Pairs of cyclic AMP analogs, that are specifically synergistic for type I and type II cAMP-dependent protein kinases, mimic thyrotropin effects on the function, differentiation expression and mitogenesis of dog thyroid cells

The role of the two different isozymes of the cAMP-dependent protein kinase is still unclear. We have investigated the potential roles for each isozyme in dog thyroid cells, a model in which the function, expression of differentiation and proliferation are positively regulated by thyrotropin acting through cyclic AMP. The dog thyroid contains both type I and type II cAMP-dependent protein kinases. These isozymes were selectively activated in vitro by type-I-directed and type-II-directed analog pairs. In thyroid slices, both type-I directed and type II-directed analog pairs synergistically activated thyroid hormone synthesis, as measured by incorporation of 131I into proteins and thyroid hormone secretion as determined by the release of butanol-extractable 131I. In primary cultures of dog thyroid cells both isozyme-directed analog pairs synergistically enhanced iodide trapping, a marker of differentiation, and DNA synthesis, as measured by the percentage of cells incorporating [3H]thymidine into their nuclei. However, DNA synthesis was more sensitive to type-I-directed pairs. The results demonstrate that both cAMP-dependent protein kinase isozymes can mediate the action of cAMP on function, differentiation expression and cell proliferation in dog thyroid cells.     

Role of protein kinase C alpha in calcium induced keratinocyte differentiation: defective regulation in squamous cell carcinoma

Calcium induces both involucrin and transglutaminase-K in normal keratinocytes (NHK) but not in squamous carcinoma cell lines (SCC). The protein kinase C (PKC) agonist phorbol myristoyl acetate potentiates and the PKC antagonist Ro31-8220 blocks the ability of calcium to stimulate the involucrin promoter in normal human keratinocytes but not in SCC4. We thus examined the ability of calcium to regulate the levels of five PKC isozymes in NHK and two SCC. In the normal keratinocytes, the levels of PKC [alpha], PKC [delta], PKC [eta], and PKC [zeta] increased over the first one to two weeks in a calcium-and time-dependent manner. PKC [epsilon] decreased in a time-and calcium-dependent fashion over the three-week period. All five isozymes showed little change during culture in SCC4 at any calcium concentration. Calcium and time of culture had partial effects on SCC12B2, a carcinoma that shows partial differentiation characteristics. Since PKC [alpha] is the only calcium responsive PKC isozyme in keratinocytes and most likely to be directly involved in calcium induced differentiation, we evaluated the effect of inhibiting its production with antisense oligonucleotides on calcium-regulated markers of differentiation. We found that the PKC [alpha] specific antisense oligonucleotide blocked calcium stimulated involucrin promoter activity as well as PKC [alpha], involucrin, and transglutaminase protein production, whereas the sense oligonucleotide control did not. We conclude that although a number of PKC isozymes are regulated during calcium-induced differentiation, PKC [alpha] plays a necessary role in mediating calcium-induced differentiation. Failure to regulate PKC [alpha] in SCC4 may underlie at least part of the failure of calcium to promote differentiation in these cells.     

Superoxide dismutase and peroxidase are coordinately regulated in differentiated and transformed tissues of Nicotiana tabacum

We used a series of normal and Agrobacterium-transformed, bacteria-free tobacco tissue cultures which differ in their levels of histodifferentiation to test the relationship of superoxide dismutase, peroxidase, and catalase to oncogenic transformation and differentiation. When compared with normal callus, tumor callus contained reduced levels of both superoxide dismutase and peroxidase, and a reduced number of isozymes of both enzymes. Teratomas characterized by limited but abnormal differentiation showed increases in superoxide-dismutase activity and isozymes, but levels of peroxidase activity lower than those found in normal callus despite an increase in the number of peroxidase isozymes. A similar disparity between low peroxidase activity and high isozyme number in the shoot suggests that there are increased levels of peroxidase inhibitors or of molecules which interfere with the spectrophotometric assay for peroxidase in more differentiated tissues. As judged by the number of isozymes of both superoxide dismutase and peroxidase in each tissue, the following conclusions are warranted: first, tobacco copper/zinc superoxide dismutases and peroxidases are encoded in several duplicated loci which are regulated independently. Second, transformation is associated with a decrease in both the specific activity and isozyme number of superoxide dismutase. Third, the partial release from the total inhibition of expression of differentiated function exhibited by teratoma is associated with an increase in both the activity and isozyme number of superoxide dismutase. Finally, the expression of superoxide dismutase and peroxidase isozymes appears to be coordinated during differentiation in a manner that is consistent with their role in an integrated mechanism for the removal of reduced oxygen species.     

