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Mouse genetics meets molecular biology at Cold Spring Harbor. Mouse Molecular Genetics sponsored by the Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, August 29-September 2, 1990.

As stated earlier, the digestion of roughly 100 short talks delivered within a few days and containing very condensed information is, even for experts, quite demanding. Sometimes one felt in danger of becoming "psyched out" by all the experiments described; perhaps smaller group discussions dealing with experiments that have not worked, as well as the elimination of redundancies in the presentations, would have added to the flavor of the meeting. But what is the "take home message" from the 1990 CSH Mouse Meeting? With all the described breakthroughs, has mouse molecular genetics and development arrived at a turning point? I am inclined to answer this question with a "yes" on the basis of the following considerations: The elegant studies of development in Drosophila and Caenorhabditis elegans were made possible by the power of genetics with the use of developmental mutations. These studies taught us that the sequential activation of a hierarchy of regulatory genes dictates the temporal and spatial patterns of expression of proteins that define cell phenotypes and the body plan, and thereby control development. With the identification of important regulatory genes responsible for many classical as well as experimentally induced mouse mutations, in conjunction with traditional transgenic studies and the power of deleting and altering genes via ES cell chimeras, the study of mouse development has now gained an important new dimension. It is feasible that the consequences of subtle but precise genetic changes, such as the modification of regulatory elements or DNA-binding domains, can be studied in the whole organism by use of ES cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Phospholipid epitopes for mouse antibodies against bromelain-treated mouse erythrocytes.

The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with phospholipid epitopes was assessed by ELISA, using four clones of monoclonal anti-BrMRBC antibodies that had idiotypes distinct from one another. The four antibodies could bind to low-density lipoproteins (LDL) from human and chicken, but not to LDL from mouse and rat. As to liposomes of natural phospholipids, all the clones reacted with liposomes of phosphatidylcholine, and some of them could react with liposomes of sphingomyelin, phosphatidylglycerol, phosphatidylic acid or cardiolipin. For liposomes of synthetic phosphatidylcholine with different fatty acids, the length of carbon chains and the number of unsaturated carbon chains of the fatty acids markedly affected the binding of each monoclonal antibody to the liposomes. The addition of dicetyl phosphate or stearylamine to phosphatidylcholine liposomes changed the reactivity of the liposomes. These results support the view that mouse anti-BrMRBC antibodies can recognize appropriately spaced phosphorylcholine residues on the surface of phospholipid liposomes, LDL and cells. The four clones had similar capacities for binding to LDL as well as to BrMRBC, but they had obviously different capacities for binding to phospholipid liposomes; the epitopes on phospholipid liposomes used in the present study were not so perfect as to react well with every anti-BrMRBC antibody.

Animals↗

Expression of two cross-reactive idiotypes on mouse antibodies against bromelain-treated mouse erythrocytes.

Two cross-reactive anti-idiotype (Id) antibodies were previously prepared from sera of rabbits immunized with mouse monoclonal antibodies against bromelain-treated mouse erythrocytes (BrMRBC). Most of the anti-BrMRBC plaque-forming cells (PFC) were suppressed by either of the two anti-Id antibodies. The Id profiles of anti-BrMRBC PFC were almost identical among various cell populations in a strain, but different among various mouse strains. Mouse sera contained both of the Id-bearing immunoglobulins Ig, and a significant part of the Id-bearing Ig were eliminated by absorption with BrMRBC. Nude BALB/c mice were almost equal to normal BALB/c mice in the Id patterns of anti-BrMRBC PFC and in the concentrations of the Id-bearing Ig. The injections of anti-Id antibodies into suckling mice suppressed, specifically, the development of the B cells to produce the homologous Id-bearing Ig, but the injection of Id-bearing monoclonal antibodies barely affected Id expression. It is suggested that the two Id are encoded in germ-line genes of mice, and are expressed independently of each other and Id-anti-Id regulations by T cells or B cells.

Animals↗

Differential sensitivity of a mouse myeloid leukemia cell line and normal mouse bone marrow cells to X-ray-induced chromosome aberrations.

