Role of mink cell focus-inducing virus in leukemias induced by Friend ecotropic virus

Recombinant viruses have been implicated in the pathogenesis of murine leukemias induced by a variety of long-latency retroviruses. Neonatal mice of several strains were inoculated with Friend ecotropic virus (F-Eco) and analyzed for the presence of mink cell focus-inducing (MCF) virus or DNA restriction enzyme fragments which were specific for Friend MCF virus (F-MCF). MCF virus was detected within 2 weeks of inoculation in NFS /N mice and at about 2 months after inoculation in BALB/c mice. Both of these strains developed erythroblastosis after inoculation with F-Eco. In contrast, MCF virus was not detected in F-Eco-inoculated C57BL mice. These mice were resistant to erythroblastosis but developed lymphoma or myelogenous leukemia or both at about 5 months after inoculation. Thus, although MCF viruses were associated with F-Eco erythroblastosis in NFS /N and BALB/c mice, they were not necessary for F-Eco-induced lymphoid or myeloid leukemias in C57BL mice. To investigate the association between resistance to erythroblastosis and absence of MCF virus, C57BL mice were inoculated with pseudotypic mixtures of F-Eco plus F-MCF; MCF virus replicated well in these mice, but the mice remained resistant to erythroblastosis. Furthermore, in genetic crosses between C57BL and NFS /N or BALB/c, some mice inherited resistance to F-Eco erythroblastosis without inheriting the C57BL resistance to the generation of MCF viruses. These results indicate that C57BL mice carry a gene for resistance to F-Eco erythroblastosis which is distinct from the C57BL genes which interfere with the generation of MCF viruses.     

Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice.

We used virus assay and in situ hybridization with a cloned fragment of the murine cytomegalovirus (MCMV) genome to study MCMV infection of circulating leukocytes harvested from 3-week-old BALB/c, C57BL/6, and C3H mice infected with MCMV intraperitoneally. Infectious virus or MCMV DNA was detected in leukocytes on days 1 through 21 of infection in BALB/c mice and on days 3 through 7 in C57BL/6 mice. On days 5 and 7, MCMV DNA or infectious virus was detected in the leukocytes of 17 (94%) of 18 BALB/c mice and 10 (59%) of 17 C57BL/6 mice. In both strains infection peaked on days 5 and 7, when as many as 0.01 to 0.1% of the circulating leukocytes contained MCMV DNA. In C3H mice, however, infectious virus was rarely recovered from leukocyte fractions and MCMV DNA was detected in the circulating leukocytes of only one animal. Circulating leukocytes may have an important role in the dissemination of CMV infections in susceptible hosts.     

Regional linkage analysis of the dioxin-inducible P-450 gene family on mouse chromosome 9

The dioxin-inducible P-450 gene family in the C57BL/6N mouse comprises two genes, P1-450 and P3-450. Restriction endonuclease-digested genomic DNA was probed with P1-450 and P3-450 full-length cDNA clones in an attempt to find species-specific fragment length differences between mouse and hamster cell lines and any restriction fragment length polymorphism among four inbred mouse strains. With this Southern blot hybridization technique, PstI fragments were used to distinguish between the mouse and hamster P1-450/P3-450 genes, and PvuII fragments were used to distinguish P3-450 differences between the AKR/J and C57L/J inbred strains. Analysis of nineteen mouse X hamster somatic cell hybrid lines and sixteen AKXL (AKR/J X C57L/J) recombinant inbred lines showed that the P1-450/P3-450 genes are located near the Mpi-1 locus, between the Thy-1 and Pk-3 loci, in the middle portion of mouse chromosome 9.     

Location of DNA sequences complementary to small nuclear repeat RNA (fr 3-RNA) in relation to DNA sequences coding for albumin and alpha-fetoprotein in rat liver cells

Rat liver nuclei contain a 29-nucleotides-long RNA (fr 3-RNA) which is transcribed from middle repetitive DNA sequences. By Southern analysis of restriction fragments of rat albumin and alpha-fetoprotein genomic clones, DNA sequences complementary to this RNA were detected on a 4.6 kbp Eco RI fragment located 600 bp downstream from the termination exon of the albumin gene and on a 2 kbp Eco RI-HindIII fragment located 10 kbp downstream from the restriction fragment containing the alpha-fetoprotein site. No sequence complementary to this RNA was found either in the introns of exons of both genes or in the regions extending 7 kbp upstream from the first albumin exon and 10 kbp upstream of the first alpha-fetoprotein exon. We concluded that sequences complementary to fr 3-RNA are present at the 3'-end flanking regions of the rat albumin and alpha-fetoprotein gene complexes.     