Protein kinase C isozymes have distinct roles in neural induction and competence in Xenopus.

The restricted ability of embryonic tissue to respond to inductive signals is controlled by a poorly understood phenomenon, termed competence. In Xenopus, dorsal ectoderm is more competent than ventral ectoderm to become induced to neural tissue. We tested whether the Xenopus protein kinase C (PKC) isozymes alpha and beta have a role in neural induction and competence. We found that PKC alpha is predominantly localized in dorsal ectoderm, whereas PKC beta is uniformly distributed. Overexpression of PKC beta conveys a higher propensity for neural differentiation to both dorsal and ventral ectoderm, but their difference in competence remains. However, ectopic expression of PKC alpha elevates the level of neural competence of ventral ectoderm to that of dorsal ectoderm. These data indicate that different PKC isozymes have distinct roles in mediating both neural induction and competence.     

Genetic effects of chronic habitat fragmentation on tree species: the case of Sorbus aucuparia in a deforested Scottish landscape

Sustainable forest restoration and management practices require a thorough understanding of the influence that habitat fragmentation has on the processes shaping genetic variation and its distribution in tree populations. We quantified genetic variation at isozyme markers and chloroplast DNA (cpDNA), analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in severely fragmented populations of Sorbus aucuparia (Rosaceae) in a single catchment (Moffat) in southern Scotland. Remnants maintain surprisingly high levels of gene diversity (HE) for isozymes (HE = 0.195) and cpDNA markers (HE = 0.490). Estimates are very similar to those from non-fragmented populations in continental Europe, even though the latter were sampled over a much larger spatial scale. Overall, no genetic bottleneck or departures from random mating were detected in the Moffat fragments. However, genetic differentiation among remnants was detected for both types of marker (isozymes Theta n = 0.043, cpDNA Theta c = 0.131; G-test, P-value < 0.001). In this self-incompatible, insect-pollinated, bird-dispersed tree species, the estimated ratio of pollen flow to seed flow between fragments is close to 1 (r = 1.36). Reduced pollen-mediated gene flow is a likely consequence of habitat fragmentation, but effective seed dispersal by birds is probably helping to maintain high levels of genetic diversity within remnants and reduce genetic differentiation between them.     

A Link between FXYD3 (Mat-8)-mediated Na,K-ATPase Regulation and Differentiation of Caco-2 Intestinal Epithelial Cells

FXYD3 (Mat-8) proteins are regulators of Na,K-ATPase. In normal tissue, FXYD3 is mainly expressed in stomach and colon, but it is also overexpressed in cancer cells, suggesting a role in tumorogenesis. We show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and a decrease in transepithelial resistance. Inhibition of cell differentiation in FXYD3-deficient cells is accompanied by an increase in the apparent Na⁺ and K⁺ affinities of Na,K-ATPase, reflecting the absence of Na,K-pump regulation by FXYD3. In addition, we observe a decrease in the maximal Na,K-ATPase activity due to a decrease in its turnover number, which correlates with a change in Na,K-ATPase isozyme expression that is characteristic of cancer cells. Overall, our results suggest an important role of FXYD3 in cell differentiation of Caco-2 cells. One possibility is that FXYD3 silencing prevents proper regulation of Na,K-ATPase, which leads to perturbation of cellular Na⁺ and K⁺ homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell differentiation.     