Cell line ML-1 was established from a myelogenous leukemia of an RFM mouse. The ML-1 cells and in vitro normal mouse bone marrow cells were analyzed to determine if there was a differential sensitivity to X-ray-induced chromosome aberrations in G1 cells and/or differences in postirradiation cell cycle progression. Cells identified as being in G1 at the time of irradiation by their staining pattern after replication in 5-bromodeoxyuridine were analyzed for all types of chromosomal aberrations following X-ray doses of 0.5, 1.0, 1.5, and 2.0 Gy. ML-1 cells showed a greater sensitivity to the induction of both chromosome-type aberrations (dicentrics and terminal deletions) and chromatid-type aberrations (exchanges and deletions) compared to normal mouse bone marrow cells, which only contained chromosome-type aberrations. The presence of chromatid-type aberrations in the ML-1 cells and not normal bone marrow cells suggested a differential progression through the cell cycle for the two cell types after irradiation. Mitotic index and flow cytometric analyses were performed and showed that both cell types have a delay in progression from G2 into mitosis, but only the normal mouse bone marrow cells have a delay in progression from G1 into S, as well as delayed progression through the S phase following X-irradiation. These results indicate that the ML-1 leukemia cells have an increased radiosensitivity. This may be due to a defect in their ability to respond to DNA damage as evidenced by their lack of a G1- and S-phase delay which allows normal cells an increased time to repair DNA damage before replication. These same characteristics have been observed in ataxia telangiectasia cells and may well represent a general feature of cells with increased radiosensitivity.

Animals↗

Enhancement of bactericidal activity of mouse peritoneal macrophages against Staphylococcus aureus by mouse interferon preparations.

The effect of mouse interferon on the bactericidal activity of macrophages against pyogenic cocci was examined. Mouse peritoneal macrophages were cultivated with Staphylococcus aureus in vitro and viable Staphylococcus was recovered by treatment of the mixed macrophage-bacteria culture with sodium dodecyl sulphate (SDS) solution. Results showed that S. aureus was phagocytized and killed by the macrophages. Mouse L cell interferon enhanced the bactericidal activity of macrophages. A mouse brain interferon preparation also enhanced this activity. However, heat-inactivated L cell interferon and heterologous rabbit RK-13 cell interferon and human leukocyte interferon did not enhance it. This suggests that interferon enhances the bactericidal activity of macrophages against S. aureus.

Animals↗

Anti-immunoglobulin autoantibodies are not preferentially induced in polyclonal activation of human and mouse lymphocytes, and more anti-DNA and anti-erythrocyte autoantibodies are induced in polyclonal activation of mouse than human lymphocytes.

Using a plaque assay with immunoglobulin (Ig)-coated SRBC, we and others have previously reported that the majority of polyclonally activated mouse lymphocytes secreted antibodies that appeared to be IgM anti-IgG autoantibodies. Careful reexamination of this assay, with application of several highly purified mouse serum and myeloma IgG and IgM preparations, revealed that IgM, which was a minor contaminant of Ig preparations, rather than IgG, was responsible for the formation of these plaques. High numbers of plaques could also be detected in assays with polyclonally activated human lymphocytes, Ig-coated SRBC, and anti-Ig developing sera. Of all IgG-, IgM- or IgA-secreting cells, 40 to 100% were detected with SRBC coated with gamma-globulin or Ig of the same isotype as the isotype to which the developing serum was specific; in general, low proportions of all PFC were detected with SRBC coated with Ig of a different isotype. Studies on the sequence of events leading to the formation of plaques with Ig-sensitized SRBC (both in humans and mice) revealed that antibodies detected in these assays were not able to bind to the Ig-coated SRBC (without the presence of developing serum), and therefore were not anti-Ig autoantibodies. It is our conclusion that the plaque assays with Ig-coated SRBC represent another type of a reverse hemolytic PFC assay that detects cells secreting antibodies regardless of their specificity, and these plaques are formed due to the cross-linking by the anti-Ig developing serum of the Ig coated on SRBC and the Ig secreted by lymphocytes. Our results confirmed preferential induction of anti-DNA antibody secreting cells in mice by showing that these antibodies indeed bind to DNA coated on SRBC. In cultures of polyclonally activated human lymphocytes, anti-DNA and anti-erythrocyte autoantibody-secreting cells were over 10 to 100 times less frequent than in mice. These results, therefore, disprove the concept of preferential induction of anti-Ig autoantibodies in the polyclonal activation of mouse and human lymphocytes, and show that anti-DNA and anti-erythrocyte autoantibodies are easily induced in the polyclonal activation of mouse, but not human, lymphocytes.

