Endogenous Mouse Mammary Tumor Virus Research Articles

Production of mouse mammary tumor virus upon transfection of a recombinant proviral DNA into cultured cells

We have investigated the intracellular proteins synthesized in rat XC and feline kidney cells transfected with endogenous mouse mammary tumor virus (MMTV) proviral DNA. The endogenous provirus GR40, associated with the Mtv-8 locus, directs the synthesis of gag proteins indistinguishable from those found in MMTV-infected cells. The env precursor Pr73 env and the mature gp52 proteins could not be detected in these cells. Instead an env-related protein of 68K is synthesized. In contrast to this endogenous provirus, a cloned exogenous proviral variant directs the synthesis of apparently normal env proteins upon transfection into the same cell lines. These results suggest that the env gene of the endogenous MMTV provirus GR40 is defective. The exogenous proviral variant is not expected to synthesize virus particles since it carries a rearrangement in the gag gene. In order to obtain an MMTV provirus capable of correctly expressing both gag and env functions, we have constructed a hybrid endogenous-exogenous provirus containing the 5′-long terminal repeat (LTR)- gag of GR40 and the pol-env-3′ LTR of the exogenous provirus. Upon transfection into feline kidney cells, this hybrid provirus directed the synthesis of apparently authentic gag and env proteins. Further, virus particles can be detected in the culture medium of the transfected cells by electron microscopy. Viral proteins obtained from viral particles banded in a sucrose gradient were detected by immunoprecipitation.

Analysis of tissue-specific methylation patterns of mouse mammary tumor virus DNA by two-dimensional Southern blotting.

We used a two-dimensional Southern blotting procedure to analyze the tissue-specific methylation patterns of the five endogenous mouse mammary tumor viruses in the GR/A mouse strain. Our findings suggest that in certain tissues (brain, kidney, and liver) all proviruses are extensively methylated. In other tissues (spleen, placenta, and testes) all proviruses are hypomethylated to some degree. In these tissues individual proviruses display both quantitative and qualitative differences in methylation. We interpret the general patterns of tissue-specific hypomethylation in terms of a "hitch-hiker" model: mouse mammary tumor virus proviral methylation patterns reflect the tissue-specific activity of neighboring sequences. The observation that certain sites on particular proviruses are differentially methylated in a tissue-specific fashion may reflect tissue-specific differences in the makeup or conformation, or both, of proviral-containing chromatin.

Alterations in chromatin structure associated with glucocorticoid-induced expression of endogenous mouse mammary tumor virus genes.

Alterations in the chromatin structure of endogenous mouse mammary tumor virus genes accompany glucocorticoid induction of viral RNA synthesis in the C57BL/6 T lymphoma cell line T1M1. These alterations are defined by the appearance of sites of DNase I hypersensitivity within proviral DNA in isolated nuclei, as well as by changes in the moderate nuclease sensitivity of entire proviral transcription units. Induced hypersensitive sites, termed type I, appear with a time course comparable to that required for induction of the rate of viral RNA synthesis and are maintained only in the continuous presence of hormone. Two such sites map to analogous positions in the 5' and 3' long terminal repeats of proviral DNA within, or very near, sequences that have been shown to comprise positions of specific binding of the glucocorticoid receptor in vitro and that are required for hormone-inducible transcription in vivo. A third type I site maps to another position of in vitro receptor binding near the 3' long terminal repeat. Some sites of DNase I hypersensitivity, termed type II, appear not to be markedly hormone dependent; two such sites are present in corresponding positions in each long terminal repeat. Comparison of the moderate DNase I sensitivity of mouse mammary tumor virus proviral DNA suggests that the three different endogenous units in T1M1 cells can be maintained in distinct chromatin conformations that are determined by factors related to the site of provirus insertion. It seems possible that altered chromatin conformations may reflect, or actually encode, important mechanistic features of these hormone-responsive genes.

Alterations in chromatin structure associated with glucocorticoid-induced expression of endogenous mouse mammary tumor virus genes.

