melanocytes may be more important in the nevogenesis. Additionally, we showed heterogeneous distribution of BRAF-mutated cells within the lesions of small congenital nevi by a combination of laser microdissection and direct sequencing. Finally, we found low frequency of BRAF(V600E) mutation (6/20, 30.0%) in medium-sized congenital nevi. Most of these nevi with wild-type BRAF had neroblastoma ras viral oncogene homolog mutations (9/14, 64.3%), suggesting different pathogenesis of medium-sized congenital nevi from acquired nevi and small congenital nevi.

Depletion of Hsp90β Induces Multiple Defects in B Cell Receptor Signaling

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.

A homozygous nonsense mutation in SOX9 in the dominant disorder campomelic dysplasia: a case of mitotic gene conversion

Campomelic dysplasia (CD; MIM 114290), an autosomal dominant skeletal malformation syndrome with XY sex reversal, is caused by heterozygous de novo mutations in and around the SOX9 gene on 17q. We report a patient with typical signs of CD, including sex reversal, who was, surprisingly, homozygous for the nonsense mutation Y440X. Since neither parent carried the Y440X mutation, possible mechanisms explaining the homozygous situation were a de novo mutation followed by uniparental isodisomy, somatic crossing over, or gene conversion. As the patient was heterozygous for six microsatellite markers flanking SOX9, uniparental isodisomy and somatic crossing over were excluded. Analysis of intragenic single-nucleotide polymorphisms suggested that the homozygous mutation arose by a mitotic gene conversion event involving exchange of at least 440 nucleotides and at most 2,208 nucleotides between a de novo mutant maternal allele and a wild-type paternal allele. Analysis of cloned alleles showed that homozygous mutant cells constituted about 80% of the leukocyte cell population of the patient, whereas about 20% were heterozygous mutant cells. Heterozygous Y440X mutations, previously described in three CD cases, have been identified in seven additional cases, thus constituting the most frequent recurrent mutations in SOX9. These patients frequently have a milder phenotype with longer survival, possibly because of the retention of some transactivation activity of the mutant protein on SOX9 target genes, as shown by cell transfection experiments. The fact that the patient survived for 3 months may thus be explained by homozygosity for a hypomorphic rather than a complete loss-of-function allele, in combination with somatic mosaicism. This is, to our knowledge, the first report of mitotic gene conversion of a wild-type allele by a de novo mutant allele in humans.

Role of Pir1 in the construction of the Candida albicans cell wall.

Searches in a Candida albicans database (http://genolist.pasteur.fr/CandidaDB/) identified two Individual Protein Files (IPF 15363 and 19968) whose deduced amino acid sequences showed 42 % and 45 % homology with Saccharomyces cerevisiae Pir4. The two DNA sequences are alleles of the same gene (CaPIR1) but IPF 19968 has a deletion of 117 bases. IPF 19968 encodes a putative polypeptide of 364 aa, which is highly O-glycosylated and has an N-mannosylated chain, four cysteine residues and seven repeats. Both alleles are expressed under different growth conditions and during wall construction by regenerating protoplasts. The heterozygous mutant cells are elongated, form clumps of several cells and are hypersensitive to drugs that affect cell wall assembly. CaPir1 was labelled with the V5 epitope and found linked to the 1,3-beta-glucan of the C. albicans wall and also by disulphide bridges when expressed in S. cerevisiae.

Defective apoptosis and B-cell lymphomas in mice with p53 point mutation at Ser 23.

Phosphorylation of the p53 tumor suppressor at Ser20 (murine Ser23) has been proposed to be critical for disrupting p53 interaction with its negative regulator, MDM2, and allowing p53 stabilization. To determine the importance of Ser23 for the function of p53 in vivo, we generated a mouse in which the endogenous p53 locus was targeted to replace Ser23 with alanine. We show that, in mouse embryonic fibroblasts generated from Ser23 mutant mice, Ser23 mutation did not dramatically reduce IR-induced p53 protein stabilization or p53-dependent cell cycle arrest. However, in Ser23 mutant thymocytes and in the developing cerebellum, p53 stabilization following IR was decreased and resistance to apoptosis was observed. Homozygous Ser23 mutant animals had a reduced lifespan, but did not develop thymic lymphomas or sarcomas that are characteristic of p53-/- mice. Instead, Ser23 mutant animals died between 1 and 2 years with tumors that were most commonly of B-cell lineage. These data support an important role for Ser20/23 phosphorylation in p53 stabilization, apoptosis and tumor suppression.