Adult↗

Redistribution of mouse spleen cell Fc receptors following treatment with mouse or human aggregated immunoglobulin G.

Mouse spleen cells treated with the Fc receptor ligands mouse IgG2b and human IgG, followed or not by a second antibody, exhibit different patterns of redistribution. In the work reported here we have examined the redistribution of Fc receptors (FcR) after binding of aggregated mouse IgG2b (Alg) or of Alg followed by anti-lg. We were particularly interested in learning whether binding of isologous Alg to FcR is followed by significant redistribution and shedding of Alg-FcR complexes. Mouse IgG2b alone will not induce capping even after 60 min at 37 degrees C. Human IgG induces some capping with minor shedding of complexes. Human IgG followed by anti-IgG readily induces capping by 15 min on 70% of the cells. This treatment also induces capping by 60 min at 20 degrees C on about 80% of the cells with a moderate degree of shedding of complexes. This is in agreement with the concept that the crosslinking required for FcR capping can be best induced with a second antibody. It is of interest, however, that heterologous IgG, unlike isologous, can induce a modest degree of capping and slight shedding even without the second antibody, suggesting that some crosslinking occurs with heterologous IgG.

Animals↗

Purification and identification of mouse lung microvessel endothelial cell-derived chemoattractant for lung-metastasizing murine RAW117 large-cell lymphoma cells: identification as mouse monocyte chemotactic protein 1.

We reported recently that medium conditioned with mouse lung microvessel endothelial cells possessed chemotactic activity for a highly lung-metastatic variant (L17) of the RAW117 murine large-cell lymphoma cell line but not for the poorly metastatic parental cells (P) or a liver-metastasizing variant (H10). The chemotactic factor was purified to homogeneity by a five-step procedure involving hydrophobic interaction, Cibacron blue F3GA affinity, metal affinity, anion exchange, and reversed phase chromatography, followed by preparative gel electrophoresis. The purified material appeared as a single broad band when analyzed by SDS-PAGE, with an average molecular weight of 26,000. The factor was cleaved by cyanogen bromide treatment, and a partial amino acid sequence of one of the cleaved polypeptides proved identical to mouse monocyte chemotactic protein 1 (mMCP-1/JE). The amino acid composition of the factor also indicated similarity to mMCP-1/JE. Separately purified mMCP-1/JE significantly stimulated the chemotactic migration of RAW117 cells (L17 >> H10, P). When recombinant human monocyte chemotactic protein 1 was compared to the purified endothelial cell chemotactic factor as a chemoattractant, similar migratory responses were observed in the RAW117 sublines. The chemotactic activity for L17 cells was significantly reduced from lung microvessel endothelial cell-conditioned medium after treatment with anti-mouse MCP-1 antibody. In contrast, the migration-stimulating activity of liver sinusoidal endothelial cell-conditioned medium to H10 cells was not affected by anti-mouse MCP-1. A major function of mMCP-1/JE is to recruit monocytes to inflammatory sites, and our results suggest that mMCP-1/JE also facilitates lymphoma lung invasion and metastasis.

Amino Acid Sequence↗

Nude mouse interim host model for human parathyroid grafts. Part IV. Human PTG xenograft rejection in a mouse B lymphocyte system.

An interim host model for human parathyroid gland (PTG) grafts has been established. In this experiment, the proliferation of mouse blood vessels and infiltration of mouse B lymphocytes into human PTG grafts were successively observed. The results showed that within 10 d of interim hosting, when PTG grafts were still nourished by permeable factors, scattered mouse B lymphocyte infiltration was already apparent. As time went by, B lymphocyte infiltration and IgG secretion became more obvious. At 60-90 d interim hosting, an intact human-mouse tissue "chimera" was formed. Thereafter, human PTG grafts were gradually fragmented and absorbed. The theoretical significance and clinical value of this rejection phenomenon are discussed.