Alterations in the chromatin structure of endogenous mouse mammary tumor virus genes accompany glucocorticoid induction of viral RNA synthesis in the C57BL/6 T lymphoma cell line T1M1. These alterations are defined by the appearance of sites of DNase I hypersensitivity within proviral DNA in isolated nuclei, as well as by changes in the moderate nuclease sensitivity of entire proviral transcription units. Induced hypersensitive sites, termed type I, appear with a time course comparable to that required for induction of the rate of viral RNA synthesis and are maintained only in the continuous presence of hormone. Two such sites map to analogous positions in the 5' and 3' long terminal repeats of proviral DNA within, or very near, sequences that have been shown to comprise positions of specific binding of the glucocorticoid receptor in vitro and that are required for hormone-inducible transcription in vivo. A third type I site maps to another position of in vitro receptor binding near the 3' long terminal repeat. Some sites of DNase I hypersensitivity, termed type II, appear not to be markedly hormone dependent; two such sites are present in corresponding positions in each long terminal repeat. Comparison of the moderate DNase I sensitivity of mouse mammary tumor virus proviral DNA suggests that the three different endogenous units in T1M1 cells can be maintained in distinct chromatin conformations that are determined by factors related to the site of provirus insertion. It seems possible that altered chromatin conformations may reflect, or actually encode, important mechanistic features of these hormone-responsive genes.

Sequence organization and molecular cloning of mouse mammary tumor virus DNA endogenous to C57BL/6 mice.

The sequence organization of mouse mammary tumor virus DNA endogenous to the C57BL/6 inbred mouse strain was characterized by Southern blot analysis, utilizing probes specific for particular regions of the mouse mammary tumor virus provirus and by molecular cloning of endogenous mouse mammary tumor virus DNA. The genome of C57BL/6 mice contains three apparently intact, endogenous proviral units; two of these units comprise the Mtv-8 (unit II) and Mtv-9 (unit III) genetic loci that are also present in the DNA of BALB/c mice. The third unit is defined by EcoRI restriction fragments of 10.0 and 8.4 kilobases that contain the 5' and 3' portions of the provirus, respectively. This unit, termed unit XI and encoded by the genetic locus Mtv-17, has not been previously recognized in C57BL/6 DNA, but it can be clearly distinguished from the proviral units at Mtv-8 and Mtv-9 by Southern blot analysis under appropriate conditions. The proviral unit at Mtv-17 is not present in BALB/c DNA. DNAs comprising the entire Mtv-8 locus and the 3' portions of Mtv-9 and Mtv-17 were cloned. Analysis of the cloned DNA revealed no obvious deletions or rearrangements that would render proviral DNA defective; however, these endogenous genes are normally not transcriptionally active.

Characterization and chromosomal distribution of endogenous mouse mammary tumor viruses of European mouse strains STS/A and GR/A

The endogenous mouse mammary tumor virus (MMTV) proviral copies in two genetically dissimilar mouse strains, STS/A, a European mouse strain, and BALB/c, were characterized. STS/A carries the same four MMTV proviral copies as GR.Mty-2-; these strains share also most of the isoenzyme markers and are therefore highly related. Cellular DNA of GR.Mty-2- contains a partial MMTV provirus that is not present in STS/A. GR.Mty-2- is derived from GR; they differ in the locus Mtv-2 that contains one MMTV provirus. Expression of this Mtv-2 endogenous MMTV provirus is directly linked to mammary tumorigenesis in GR. MMTV proviral loci were studied using restriction enzyme analysis and the Southern transfer procedure using liver DNAS from recombinant inbred strains between BALB/c and STS/A. All segregating MMTV-specific EcoRI fragments were identified to MMTV proviral loci and most of these were localized by studying the cosegregation of the Mtv units and known chromosomal markers. Since STS/A, GR.Mty-2-, and GR are highly related, the five complete endogenous MMTV proviruses of GR were located on the following chromosomes: Mtv-2 on chromosome 18, Mtv-3 on 11, Mtv-19 on 1, Mtv-20 on 4, whereas Mtv-8 has tentatively been located on chromosome 18 by Callahan et al. ( R. Callahan, D. Gallahan, and Ch. Kozak (1984), J. Virol., 49, 1005–1008). GR and GR.Mty-2 furthermore contain two incomplete MMTV proviral elements, one of which is also present in STS/A.

Mammary tumorigenesis in C3Hf/Ki mice: examination of germinal mouse mammary tumor viruses and the int-1 and int-2 putative proto-oncogenes.

