Peromyscus maniculatus--Mus musculus chromosome homology map derived from reciprocal cross species chromosome painting.

The Mus musculus and Rattus norvegicus genomes have been extensively studied, yet despite the emergence of Peromyscus maniculatus as an NIH model for genome sequencing and biomedical research much remains unknown about the genome organization of Peromyscines. Contrary to their phylogenetic relationship, the genomes of Rattus and Peromyscus appear more similar at the gross karyotypic level than either does to Mus. We set out to define the chromosome homologies between Peromyscus, Mus and Rattus. Reciprocal cross-species chromosome painting and G-band homology assignments were used to delineate the conserved chromosome homology map between P. maniculatus and M. musculus. These data show that each species has undergone extensive chromosome rearrangements since they last shared a common ancestor 25 million years ago (mya). This analysis coupled with an inferred homology map with Rattus revealed a high level of chromosome conservation between Rattus and Peromyscus and indicated that the chromosomes of Mus are highly derived.

Erythroid cell fusion in the early phase of Friend virus leukemogenesis.

Infection of susceptible strain mice with the oncogenic Friend erythroleukemia virus initially results in fulminant erythroid hyperplasia. Several weeks later a frank erythroid leukemia develops. At the earliest stages of Friend disease there is extensive cell fusion involving erythroid cells but not platelets and granulocytes. Fusion was detected in experiments with allophenic (chimeric) mice whose component strains express electrophoretically distinct forms of the dimeric enzyme glucose phosphate isomerase (GPI). Infection of such animals with the polycythemic strain of Friend virus resulted in the rapid development of Friend disease. Concomitant with the appearance of early disease symptoms was the appearance in the red cells of the heterodimeric form of GPI, an unequivocal consequence of cell fusion. Platelet and granulocyte samples from the same infected animals failed to exhibit the hybrid GPI form. Furthermore, no hybrid dimer was evident in red cells from chimeric mice in which blood formation had been stimulated by phenylhydrazine treatment. These observations suggest that the occurrence of cell fusion early after infection by Friend virus is a significant aspect in the rapid development of neoplastic disease.

Single locus (rol) control of extreme resistance to red cell osmotic lysis: intrinsic mode of gene action.

Previous work has indicated that inbred mouse strains C57BL/6 and DBA/2 produce red cells differing in their sensitivity to osmotic lysis and that the trait is under multigene control. A recombinant inbred strain (BXD-31), produced from C57BL/6 and DBA/2, has red cells manifesting resistance to osmotic lysis far greater than that of either progenitor. We demonstrate here that the fragility difference between BXD-31 and DBA/2 is the consequence of allelic variation at a single autosomal locus, termed rol. The resistance allele (rol') is almost completely recessive to the sensitive one (rols). Results of bone marrow chimera analyses indicate that (1) the mode of rol gene action is by a direct influence on the properties of the red cells rather than an indirect influence on their extracellular milieu, and (2) rol does not affect erythrocyte production and turnover. The fragility difference caused by rol variation is likely to involve the erythrocyte membrane or underlying cytoskeleton, since various red cell properties sensitive to ion metabolism differences are unaffected by the gene.

Genotype-limited changes in platelet and erythroid kinetics in Friend-virus-infected allophenic mice.