Animals↗

The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor. Comparison of all 10 variable exons between mouse, human, and rat.

The human CD44 cell surface glycoprotein family which has been implicated in lymphocyte homing, tumor metastasis, and extracellular matrix attachment consists of a large number of related isoforms that derive from the differential splicing of a single CD44 gene transcript. Recent mapping of the CD44 locus in man indicated the presence of at least nine alternative exons within the region of the gene encoding the variable membrane proximal extracellular domain. Here we report the identification of a 10th alternative exon (termed V1) in the mouse, human, and rat CD44 genes. We demonstrate tissue-specific patterns of expression for transcripts containing exons V1-V10 in the mouse and a highly restricted usage of exon V1 in transcripts from mouse gastric tissue. Close sequence homology between exons V1-V10 from mouse rat and human points to a specific functional role rather than a purely structural role for the membrane proximal extracellular domain of the CD44 molecule.

Alternative Splicing↗

Prevalence of enterotropic and polytropic mouse hepatitis virus in enzootically infected mouse colonies.

Mouse hepatitis virus (MHV) causes the most prevalent viral infection in contemporary laboratory mouse colonies. According to their primary replication site, different strains of MHV segregate into two overlapping biotypes, enterotropic and polytropic. These two groups vary greatly in disease pattern, pathogenicity, immune response, and duration of infection. Historically, the polytropic MHV strains represent the extensively studied prototype strains that have minimal enterotropism, whereas enterotropic MHV strains have been less characterized. Anecdotal reports suggest that most MHV strains encountered today belong to the enterotropic biotype. We have identified 15 isolates of MHV from 19 independent enzootically infected mouse colonies. Sequencing of a variable region of the nucleoprotein (N) gene of each isolate confirmed that all were independent genetic variants. The principal tissue tropism of the new isolates was determined by experimental inoculation of infant mice and examination of intestine, liver, spleen, and brain for lesions. Nine isolates infected only intestine; four isolates infected intestine and liver; one isolate infected intestine, liver, and brain; and one isolate infected liver. These results confirm that the enterotropic MHV biotype is predominant in contemporary laboratory mouse colonies. The MHV biotype features need to be taken into consideration when dealing with MHV infections.

Animals↗

Cloning of an infectious milk-borne mouse mammary tumor virus (MMTV) DNA from a mammary tumor that developed in an endogenous MMTV-free wild mouse.

Molecular characterization of infectious mouse mammary tumor viruses (MMTVs) has been hampered due to the problem of cloning a full-length exogenous virus into a plasmid. The present report describes our strategy for obtaining a full-length clone of an exogenous MMTV from a mouse mammary tumor that arose spontaneously in a wild Chinese mouse free of endogenous MMTV and shows that the cloned virus (JYG-MMTV) is expressed in rat RBA cells. Four-week-old C58/J x CBA/CaJ female mice, free of both endogenous and exogenous MMTVs, were injected with virus-secreting RBA cells. The progeny of these mice were bred, and their offspring were tested for the presence of MMTV. These third-generation mice were found to actively produce MMTV that was shed in their milk and transmitted to their offspring. The virus was detected not only in the mammary glands of these young mice, but also in their spleens and bone marrow. These results suggest that our plasmid-cloned exogenous JYG-MMTV is infectious. This virus can now be used effectively in manipulating the various genes of JYG-MMTV and other MMTV strains to understand their structure/function relationships.

Animals↗

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.Mtv-2-; these strains share also most of the isoenzyme markers and are therefore highly related. Cellular DNA of GR.Mtv-2- contains a partial MMTV provirus that is not present in STS/A. GR.Mtv-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.Mtv-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.Mtv-2 furthermore contain two incomplete MMTV proviral elements, one of which is also present in STS/A.

Animals↗

Structure, stability, methylation, expression and glucocorticoid induction of endogenous and transfected proviral genes of mouse mammary tumor virus in mouse fibroblasts.