The organization and expression of endogenous mouse mammary tumor virus (MMTV) proviruses in normal and neoplastic C3Hf/Ki tissues were examined. MMTV-containing EcoRI, HindIII, BamHI and PstI restriction fragments of C3Hf/Ki DNA were identical to those of C3H/StWi DNA. The full-length endogenous MMTV Units Ia (Mtv-7), II (Mtv-8), III (Mtv-9) and IV (Mtv-10), in addition to the subgenomic endogenous MMTV Units I (Mtv-6) and IX (Mtv-14), were germinally transmitted in C3Hf/Ki DNA. The previously uncharacterized Mtv-7 was contained in EcoRI fragments of 16.7 and 11.7 kbp. The endogenous MMTV Unit V (Mtv-1), which is responsible for virus production and mammary tumorigenesis in C3Hf/He mice, was absent from C3Hf/Ki DNA. The 9.0 kb gag-pol, the 3.8 kb env and the 1.7 kb LTR MMTV RNA transcripts were present in C3Hf/Ki mammary glands. MMTV proviruses, in addition to the endogenous C3Hf/Ki MMTV complement, were not detected in C3Hf/Ki mammary tumor DNA. The DNA organization and RNA expression of the putative mammary proto-oncogene regions int-1 and int-2 were also examined in C3Hf/Ki mammary tumors. The int-1 and int-2 regions did not appear rearranged, amplified, or expressed in C3Hf/Ki mammary tumors. These studies indicate that MMTV proviral activation of the int proto-oncogenes is not necessary for C3Hf/Ki mammary tumorigenesis.

Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure.

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.

Rearrangements in the Long Terminal Repeat of Extra Mouse Mammary Tumor Proviruses in T-Cell Leukemias of Mouse Strain GR Result in a Novel Enhancer-Like Structure

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.

Interaction of the heterozygous nude gene with the asplenia trait in mammary tumorigenesis.

The BALB/c mouse strain has been shown to contain endogenous mouse mammary tumor virus (MMTV) proviral sequences. However, no exogenous MMTV particles have been detected in their tissues. Female BALB/c mice from our colonies exhibit a very low incidence of spontaneous mammary tumors (SMT); less than 1% at up to 20 mo of age. Immunodeficient BALB/c mice heterozygous for the nude gene (nu/+, +/+), for the dominant hemimelia gene associated with asplenia (+/+, Dh/+), or for both traits (nu/+, Dh/+) have been examined for SMT incidence and the presence of MMTV proviruses. Based on restriction digestion with Eco RI, Bam HI, and Pst I, the immunodeficient mice have an MMTV provirus copy number and organization identical to the BALB/cCrgl strain. This MMTV DNA pattern is distinct from the MMTV proviruses in C3H/He, C57BL/6J and CBA/CaJ mice, which were parental strains of the immunodeficient mutants. Normal female BALB/c or BALB/c heterozygous for the asplenic trait do not develop significant numbers of SMT at up to 19 mo of age. In contrast, an incidence of 23.8% and 57.7% SMT was observed in BALB/c nu/+ heterozygotes, and in BALB/c nu/+, Dh/+ heterozygotes, respectively. These results indicate that agenesis of the spleen, concomitant with the presence of the heterozygous nude gene, contribute to a high incidence of SMT in the low-SMT BALB/c mouse strain.

The methylation pattern of endogenous mouse mammary tumor virus proviral genes is tissue specific and stably inherited

The methylation pattern of mouse mammary tumor virus (MMTV) proviral genes endogenous to the mouse strains C3H, 020, FM/JmsA, C57BL6, and BALB/c were investigated in various organs and mammary tumor tissue. Digestion of DNA with EcoRI or with EcoRI and HpaII followed by Southern blotting analysis and hybridization to a nick-translated MMTV DNA, allowed the distinction between the fully methylated and hypomethylated gene copies. MMTV proviral gene methylation was found to be organ specific, and the methylation pattern is stably inherited. The same proviral units present in different strains of mice exhibit the same organ-specific methylation patterns. Although proviral genes are normally heavily methylated in all tissues, hypomethylation of endogenous proviral genes was found in organs not known to express MMTV.

Dexamethasone Increases the Number of RNA Polymerase II Molecules Transcribing Integrated Mouse Mammary Tumor Virus DNA and Flanking Mouse Sequences

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.

Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.

Characterization and chromosomal location of endogenous mouse mammary tumor virus loci in GR, NFS, and DBA mice

Endogenous mouse mammary tumor virus (MMTV) proviral copies were characterized in three genetically dissimilar mouse strains: GR, a high-tumor-incidence strain bred in Europe that carries an MMTV proviral copy associated with early mammary tumors; DBA, a high-tumor-incidence laboratory strain bred in the USA with an endogenous copy that is associated with MMTV antigen expression in the milk; and NFS, a recently inbred line of the low-tumor-incidence NIH Swiss mouse. MMTV proviral loci were studied using restriction endonuclease analysis and the Southern transfer procedure in genetic crosses and in somatic cell hybrids. By studying the segregation of MMTV-specific EcoRI, BamHI, and PstI fragments, the organization of these fragments into MMTV proviral loci was determined and it was shown that (1) many homologous proviral loci are present in these three mouse strains, (2) these MMTV proviruses differ in their pattern of internal restriction sites, and (3) the MMTV loci are distributed on multiple chromosomes including 1 and 7.

Amplification and novel locations of endogenous mouse mammary tumor virus genomes in mouse T-cell lymphomas

Endogenous mouse mammary tumor virus genomes are amplified and located in novel cell DNA sequences in many mouse T-cell lymphomas. Transplanted tumors recovered from the same mouse strain and shown to be of independent origin by chromosomal analysis, by the presence of JH immunoglobulin gene rearrangements, or by the integration patterns of exogenous Moloney MuLV genomes frequently showed similar patterns of novel mouse mammary tumor virus-containing cell DNA fragments. This process of amplification and relocation can occur within a limited number of cell generations and in C57BL/6 mice does not lead to the synthesis of mature virus-encoded proteins. In some instances, amplified mouse mammary tumor virus genomes contained novel restriction cleavage sites in the gag-pol region. The restricted time course of occurrence, lack of synthesis of mature virion proteins, and apparent site specificity indicate that this process of retrovirus amplification differs significantly from virus replication after exogenous infection.

Mouse mammary tumor virus: specific methylation patterns of proviral DNA in normal mouse tissues.

The methylation state of endogenous mouse mammary tumor virus (MuMTV) proviral DNA was examined in normal mouse tissues. DNAs from various tissues were cleaved with the methylation-sensitive enzymes HhaI and HpaII and analyzed by Southern blotting. Tissue-specific MuMTV proviral DNA methylation patterns were found in the BALB/c, C3H, C57BL, GR/A, and GR-Mtv-2- mouse strains. MuMTV proviral DNA was hypomethylated in DNAs from the spleens and testes of all strains examined. The GR/A mouse strain, which was most thoroughly studied, also showed hypomethylation of MuMTV proviral DNA in bone marrow and placental tissues. Analysis of RNAs extracted from GR/A liver, mammary tumor, testes, placenta, and spleen tissues demonstrated that MuMTV proviral hypomethylation need not reflect significant proviral transcription.

Further Evidence for the Protein Coding Potential of the Mouse Mammary Tumor Virus Long Terminal Repeat: Nucleotide Sequence of an Endogenous Proviral Long Terminal Repeat

The 3' half of an endogenous mouse mammary tumor virus from a C3H mouse was cloned in the Charon 4A vector phage. A comparison of the proviral clone with previously published endogenous mouse mammary tumor virus restriction maps identified it as endogenous unit II (J. Cohen and H. Varmus, Nature [London] 278:418-423, 1979), which is present in all inbred mouse strains derived from the original Bagg albino x DBA cross. The nucleotide sequence of the unit II long terminal redundancy (LTR) was determined and compared with the sequence previously determined for the exogenous C3H virus LTR (Donehower et al., J. Virol. 37:226-238, 1981). Virtually all sequence differences between the two LTRs were base substitutions. The total amount of sequence divergence was 6.6%. The large open reading frame reported previously in the exogenous LTR was preserved in the endogenous LTR. In addition, the pattern of sequence divergence was highly nonrandom with respect to the putative amino acid codons of the two open reading frames. Most of the base substitutions in this region resulted in silent or conservative amino acid codon changes. The nonrandom divergence pattern indicates that selective forces are operating on this segment of DNA and argues that the putative protein is functional in the life cycle of mouse mammary tumor virus. Possible roles for the protein and its mode of expression are discussed.