We report here results suggesting that cells of the megakaryocytic lineage or uncommitted precursor cells may be targets for Friend-virus-induced proliferation, and that genetic differences (other than Fv-2) between strains C57BL/6 and DBA/2 affect the susceptibility of these cells to Friend virus. The evidence suggesting this was derived from experiments with C57BL/6 in equilibrium DBA/2 allophenic mice. Within the first few weeks following infection of these mice with the polycythemic NB-tropic strain of Friend virus (FV-P), we observed a rapid shift in the genotypic composition of both red cells and platelets in favor of those of the DBA/2 genotype. Infection with the anemia-inducing strain of Friend virus (FV-A) also resulted in preferential production of DBA/2 strain erythrocytes, but its effect on platelet kinetics was nil. The FV-P- and FV-A-induced change in red cell composition is consistent with the view that erythroid precursors are target cells for Friend virus and that viral infection preferentially stimulates proliferation of susceptible strain (DBA/2) erythroid precursors. As for the platelet shifts induced by FV-P (and not FV-A), we believe the changes in platelet mosaicism also could be caused by viral-induced proliferation of DBA/2 platelet precursors, or more primitive progenitors, over the C57BL/6 ones. Thus, these results implicate the existence of nonerythroid target cells for FV-P-induced proliferation, as well as the existence of genetic differences between strains C57BL/6 and DBA/2 that modulate the responsiveness of such cells to infection.

Cellular site and mode of Fv-2 gene action. II. Conditional protection of Fv-2ss cells by admixture with Fv-2rr cells.

Fv-2ss marrow cells are protected in vivo from Friend virus-induced erythroleukemia by admixture with a preponderance of Fv-2rr marrow cells. This was demonstrated both in allophenic (mosaic) mice and in bone marrow chimeras constructed from C57BL/6 strains differing at Fv-2 and an enzyme marker (glucose phosphate isomerase). The bone marrow chimeras were constructed by injection of marrow cells from Fv-2ss mice into unirradiated Fv-2rr mice. Bone marrow chimeras derived from this procedure produced 1%-2% donor (Fv-2ss) erythrocytes; this level of chimerism was maintained indefinitely. All the bone marrow chimeras as well as allophenic mice with less than 20% Fv-2ss red cells failed to develop any of the symptoms of Friend disease after infection with the polycythemic strain of Friend virus. The Fv-2rr-mediated protection of Fv-2ss marrow cells could be reversed by pretreatment of the two types of chimeras with either monoclonal or polyclonal antithymocyte antisera. Chimeras treated with either reagent and infected with Friend virus developed symptoms of Friend disease and experienced a shift in their erythrocyte mosaic composition favoring cells of the susceptible genotype. These results are consistent with the notion that a functioning immune system plays a role in the Fv-2rr-mediated protection of Fv-2ss Friend virus target cells. Furthermore, these studies establish conditions whereby a small population of sensitive strain cells in an overwhelming background of resistant strain cells can be selectively expanded. Such conditions could be useful in efforts to clone the Fv-2 gene.

Friend viral pathogenesis in C57BL/6 reversible DBA/2 allophenic mice.

Infection of mice with Friend erythroleukemia virus initially causes massive proliferation of erythroid precursors accompanied by splenomegaly and reticulocytosis. Strains of mice differ among themselves in susceptibility to Friend virus and one of the major genes affecting the early response to viral infection is Fv-2. Allophenic mice compounded from a resistant strain C57BL/6 (Fv-2rr) and a susceptible one DBA/2 (Fv-2ss) were infected with the polycythemic strain of Friend virus to determine whether susceptibility/resistance was limited to cells of the respective genotypes or if there was an influence across the genotypic barriers. The manifestations of viral pathogenesis monitored were splenomegaly, reticulocytosis and leukocytosis. In addition, the proportion of red cells of the two genotypes in each animal was monitored before and after viral infection by analyses for strain specific electrophoretic variants of hemoglobin and glucose phosphate isomerase. The group of allophenic mice with 25% or more susceptible-strain red blood cells all developed symptoms of virus-induced disease and also revealed dramatic increases in the number of red cells of the susceptible-strain genotype. Thus, no evidence for protection of susceptible-strain cells by ones of the resistant strain could be observed and the disease developed primarily in susceptible strain cells. On the other hand infected animals with 15% or less DBA/2 red cells were severely retarded in the development of Friend disease. Under these circumstances susceptible strain target cells might fail to undergo viral induced replication as a result of direct protection by resistant strain cells. Alternatively, other more complex mechanisms might be involved such as protective anti-viral immune reactions.