Location of DNA sequences complementary to small nuclear repeat RNA (fr 3-RNA) in relation to DNA sequences coding for albumin and α-fetoprotein in rat liver cells

Rat liver nuclei contain a 29-nucleotides-long RNA (fr 3-RNA) which is transcribed from middle repetitive DNA sequences. By Southern analysis of restriction fragments of rat albumin and α-fetoprotein genomic clones, DNA sequences complementary to this RNA were detected on a 4.6 kbp EcoRI fragment located 600 bp downstream from the termination exon of the albumin gene and on a 2 kbp EcoRI-HindIII fragment located 10 kbp downstream from the restriction fragment containing the α-fetoprotein site. No sequence complementary to this RNA was found either in the introns of exons of both genes or in the regions extending 7 kbp upstream from the first albumin exon and 10 kbp upstream of the first α-fetoprotein exon. We concluded that sequences complementary to fr 3-RNA are present at the 3′-end flanking regions of the rat albumin and α-fetoprotein gene complexes.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Antibody response against hamster red blood cells (H-RBC) was examined in inbred strains of C57BL/6, AKR, C3H/He, DDD and SL mice, and outbred CF1 mice. 1) There were strain differences in antibody response after a primary intravenous injection of H-RBC. DDD, SL and CF1 mice belonged to high-responder strains, while C57BL/6, AKR and C3H/He to low-responder strains. In the spleens of immunized CF1 and SL, 40 to 70 times as many plaque-forming cells (PFC) as those in C57BL/6 mice were detected. The magnitudes of the response were: CF1 ≒ SL>DDD>>C3H/He ? AKR>C57BL/6. 2) 2-mercaptoethanol resistant (MER) antibody was detected in neither low- nor high-responders after a primary intravenous antigen-injection. 3) After a secondary intravenous antigen-injection, MER antibody was detected in all the SL mice, but only in 30 to 50% of AKR and C57BL/6 mice. 4) A subcutaneous injection of H-RBC in Freund's complete adjuvant (FCA) did not elicit antibody production within 10 days. When mice pre-sensitized 7 days in advance w^Tith H-RBC in FCA were intravenously injected with H-RBC, enhanced antibody production of the primary type was observed in all the mouse strains. 5) In pre-sensitized mice, the extent of the enhancement of antibody production was the highest in low-responder C57BL/6 mice and the lowest in high-responder SL and CF1 strains. Thus, there was no strain difference in antibody titers or the numbers of PFC after the booster.     

In vitro splenic T cell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain JHM.

Mortality rates among BALB/cByJ, A/JCr, C3H/HeSnJ, and C57BL/6NCr mice inoculated oronasally with mouse hepatitis virus (MHV) strain JHM, ranged from 25 to 67%. Spleen cells harvested from the first three genotypes at 5 days postinoculation proliferated poorly in response to concanavalin A stimulation and produced significantly less interleukin (IL) 2 than cells from uninfected control mice. The function of spleen cells harvested at 14 days postinoculation varied and was host genotype-dependent. Despite clinical signs among some infected C57BL/6NCr mice, spleen cell function was relatively unaffected. C57BL/10ScNCr, B10.A, and SJL/JCr mice remained clinically normal after MHV inoculation. Proliferation and IL2 production by cells from inoculated C57BL/10ScNCr and B10.A mice were similar to responses of their respective controls. In contrast, cells from inoculated SJL/JCr mice were hyper-responsive and produced peak levels of IL2 earlier than control cells. Among the seven genotypes tested, only BALB/cByJ and C3H/HeSnJ spleen cells produced detectable IL4 after primary stimulation with concanavalin A or after priming and restimulation. Primary IL4 production by cells from these two genotypes was significantly reduced if donors were inoculated with MHV 5 days prior to spleen harvest. IL4 production by cells from acutely infected BALB/cByJ mice was considerably enhanced by priming and restimulation.     