Integration of new endogenous mouse mammary tumor virus proviral DNA at common sites in the DNA of mammary tumors of C3Hf mice and hypomethylation of the endogenous mouse mammary tumor virus proviral DNA in C3Hf mammary tumors and spleens.

To understand the molecular mechanisms by which the endogenous murine mammary tumor virus (MuMTV) proviruses are expressed and produce late-occurring mammary tumors in C3Hf mice, we analyzed, by the use of restriction enzymes and the Southern transfer procedure, genomic DNA from normal organs of mammary tumor-bearing and tumor-free mice and from 12 late-occurring C3Hf mammary tumors. We found, by using the restriction enzymes EcoRI and HindIII, that in addition to the preexisting endogenous MuMTV proviruses, new MuMTV-specific proviral DNA was integrated into new sites in the host genome in all 12 of the tumors that we examined. PstI digests of C3Hf tumor DNA revealed that the new proviral DNA found in C3Hf tumors was of endogenous origin. Moreover, the respective sizes of at least one of the new DNA fragments generated by EcoRI or HindIII digestion were the same in at least 50% of the C3Hf tumors analyzed, suggesting that the integration site of this new proviral DNA could be at the same location in the host genome of these tumors. Our results may imply that mammary tumorigenesis in C3Hf mice results from activation of cellular oncogenes by an MuMTV proviral DNA promoter. Specific hypomethylation of MuMTV proviral DNA was detected in the mammary tumors and spleens of C3Hf tumor-bearing mice. Our results indicated that most, if not all, of the hypomethylated MuMTV proviral DNA sequences were derived from the endogenous MuMTV provirus located at the MTV-1 locus, a locus responsible for the production of MuMTV antigens and increased incidence of mammary carcinoma in C3Hf mice. In spleens of non-tumor-bearing mice of ages 3, 6, 9, and 12 months, there was progressive hypomethylation of proviral DNA with increasing age, suggesting a possible correlation between demethylation of MuMTV proviral DNA in the spleens of C3Hf mice and the expression of endogenous MuMTV.

Cloned mouse mammary tumor virus DNA exhibits glucocorticoid-dependent expression in simian virus 40-transformed mink cells.

Mink lung epithelial cells were transfected with two cloned mouse mammary tumor virus (MMTV) DNAs, a 9-kilobase clone derived from an unintegrated exogenous viral genome and a 14-kilobase clone containing an integrated endogenous provirus along with cellular flanking sequences. Mink lung cells were chosen because they do not contain endogenous MMTV sequences. On the basis of our observation that simian virus 40 DNA efficiently transforms these cells, we isolated cell clones containing MMTV DNA by using transformation with simian virus 40 DNA as a selective marker in cotransfection experiments. Levels of the 9-kilobase MMTV mRNA representing the entire viral genome and of the spliced 4.4-kilobase mRNA which codes for the viral envelope proteins were glucocorticoid dependent in transformed cells. Expression of low levels of Pr77gag, the precursor of the group-specific viral core proteins, and of gPr73env, the precursor of the viral envelope proteins, was also hormone dependent. We conclude that these cloned MMTV DNAs contain all the information necessary for the synthesis of normal viral RNAs and proteins. These findings also provide further evidence that the DNA sequences involved in the hormone responsiveness of MMTV expression are contained within the viral genome.

Cloned endogenous mouse mammary tumor virus DNA is biologically active in transfected mouse cells and its expression is stimulated by glucocorticoid hormones

We have isolated an intact copy of endogenous mouse mammary tumor virus (MMTV) DNA with its adjacent cellular sequences from a gene library of A/J mouse parotid DNA. The bacteriophage λ DNA containing the viral genome was introduced into Ltk − mouse cells together with a plasmid containing the thymidine kinase ( tk) gene of herpes simplex virus. Tk + L cells containing the cloned MMTV DNA-synthesized virus-specific RNA and proteins. In all except one cell clone the viral gene expression was stimulated by the addition of dexamethasone. The degree of methylation of the active transfected viral DNA was much lower than that observed for the inactive resident endogenous proviruses present in untransfected and transfected mouse L cells.