宽叶韭种内分化的同工酶及可溶性蛋白的研究

采用聚丙烯酰胺凝胶电泳（ＰＡＧＥ）对宽叶韭的３个代表居群内不同植株间的过氧化物酶、酯酶和１６个居群间的酯酶（ＥＳＴ）、过氧化物酶（ＰＯＸ）、葡萄糖－６－磷酸脱氢酶（Ｇ６ＰＤＨ）、苹果酸酶（ＭＥ）、苹果酸脱氢酶（ＭＤＨ）、天冬氨酸转移酶（ＡＡＴ）、腺苷三磷酸酶（ＡＴＰａｓｅ）、乳酸脱氢酶（ＬＤＨ）８种同工酶系统以及可溶性蛋白进行了分析。结果发现：居群内不同植株的酶谱无差异;居群间的酶谱存在多态性,酶谱差异明显,且各居群均有其特征酶谱。采用“排序”方法和聚类分析,对宽叶韭１６个居群酶谱的相似系数和酶谱距离进行了研究。同工酶谱表型差异彼此显著的３个代表居群的可溶性蛋白双向电泳图谱也表现出明显差异,印证了同工酶的分析结果。结合细胞学的最新核型证据,从生物化学角度确定了这１６个居群间的亲缘关系和彼此的分化程度。结果表明：宽叶韭明显分为两大类,即冬季不倒苗和冬季倒苗两类,在同一类型中各居群之间彼此近缘,且冬季不倒苗的类型分化程度较高。本研究首次将多种同工酶的综合分析与聚类分析及可溶性蛋白双向电泳用于葱属植物的种内分化研究,为植物分子进化和系统学研究提供了科学依据     

Isozymes and DNA markers in gene conservation of forest trees

For long-lived plants that have to cope with high temporal and spatial environmental heterogeneity, genetic diversity is of prime importance for species persistence. Detrimental anthropogenic impact on the gene pool of forest trees calls for conservation of genetic resources. Potentials and limitations of isozymes and DNA markers in forest genetic conservation are reviewed. These markers can contribute to conservation with respect to the delimitations of species and hybrid zones, as well as the assessment of genetic diversity within and among populations. Markers are valuable to identify resource populations, since today‘s genetic diversity in forest trees is predominantly the result of plant history (e.g. glacial refuges, migration). Several suggestions have been put forward to optimize sampling of in situ or ex situ populations on the grounds of marker data. Restraint in this area is recommended. Different types of genetic markers (terpenes, isozymes, nuclear and extrachromosomal DNA polymorphisms) and quantitative traits yield different information about genetic diversity and population differentiation. Hence identification of resource populations should not solely be based upon a certain marker type or on quantitative traits alone. The capability of available markers to predict or assess adaptive potentials in forest tree populations is still very limited. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression and catalysis of sex-specific cytochrome P450 isozymes in rat liver

Research interest in the study of cytochromes P450 has recently been shifting to the characterization of "constitutively" expressed isozymes from that of the inducible forms. Several "constitutive" cytochrome P450 isozymes have been purified from rat liver including five immunochemically related proteins designated cytochromes P450f, P450g, P450h, P450i, and P450k. These hemoproteins have been identified as distinct isozymes on the basis of spectral, electrophoretic, and catalytic properties and NH2-terminal sequence analysis. Purification and immunoquantitation studies have indicated that these isozymes are expressed in a developmental as well as sex-related manner, and are relatively refractory to induction by xenobiotics. Cytochromes P450h and P450g are male-specific proteins, cytochrome P450i is a female-specific isozyme, while cytochromes P450f and P450k are present in both male and female adult rats. In addition, the expression of cytochrome P450g was shown to segregate into two phenotypes in outbred rats. Genetic studies utilizing inbred strains have indicated that the gene responsible for inheritance of high levels of cytochrome P450g is autosomal. Although considerable progress has been made in understanding the role of gonadal hormones and growth hormone in the hepatic regulation of cytochromes P450g, P450h, and P450i in particular, the physiological significance of the "constitutive" isozymes in the liver remains largely unresolved.     