Neospora caninum: high susceptibility to the parasite in C57BL/10ScCr mice

C57BL/10ScCr mice, lack Toll-like receptor 4 and a functional Interleukin-12 receptor. Taking this into account, susceptibility of these mice to Neospora caninum infection was assessed comparatively to that of immunocompetent C57BL/10ScSn mice. C57BL/10ScCr mice inoculated intraperitoneally with 5x10(5)N. caninum tachyzoites showed a high susceptibility to this parasite. All infected C57BL/10ScCr mice were dead by day 8 post-infection whereas all control C57BL/10ScSn mice survived this parasitic challenge. Immunohistochemical analysis of infected C57BL/10ScCr mice showed N. caninum tachyzoites spread in the pancreas, liver, lung, intestine, heart and brain whereas no parasites were detected in similarly infected C57BL/10ScSn controls. The higher susceptibility of C57BL/10ScCr mice to neosporosis correlates with reduced interferon-gamma mRNA expression and increased IL-4 mRNA expression, comparatively to C57BL/10ScSn controls, detected in the spleen after the parasitic challenge. C57BL/10ScCr mice could thus be used as a new experimental model where to study immunobiological mechanisms associated with host susceptibility to neosporosis.     

Molecular cloning, characterization, and genetic mapping of an endogenous murine mammary tumor virus proviral unit I of C3H/He mice.

We have cloned and characterized a novel endogenous murine mammary tumor virus proviral unit of the C3H/He strain of mice. The cloned proviral unit is 16 kilobase pairs (kbp) in size and is composed of a 5.6-kbp 5' EcoRI segment of an endogenous provirus with 10.4-kbp flanking cellular sequences. A comparison of the restriction map of the cloned proviral DNA with that of an endogenous provirus of the GR strain of mice has revealed minor differences in restriction sites on the two proviruses. The restriction enzyme SstI, which does not cleave the 5' EcoRI fragment of GR DNA, cleaves the C3H/He proviral sequences once; MspI has an additional site in the C3H/He proviral sequences. By using a subcloned fragment containing unique cellular sequences as a hybridization probe, we (i) mapped the C3H/He proviral unit to chromosome 14 by using mouse-hamster somatic cell hybrids, and (ii) demonstrated that this proviral unit is also present in the genome of DBA/2 mice. From these results we conclude that the C3H/He strain of mice acquired this proviral unit from DBA stock by genetic transmission. Our data also indicate that the murine mammary tumor virus sequences present in the gag-specific proviral unit of C3H/He mice extend at least 2.45 kbp downstream of the EcoRI site in the genomic DNA. Since the structural organization and chromosomal location of this proviral unit are distinct from those of previously reported proviral units represented by similar-sized (16.7-kbp) EcoRI fragments, we tentatively propose to designate this proviral unit Mtv-7a.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

It was confirmed by passive transfer experiments that the function of thymus-derived cells specific for hamster erythrocytes (HRBC) was deficient in the low-responder mouse strains. 1) Antibody production against HRBC was enhanced by passive transfer of thymus cells from normal SL mice (high-responder) to normal C57BL/6 mice (low-responder). 2) The enhancing effect of passive transfer of thymus cells from SL mice was abrogated by pre-sensitization of the recipients (C57BL/6) with thymus cells from SL mice. 3) In C57BL/6 mice, antibody production against HRBC was enhanced by the transfer of lymph node or spleen cells from C57BL/6 mice which had been sensitized with HRBC in Freund's complete adjuvant or hamster lymphoma cells.     

Incomplete proviral genome of mouse mammary tumour virus is present on chromosome Y of NIH Swiss and some genetically related mouse strains.

An incomplete proviral genome of endogenous mammary tumour virus (MMTV) was found in DNA of several strains of mice. This MMTV-related sequence was assigned to the Y chromosome since it was clearly observed in male mice only. This MMTV provirus contained a sequence related to LTR (long terminal repeat), but not to gag-pol and env genes. NFS, NIH Swiss/S, STS/A, and DD/Tbr mice have this sequence but BALB/cHeA, SHN, SLN, C57BL/6NJcl, C3H/HeNJcl and CBA/JJcl mice are negative. In the strains containing this sequence, a DNA test for the sequence makes it possible to easily distinguish the DNAs of male or female mice.     

Segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine IFN-beta gene to the murine IFN-alpha genes.

Southern blot analysis with murine (Mu) interferon (IFN)-alpha cDNA of restricted genomic DNA of three inbred strains of mice belonging to the species Mus musculus domesticus (BALB/c, C57BL/6, and DBA/2) revealed only a limited degree of polymorphism. For example, with HindIII there were only two polymorphic bands out of 14 hybridizing fragments. With Mu IFN-beta cDNA there was no polymorphism at all between BALB/c and C57BL/6 in DNA restricted with seven different enzymes. In contrast, HindIII-restricted DNA of an inbred strain of wild mice (M. spretus Lataste) hybridized with the IFN-alpha probe displayed a high degree of polymorphism compared with the three strains of laboratory mice and was also polymorphic when probed with IFN-beta cDNA. Although M. musculus domesticus and M. spretus Lataste represent different species, certain interspecies crosses are possible in the laboratory. This enabled us to follow segregation of restriction fragment length polymorphism in HindIII-restricted DNA obtained from 18 backcross progeny of a (DBA/2 X M. spretus)F1 X DBA/2 interspecies cross. There was complete coincidence between the segregation of parental (DBA/2) and (DBA/2 X M. spretus)F1-type IFN-beta and IFN-alpha restriction fragment length polymorphism, indicating tight linkage of the IFN-beta and IFN-alpha genes. In addition, in 15 of 18 progeny the segregation coincided with that of the brown locus on chromosome 4, in accord with previous results obtained with the IFN-alpha probe in strains derived from crosses between BALB/c and C57BL/6 mice. Thus, the Mu IFN-beta gene is tightly linked to the Mu IFN-alpha gene cluster on chromosome 4 near the brown locus.     

Genetic basis for unresponsiveness to lipopolysaccharide in C57BL/10Cr mice

The unresponsiveness to LPS detected in C57BL/10Cr mice is inherited as a recessive trait and is determined by an autosomal gene linked to theMup-1 locus on chromosome 4. Since no complementation for LPS responsiveness was observed in F₁ hybrid mice between C3H/HeJ and C57BL/10Cr, we conclude that C57BL/10Cr mice carry a defective allele at the sameLps locus, previously identified by the mutation detected in the C3H/HeJ strain.     

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.     

Differences in interleukin-12 and -15 production by dendritic cells at the early stage of Listeria monocytogenes infection between BALB/c and C57 BL/6 mice

The mechanisms responsible for the resistance of C57BL/6 mice and for the susceptibility of BALB/c mice to infection with Listeria monocytogenes were studied by comparing early IL-12 and IL-15 production by dendritic cells (DC) after infection with L. monocytogenes. Splenic DC expressing CD11b(low) and CD11c(+) obtained from C57BL/6 mice at 3 and 6 h after L. monocytogenes infection expressed higher levels of IL-12 p40 mRNA and IL-12 p40 protein than did those from BALB/c mice. Concurrently, a larger amount of IFN-gamma was produced by the splenic T cells from C57BL/6 mice in response to immobilized anti-TCRalphabeta mAb than by those from BALB/c mice, while the splenic T cells from BALB/c mice produced a higher level of IL-4 upon TCR alphabeta stimulation than did those of C57BL/6 mice. IL-15 mRNA and intracellular IL-15 protein were detected more abundantly in the DC from C57BL/6 mice than in those from BALB/c mice on day 3 after infection. CD3(+) IL2Rbeta (+) cells in the spleen were increased in C57BL/6 mice but not in BALB/c mice at the early stage after infection. Furthermore, IL-12Rbeta2 gene expression was up-regulated in T cells from C57BL/6 mice but not in those from BALB/c mice at the early stage after listerial infection. These results suggest that the difference in early production of IL-12 and IL-15 by DC may at least partly underlie the difference in susceptibility to L. monocytogenes between C57BL/6 and BALB/c mice.     