Human tissue kallikrein induces hypotension in transgenic mice.

We investigated the role of the kallikrein-kinin system in blood pressure control by developing transgenic mice overexpressing human tissue kallikrein. Two lines of transgenic mice carrying the human tissue kallikrein gene under the control of the mouse metallothionein metal-responsive promoter were established. Human tissue kallikrein was identified in pancreas, salivary gland, kidney, liver, and spleen of the transgenic mice by a specific radioimmunoassay for human tissue kallikrein. The immunoreactive human tissue kallikrein reached high levels in the circulation. The linear displacement curves for the transgenic product were parallel with the human tissue kallikrein standard curve, indicating their immunologic identity. The expression of human tissue kallikrein transcript in the transgenic mice was further confirmed by Northern blot analysis and by reverse transcription-polymerase chain reaction followed by Southern blot. Both lines of transgenic mice had significantly lowered blood pressure (86.4 +/- 13.5 mm Hg [mean +/- SD], n = 8 and 78.9 +/- 12.4 mm Hg, n = 8) compared with control mice (100.9 +/- 5.0 mm Hg, n = 8). Induction with zinc did not lower the blood pressure further despite elevated expression of the transgene. Administration of aprotinin, a potent tissue kallikrein inhibitor, restored the blood pressure of the transgenic mice but had no significant effect on control littermates. Our findings raise the possibility of tissue kallikrein being a powerful modulator of blood pressure and provide a new animal model for the study of blood pressure regulation.

Ten kilobases of 5'-flanking region confers proper regulation of the mouse alcohol dehydrogenase-1 (Adh-1) gene in kidney and adrenal of transgenic mice.

The expression profile of the mouse Adh-1 gene, which encodes class I alcohol dehydrogenase enzyme (ADH), is complex and includes tissue specificity and differential hormone responsiveness. Whereas kidney Adh-1 transcription rate is stimulated six- to sevenfold by testosterone treatment, adrenal gland ADH-1 mRNA is reduced to less than 5% of control level within 18 h following hormone administration. Androgen receptor is required for both responses since neither occurs in Tfm mutant mice lacking receptor. Hormonal and tissue-specific aspects of Adh-1 regulation were studied in transgenic mice harboring either of two constructs containing either -2.5 kb or -10 kb of 5'-flanking sequence attached to an Adh-1 minigene. The minigene transcript was expressed in kidney and adrenal tissues, but not liver, in five independent lines harboring a transgene with -2.5 kb of 5'-flanking sequence. Androgen treatment repressed the level of the minigene transcript in adrenal gland, but did not cause induction in kidney. In four lines of transgenic mice carrying the construct with -10 kb of 5'-flanking sequence, the minigene transcript was both repressed in adrenal and induced in kidney by testosterone. These lines have no detectable transgene expression in liver tissue. The -10 kb region in the mouse Adh-1 gene contains necessary controlling regions for proper tissue expression and hormonal regulation in kidney and adrenal; however, this region does not contain all essential elements necessary for expression in liver.

Enhanced lung colonization and tumorigenicity of fused cells isolated from primary MCA tumors.

Cells derived from cell-cell fusion events were clonally isolated from primary methylcholanthrene (MCA)-induced tumors in allophenic mice. Compared with non-fused cells isolated from the same cultures, the fused cells had markedly greater experimental metastasizing (lung colonizing) activity, but only slightly greater tumorigenicity and the same cloning efficiency in soft agar. Cell-cell fusion may thus contribute to the generation of tumor heterogeneity that underlies the process of tumor progression.

Silent polymorphisms within the coding region of human sodium/hydrogen exchanger isoform-1 cDNA in peripheral blood mononuclear cells of leukemia patients: A comparison with healthy controls.