Isoperoxidases as markers of the wound-induced differentiation pattern in potato tuber

Isoperoxidases induced by wounding in potato tuber tissue were separated by starch gel electrophoresis and found to be distributed in a highly specific spatial pattern. This pattern of molecular differentiation correlates well with the pattern of cell differentiation associated with wound healing. Although wound induced isoperoxidases were found to vary between three varieties of potato, they were distributed in the same spatial pattern. The combination of isozymes extracted from various parts of a potato plant was specific for each of nine organs and tissues, and all combinations were different from the isozymes from wounded tuber tissue. Isoperoxidases can thus be considered as highly specific molecular markers of cell differentiation.     

Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific

We have studied the expression of mRNA encoding all known protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon, zeta, and eta) in murine tumor cell lines that exemplify hemopoietic cells arrested at different stages of development as well as in normal hemopoietic cells. We demonstrate that some of the isozymes, PKC-alpha, -beta, and -eta, are differentially expressed in different lineages. PKC-alpha and -beta generally are not detectable in myeloid cell lines, where PKC-delta is the predominant isoform. Both PKC-alpha and -beta are abundant in most T and B lymphocytic lines, but steady state levels of PKC-beta mRNA are lowest in plasma cell tumors, which exemplify the terminally differentiated B lymphocyte. In contrast, the levels of PKC-alpha mRNA remain high in plasma cell tumors, and a novel, 2.5-kb PKC-alpha mRNA gains prominence. PKC-eta mRNA is the major PKC isoform expressed in T lymphocytes, but it also is highly abundant in some myeloid lines. PKC-delta is expressed at high levels in all the lines we studied, whereas PKC-epsilon and -zeta are found in most cells but only at rather low levels. Analysis of myeloid clones derived from bipotential B lineage progenitor cell lines suggests that the B cell phenotype is associated with the expression of PKC-alpha. The close correlation of protein levels with mRNA levels indicates that PKC expression in hemopoietic cells is mainly regulated at the level of mRNA. The lineage- and differentiation stage-specific patterns of PKC-isozyme expression presented here suggest the involvement of specific PKC isozymes in differentiation as well as lineage determination of hemopoietic cells.     

秋茄（Kandelia candel）的区域性种群（population）遗传结构

在台湾北部淡水河河口沼泽地带的秋茄（水笔仔）,依其林冠高度略可分成高、矮两个亚种群（Subpopulation）。矮生秋茄长在地面较高的河边,高生秋茄则沿着一条潮溪,长在较低处。使用淀粉凝胶方法研究同功酶组合型式,判断这个地区的区域种群的遗传结构,发现生长在沼泽区内较干燥地带的秋茄和长在潮湿地带的秋茄遗传上的分化程度很低（Fst= 0.043）。这可能是因为区内的基因流传很顺畅,抵消了可能发生的种群分化。     

Specific genetic markers for wheat, spelt, and four wild relatives: comparison of isozymes, RAPDs, and wheat microsatellites.

Three types of markers-isozymes, RAPDs (random amplified polymorphic DNAs), and wheat microsatellites- were tested on wheat, spelt, and four wild wheat relatives (Aegilops cylindrica, Elymus caninus, Hordeum marinum, and Agropyron junceum). The aim was to evaluate their capability to provide specific markers for differentiation of the cultivated and wild species. The markers were set up for subsequent detection of hybrids and introgression of wheat DNA into wild relatives. All markers allowed differentiation of the cultivated from the wild species. Wheat microsatellites were not amplified in all the wild relatives, whereas RAPDs and isozymes exhibited polymorphism for all species. The dendrograms obtained with RAPD and isozyme data separated Swiss wheat cultivars from those collected in Austria and England, while no difference was found between Swiss spelt and wheat. RAPD data provided a weak discrimination between English and Austrian E. caninus. The microsatellite-based dendrogram discriminated populations of Ae. cylindrica, but no clear separation of H. marinum from E. caninus was revealed. The similarity matrices based on the three different sets of data were strongly correlated. The highest value was recorded between the matrices based on RAPDs and isozymes (Mantel's test, r = 0.93). Correlations between the similarity matrix based on microsatellites and matrices based on RAPDs and isozymes were lower: 0.74 and 0.68, respectively. While microsatellites are very useful for comparisons of closely related accessions, they are less suitable for studies involving less-related taxa. Isozymes provide interesting markers for species differentiation, but their use seems less appropriate for studies of within-species genetic variation. RAPDs can produce a large set of markers, which can be used for the evaluation of both between- and within-species genetic variation, more rapidly and easily than isozymes and microsatellites.     