Characterization, expression pattern and chromosomal localization of the spermatogenesis associated 6 gene (Spata6).

We report the cloning and characterization of the spermatogenesis associated 6 gene (Spata6) encoding a predicted protein of 488 amino acids. It exhibits similarity with the motor domain of kinesin related proteins and with the Caenorhabditis elegans neural calcium sensor protein (NCS-2). The gene encodes three mRNAs of approximately 2.6, approximately 1.8 and approximately 1.2 kb. The expression of the 2.6 kb mRNA is detected at low levels in testis, ovary, thymus and placenta, while the 1.8 and 1.2 kb transcripts are exclusively expressed in testis. The 1.8 and 1.2 kb transcripts are specifically expressed in haploid germ cells. Data from in situ hybridization experiments suggested that mRNA expression of Spata6 in spermatids is higher than in spermatocytes and spermatogonia. RT-PCR analysis and whole mount in situ hybridization demonstrate that the Spata6 transcript is expressed during embryonic development and is localized in neural tube, somites and limb buds of mouse embryo. The Spata6 gene consists of 15 exons ranging in size between 40 and 596 bp. The 2.6 and 1.8 kb transcripts have different 5' untranslated sequences but have the same translational initiation site and therefore may encode the same protein with a predicted molecular weight of 49.7 kDa. The 1.2 kb transcript is derived from a proximal promoter between exons 7 and 8, and contains a translation initiation codon AUG, which is in frame with initiator AUG codon of the 2.6 and 1.8 kb transcripts. Therefore, the 1.2 kb transcript may code for a truncated protein of 32 kDa. Western blot analysis with the antiserum raised against a synthetic peptide from the C-terminal of the deduced Spata6 protein detects only a single protein of 53 kDa in all tissues studied. The Spata6 gene was localized to chromosome 5, region q34-35 in the rat and to chromosome 1, region p32-35 in the human. In an effort to determine the function of Spata6, we inactivated the mouse gene in embryonic stem cells through homologous recombination. Although the heterozygous mutant cells were able to generate low coat colour chimeric mice, all chimeras did not transmit the targeted allele to their progeny suggesting that a high contribution of Spata6(+/-) cells lead to the lethality of the chimeric embryos.

Stress-induced gene expression requires programmed recovery from translational repression.

Active repression of protein synthesis protects cells against protein malfolding during endoplasmic reticulum stress, nutrient deprivation and oxidative stress. However, long-term adaptation to these conditions requires synthesis of new stress-induced proteins. Phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation in diverse stressful conditions. GADD34 is a stress-inducible regulatory subunit of a holophosphatase complex that dephosphorylates eIF2alpha, and has been hypothesized to play a role in translational recovery. Here, we report that GADD34 expression correlated temporally with eIF2alpha dephosphorylation late in the stress response. Inactivation of both alleles of GADD34 prevented eIF2alpha dephosphorylation and blocked the recovery of protein synthesis, normally observed late in the stress response. Furthermore, defective recovery of protein synthesis markedly impaired translation of stress-induced proteins and interfered with programmed activation of stress-induced genes in the GADD34 mutant cells. These observations indicate that GADD34 controls a programmed shift from translational repression to stress-induced gene expression, and reconciles the apparent contradiction between the translational and transcriptional arms of cellular stress responses.

Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. This unit provides a protocol in which the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker. A negative selectable marker is included in the construct outside the region of target gene homology in order to enrich for clones in which the target gene has undergone homologous recombination. The altered target gene is then expressed in embryonic stem cells. A support protocol describes a method for transient expression of Cre recombinase to remove sequences between lox sites, which can also be used as a selection method.