A sequence of mouse genomic DNA containing an endogenous mouse mammary tumor virus (MMTV) provirus, which was isolated by molecular cloning in lambda vector phage, has been reintroduced into cultured mouse L cells, using the thymidine-kinase cotransfection technique. Individual cell clones that acquired the transfected MMTV proviral DNA have been isolated. The transfected DNA remains stable in these cells and does not become methylated. It is transcribed into MMTV RNA, And the transcription is stimulated by glucocorticoid hormones.

Animals↗

Mouse transferrin receptor 1 is the cell entry receptor for mouse mammary tumor virus.

Enveloped viruses enter cells by binding to their entry receptors and fusing with the membrane at the cell surface or after trafficking through acidic endosomal compartments. Species-specific virus tropism is usually determined by these entry receptors. Because mouse mammary tumor virus (MMTV) is unable to infect Chinese hamster cells, we used phenotypic screening of the T31 mouse/hamster radiation hybrid panel to map the MMTV cell entry receptor gene and subsequently found that it is transferrin receptor 1. MMTV-resistant human cells that expressed mouse transferrin receptor 1 became susceptible to MMTV infection, and treatment of mouse cells with a monoclonal antibody that down-regulated cell surface expression of the receptor blocked infection. MMTV, like vesicular stomatitis virus, depended on acid pH for infection. MMTV may use transferrin receptor 1, a membrane protein that is endocytosed via clathrin-coated pits and traffics through the acidic endosomes, to rapidly get to a compartment where acid pH triggers the conformational changes in envelope protein required for membrane fusion.

Animals↗

Wnt-3, a gene activated by proviral insertion in mouse mammary tumors, is homologous to int-1/Wnt-1 and is normally expressed in mouse embryos and adult brain.

We have isolated a common insertion site, Wnt-3, for proviruses of the mouse mammary tumor virus (MMTV). Of mammary tumors induced by the GR variant of MMTV, 5% contains a provirus at Wnt-3, which is located on mouse chromosome 11. The gene is transcribed into a 3.8-kilobase (kb) mRNA in tumors with nearby proviral insertions but not in tumors with proviruses at other loci or in most adult tissues. Normal expression of Wnt-3 is detected in mouse embryos (with a peak around day 12 of gestation) and at low levels in adult brain. The transcriptional unit of the Wnt-3 gene spans approximately 55 kb, with a first intron of 36 kb. The deduced amino acid sequence of the Wnt-3 protein is 47% identical to the int-1/Wnt-1 gene product.

Amino Acid Sequence↗

The endogenous proviral mouse mammary tumor virus genes of the GR mouse are not identical and only one corresponds to the exogenous virus.

The endogenous proviral copies of mouse mammary tumor virus (MMTV) were selected from a gene library of GR mouse DNA. We obtained five different lambda. MMTV recombinant clones. Four of them correspond to the 3' Eco RI fragments of the endogenous proviruses an one comprises an intact MMTV provirus with 2 to 3 kb of flanking mouse genomic DNA. Heteroduplex formation followed by S1 digestion under stringent conditions shows that there is nucleotide sequence heterology among the cloned endogenous proviral copies. Only one endogenous proviral copy, associated with the mtv-2 locus, was found to be totally homologous to the exogenous proviral DNA.

Animals↗

Molecular biological characterization of a highly leukaemogenic virus isolated from the mouse. III. Identity with mouse mammary tumour virus.

A highly leukaemogenic virus isolate (DMBA-LV) endogenous to the CFW/D mouse has been found to contain two viral genomes. One was closely related to the type B milk-borne mouse mammary tumour virus (MMTV) and present in tenfold excess over a type C viral genome which was only partially related to xenotropic and polytropic isolates from the CFW/D mouse as well as to the ecotropic Moloney murine leukaemia virus isolate. The thymic lymphoma cell line that produced DMBA-LV expressed high levels of MMTV viral RNA (35S and the 24S envelope mRNA). Both the virus and the virus-producing cell line expressed multiple species of type C viral RNA. Similar species of type C viral RNA were also associated with non-infectious, non-leukaemogenic viral particles present in both normal lymphoid cells and in a MMTV-free thymic lymphoma cell line established from a second chemical carcinogen-induced tumour.

Animals↗