LMNA Knock-Down Affects Differentiation and Progression of Human Neuroblastoma Cells

Background

Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma.

Methodology/Principal Findings

Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases.

Conclusions/Significance

We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.     

Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle

Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor.     

Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.     

Dynamic Status of REST in the Mouse ESC Pluripotency Network

Background

REST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network.

Methods

Employing biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature.

Results

We show that Rest+/− and Rest−/− AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/− and Rest−/− AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a REST-dependent pluripotency when cultured under feeder-free conditions, as well as Rest−/− AB-1 ESCs, showed no REST-dependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST''s role in ESC pluripotency.

Conclusions

REST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner.     

Genetic variation and differentiation within a natural community of five oak species (Quercus spp.)

Chloroplast DNA and two categories of nuclear markers - isozymes and microsatellites - were used to examine a very rich natural community of oaks (Quercus spp.) situated in west-central Romania. The community consists of five oak species: Q. robur, Q. petraea, Q. pubescens, and Q. frainetto - that are closely related -, and Q. cerris. A total of five chloroplast haplotypes was identified. Q. cerris was fixed for a single haplotype. The other four species shared the two most common haplotypes. One haplotype was confined to Q. robur and a very rare one was restricted to Q. petraea. Both types of nuclear markers revealed a larger genetic variation for Q. pubescens and Q. petraea than for Q. frainetto and Q. robur, although the differences between species are in most cases not significant. At the nuclear level, Q. cerris could be clearly separated from the other four oak species confirming the taxonomic classification. Regardless of the estimate used, the levels of polymorphism revealed by microsatellites were much higher than those based on isozymes. For the four closely related species the overall genetic differentiation was significant at both categories of nuclear markers. Several loci, such as Acp-C for isozymes, and ssrQpZAG36 and ssrQrZAG96 for microsatellites were very useful to discriminate among species. However, the level of differentiation varied markedly between pairs of species. The genetic affinities among the species may reflect different phylogenetic distances and/or different rates of recurrent gene flow at this site.     

Do molecular markers reflect patterns of differentiation in adaptive traits of conifers?

We have examined patterns of variation of several kinds of molecular markers (isozymes, RFLPs of ribosomal DNA and anonymous low-copy number DNA, RAPDs and microsatellites) and an adaptive trait [date of bud set in Scots pine (Pinus sylvestris L.)]. The study included Finnish Scots pine populations (from latitude 60°N to 70°N) which experience a steep climatic gradient. Common garden experiments show that these populations are adapted to the location of their origin and genetically differentiated in adaptive quantitative traits, e.g. the date of bud set in first-year seedlings. In the northernmost population, bud set took place about 21 days earlier than in the southernmost population. Of the total variation in bud set, 36.4% was found among the populations. All molecular markers showed high levels of within-population variation, while differentiation among populations was low. Among all the studied markers, microsatellites were the most variable (H_e=0.77). Differences between populations were small, G_ST was less than 0.02. Our study suggests that molecular markers may be poor predictors of the population differentiation of quantitative traits in Scots pine, as exemplified here by bud-set date.     

Unusual glucose phosphate isomerase isozyme patterns in lymphocytes from two C57BL/6J in equilibrium A/J allophenic mice

Analysis of C57BL/6J in equilibrium A/J allophenic mice for their lymphocyte composition, using H-2 antigens as external markers, and glucose phosphate isomerase (GPI) isozymes as internal markers, has led to the discovery of two unusual mice. Both mice showed heterodimers of GPI isozymes upon electrophoresis of the lymphocyte lysate. Specific anti-H-2 antisera confirmed that the cells of the mice were of C57BL/6J and A/J origin, as expected, but that the "A/J" cells seemed to behave as F1 hybrids containing the Gpi-1a and Gpi-1b alleles. Possible origins of the Gpi-1b allele in the "A/J" cells are discussed.