Chimeric analysis of Notch2 function: a role for Notch2 in the development of the roof plate of the mouse brain.

Notch proteins are transmembrane receptors involved in cell-fate determination throughout development. Targeted disruption of either the Notch1 or Notch2 gene in mice results in embryonic lethality around embryonic day (E) 10.5 with widespread cell death. Although Notch1-deficient mice show disorganized somitogenesis, Notch2 mutants did not show definitive abnormalities in any tissue expressing high levels of the Notch2 gene, including the central nervous system. To study Notch2 function in development beyond the embryonic lethal stage, we performed chimeric analysis between Notch2 mutant and wild-type mouse embryos. Chimeric embryos developed normally and homozygous Notch2 mutant-specific cell death was not observed. Although chimeric embryos showed normal mosaicism until E9.5 in all tissues studied to date, Notch2 homozygous mutant cells failed to contribute to formation of the roof plate of the diencephalon and mesencephalon at later developmental stages, when Notch2 is normally expressed at high levels at there. Furthermore, Notch2 heterozygous mutant cells were also excluded from the roof plate of the chimera, however, Notch2 heterozygous mutant mice developed normally. We also showed that Wnt-1 and Mash1 expression patterns at the roof plate were disorganized in Notch2 homozygous mutant embryos. These results indicate that Notch2 plays an important role in development of the roof plate of the diencephalon and mesencephalon, and suggest that cellular rearrangement is involved in this process.

Targeted disruption of one allele of the Y-box protein gene, Chk-YB-1b, in DT40 cells results in major defects in cell cycle

Y-box or inverted CCAAT box-binding proteins are multifunctional regulators of transcription and translation of several genes. Although YB-1 has been shown to play a key role in cell cycle, to date, there is no direct evidence. We disrupted one allele of Chk-YB-1b in a chicken pre-B lymphocyte cell line, DT40. Compared to wild-type DT40 cells, these heterozygous DT40YB1b +/− cells with one copy of the wild-type Chk-YB-1b allele showed multiple abnormalities, which include slower rate of growth, abnormal cell morphology, increased cell size, and increased genomic DNA content. These phenotypic defects resemble those cells that have a block in G2 and/or mitosis (G2/M). In addition, we have observed that a fraction of these heterozygous DT40YB1b +/− cells undergo apoptosis. In conclusion, we have discovered major defects in the G2/M phase of cell cycle in YB-1 knocked-out heterozygous mutant cells, providing for the first time direct evidence establishing a crucial role for YB-1 in cell proliferation.

Production of a Homozygous Mutant Embryonic Stem Cell Line (Double Knockout)

AbstractHomozygous mutant embryonic stem (ES) cell lines have proven very useful in studying gene and protein function, and there are several reasons why this system might be preferred. Because many genes express their phenotype at the level of individual cells, using these cell lines eliminates the time and expense of producing a mutant animal. They can be isolated directly from a heterozygous mutant cell line without rederiving a cell line or using primary tissues from a mutant animal. In some cases, analysis of the phenotype of homozygous mutant cells can be performed more easily in culture. In addition, homozygous mutant ES cell lines may be produced even when a homozygous mutation is lethal to the animal. Finally, microinjection of homozygous mutant ES cells into blastocysts may allow investigation of the developmental potential of cells with such lethal mutations. This unit was moved from Chapter 9 to reflect a more appropriate placement, and has been updated and revised.

Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

AbstractFormerly UNIT 9.16, this unit takes a more appropriate place in Chapter 23, and has been updated and revised for this publication. Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. In this unit, the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker (e.g., neo). Expression of neo is obtained by including the phosphoglycerate kinase (PGK) promoter in the construct. To enrich for clones in which the target gene has undergone homologous recombination over those in which random integration of the construct has occurred, a negative selectable marker, herpes simplex virus thymidine kinase (HSV‐TK), is included in the construct outside the region of homology to the target gene. Depending upon the target gene, it may be easier to assemble the construct by adding the neo and TK genes to the cloned target gene or by adding two fragments of the target gene to a plasmid containing the neo and TK genes.