Maternal contamination of some NZB sublines and genetic profiles of NZ-strains

Six sublines of NZB mice bred in Japan were collected and their mitochondrial DNA (mtDNA) was examined by restriction analysis. The phenotypes of at least three of these sublines (NZB/Nrs, NZB/Nga and NZB/KlJms) differed from a standard one (NZB/BlWehi). Since mtDNA is inherited maternally, all sublines of a single inbred strain should share the same mtDNA phenotype. Therefore, b-type of mtDNA should be observed in all NZB sublines. Nevertheless, the above-mentioned sublines showed d-type mtDNA. These results suggested a genetic contamination of these sublines. This was confirmed by the finding that six aberrant alleles were detected also in their nuclear genomes using biochemical markers. For elucidation of the cause of contamination, we characterized the genetic profiles of four standard NZ-strains, NZB/BlWehi NZO/BlWehi, NZC/BlWehi and NZX/BlWehi, and of common inbred strains with black coat color, C57BL/6J, C57BL/10Sn, C57BL/Ks, C58/J and AU/SsJ. We found that five of the six aberrant alleles most strongly corresponded with those of C57BL/Ks. These results suggest that this contamination was ascribable to cross of NZB mice with a certain C56BL strain. We also deduced that NAB/BlPt and NZB/Füll also probably were contaminated strains, suggesting that this contamination was not restricted to Japan.     

High-throughput sequencing of Astrammina rara: Sampling the giant genome of a giant foraminiferan protist

Background

C57BL/6J mice possess a single intelectin (Itln) gene on chromosome 1. The function of intelectins is not well understood, but roles have been postulated in insulin sensitivity, bacterial recognition, intestinal lactoferrin uptake and response to parasites and allergens. In contrast to C57BL/6J mice, there is evidence for expansion of the Itln locus in other strains and at least one additional mouse Itln gene product has been described. The aim of this study was to sequence and characterise the Itln locus in the 129S7 strain, to determine the nature of the chromosomal expansion and to inform possible future gene deletion strategies.

Results

Six 129S7 BAC clones were sequenced and assembled to generate 600 kbp of chromosomal sequence, including the entire Itln locus of approximately 500 kbp. The locus contained six distinct Itln genes, two CD244 genes and several Itln- and CD244-related pseudogenes. It was approximately 433 kbp larger than the corresponding C57BL/6J locus. The expansion of the Itln locus appears to have occurred through multiple duplications of a segment consisting of a full-length Itln gene, a CD244 (pseudo)gene and an Itln pseudogene fragment. Strong evidence for tissue-specific distribution of Itln variants was found, indicating that Itln duplication contributes more than a simple gene dosage effect.

Conclusions

We have characterised the Itln locus in 129S7 mice to reveal six Itln genes with distinct sequence and expression characteristics. Since C57BL/6J mice possess only a single Itln gene, this is likely to contribute to functional differences between C57BL/6J and other mouse strains.     

Interleukin 1beta (IL1B) signaling is a critical component of radiation-induced skin fibrosis

Interleukin 1 beta (IL1B), a potent pro-inflammatory cytokine, is directly up-regulated by radiation and is known to regulate other inflammation-related molecules, such as the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). However, the nature of the interaction of IL1B with MMPs and TIMPs in radiation-induced skin fibrosis is unknown. We examined the response of primary dermal keratinocytes, fibroblasts and endothelial cells to single-fraction radiation (10 Gy) and compared the results to a temporal sequence of histology from irradiated C57BL/6 and IL1R1 knockout mice. These studies showed that keratinocytes are the major IL1-producing cells in vitro and that radiation induces an immediate and chronic elevation in the expression of IL1B mRNA in the skin of C57BL/6 mice. This elevation was principally early and was less pronounced in the IL1R1 knockout strain, which also demonstrated reduced late radiation fibrosis. Radiation also increased expression of MMP mRNA in C57BL/6 mice. Finally, exogenous IL1B protein induced robust endogenous IL1B mRNA expression, along with a brisk increase in MMPs and collagen III, but only in the C57BL/6 mice. In conclusion, these data suggest that IL1B plays a critical role in radiation-induced fibrosis and that the increased MMPs fail to block the IL1-related collagen accumulation.