SNF2beta-BRG1 is essential for the viability of F9 murine embryonal carcinoma cells.

The yeast and animal SNF-SWI and related multiprotein complexes are thought to play an important role in processes, such as transcription factor binding to regulatory elements, which require nucleosome remodeling in order to relieve the repressing effect of packaging DNA in chromatin. There are two mammalian homologs of the yeast SNF2-SWI2 subunit protein, SNF2alpha-brm and SNF2beta-BRG1, and overexpression of either one of them has been shown to enhance transcriptional activation by glucocorticoid, estrogen, and retinoic acid (RA) receptors in transiently transfected cells. We have investigated here the function of SNF2beta-BRG1 in the RA receptor-retinoid X receptor-mediated transduction of the retinoid signal in F9 embryonal carcinoma (EC) cells which differentiate into endodermal-like cells upon RA treatment. The two SNF2beta-BRG1 alleles have been targeted by homologous recombination and subsequently disrupted by using a conditional Cre recombinase. We show that F9 EC cells inactivated on both SNF2beta alleles are not viable and that heterozygous mutant cells are affected in proliferation but not in RA-induced differentiation. Thus, in F9 EC cells, SNF2beta-BRG1 appears to play an essential role in basal processes involved in cell proliferation, in addition to its putative role in the activation of transcription mediated by nuclear receptors.

The chicken HMG-17 gene is dispensable for cell growth in vitro.

HMG-17 is a highly conserved and ubiquitous nonhistone chromosomal protein that binds to nucleosome core particles. HMG-17 and HMG-14 form a family of chromosomal proteins that have been reported to bind preferentially to regions of active chromatin structure. To study the functional role of the single-copy chicken HMG-17 gene, null mutants were generated by targeted gene disruption in a chicken lymphoid cell line, DT40. Heterozygous and homozygous null mutant cell lines were generated by two independent selection strategies. Heterozygous null mutant lines produced about half the normal level of HMG-17 protein, and homozygous null lines produced no detectable HMG-17. No significant changes in cell phenotype were observed in cells harboring either singly or doubly disrupted HMG-17 genes, and no compensatory changes in HMG-14 or histone protein levels were observed. It is concluded that HMG-17 protein is not required for normal growth of avian cell lines in vitro, nor does the absence of HMG-17 protein lead to any major changes in cellular phenotype, at least in lymphoid cells.

An assay for loss of heterozygosity in vivo at the Dlb-1 locus.

Loss of heterozygosity (LOH) is a frequent event in many tumours, resulting in the loss of tumour suppressor genes and ultimately magnifying the number of cells with a mutant phenotype. We have used the Dlb-1 locus as a simple quantitative assay for LOH in vivo. Mutations of the dominant Dlb-1b allele are readily detected in heterozygous (Dlb-1a/Dlb-1b) mice by the loss of histochemical staining, which results in unstained, white (Dlb-1a/Dlb-1-) ribbons on a stained background. Such ribbons are extremely rare in untreated C57BL mice which are homozygous for the dominant allele, as would be expected when two independent mutations are required. To test for LOH, we first treated the animals with a high dose of ethylnitrosourea (ENU) which induces many mutations and thus many heterozygous cells, and allowed 2 weeks for gene expression. Then the animals were treated with the test agent to determine if it could cause LOH and thus convert heterozygous mutant cells, which would not produce detectable ribbons, into homozygotes that would produce white, non-staining ribbons. Treatment with ENU alone produced a low, but detectable, frequency of mutant ribbons. Treatment with X-rays alone produced no detectable increase in the frequency of mutant ribbons. Combinations of these treatments produced additive effects, thus showing that no significant LOH was induced. The additivity of the two equal ENU treatments was unexpected, since double mutants should increase as the square of the mutant frequency.(ABSTRACT TRUNCATED AT 250 WORDS)