We have examined the sequence of the cDNA encoding the sodium/hydrogen exchanger isoform 1 (NHE1), from 23 bases upstream of the start codon to 28 bases downstream of the stop codon. Template was prepared from (1) peripheral blood mononuclear cells (PBMC) isolated from 10 healthy unrelated Caucasian volunteers; (2) PBMCs isolated from 6 leukemic patients (acute lymphoblastic leukemia [ALL], n = 3; chronic lymphocytic leukemia [CLL], n = 1; chronic myelogenous leukemia [CML], n = 2); and (3) samples of 4 leukemic cell lines (ALL: CEM, MOLT4; AML: KG1a; CML: K562). NHE1 cDNA in normal PBMCs showed silent polymorphism of nucleotides 112 (N1: T, frequency 0.70; C, frequency 0.30; prevalence of heterozygosity 0.42); 2248 (N2: G, frequency 0.90; A, frequency 0. 10; heterozygosity 0.18); and 2493 (N3: G, frequency 0.90; A, frequency 0.10; heterozygosity 0.18). Deduced primary structure of NHE1 protein in all normal volunteers was identical to that previously published for NHE1 from renal and cardiac tissue. Similar to normal PBMCs, NHE1 cDNA from leukemic cells showed polymorphism of nucleotides N1, N2, and N3, but failed to demonstrate leukemia-specific sequence differences. We conclude that the coding region of NHE1 cDNA shows a greater level of polymorphism than is currently recognized, but that sequence mutation of NHE1 is not a key event in the pathogenesis of leukemia.

Audiogenic seizure susceptibility of C57BL/6 in equilibrium DBA/2 allophenic mice.

Allophenic mice composed of cells from a strain (DBA/2) susceptible to sound-induced seizures and cells from a strain (C57BL/6) resistant to them were produced by embryo aggregation techniques. Twenty-eight allophenic mice were tested for audiogenic seizure susceptibility. The results were compared with the genotypic composition of the coat melanocytes. For those animals with a predominance of one genotype or the other in the coat, their seizure phenotype was the same as that of the strain most represented in the coat. In contrast, those animals with major contributions of both genotypes in the coat demonstrated the entire spectrum of susceptibility phenotypes. Such results are likely to be a manifestation of a relatively small target tissue for the genes influencing the development of audiogenic seizure susceptibility.

Cell and tissue-specific expression of a heterologous gene under control of the myelin basic protein gene promoter in transgenic mice.

Myelin basic protein (MBP) is the second most abundant protein in CNS myelin. We have used transgenic mice to investigate the ability of the 5' flanking sequence of the mouse MBP gene to regulate the cell-type-specific- and temporal expression of a heterologous gene under its control. Transgenic mice were produced with a construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene down-stream of the MBP 5' flanking sequence and CAT expression was monitored both enzymatically and histochemically. The results indicate that 1323 bp of 5' flanking sequence is sufficient to direct CAT expression specifically to the tissue and cell-type, in which MBP is normally synthesized. Additionally, this length of sequence also retains the ability to temporally regulate CAT levels in a manner analogous to endogenous MBP levels.

Effect of laboratory error on the identification of persons with hypercholesterolemia in an employee health service.

Cholesterol is a major risk factor for coronary heart disease. Of 117 employees seen consecutively in the Tennessee State Employee Health Service voluntary screening program, 86 (74%) had cholesterol levels above the reference range reported by a commercial clinical laboratory. This was three times greater than the calculated expected number of 29 (25%, 17 at moderate risk, 12 at high risk for coronary disease). Age and sex adjustment using Lipid Research Clinic guidelines reduced the number with elevated cholesterol to 55 (47%, 24 at moderate risk, 31 at high risk). Split sample cholesterol assays run independently by the commercial laboratory and a university laboratory showed excellent correlation (r = 0.99, commercial laboratory = 1.0 (university laboratory) + 10.8), but a systematic difference of 12.4 mg/dL (SD = 6.4 mg/dL, paired-t = 9.63, p less than 0.00001) between the two laboratories. Further adjustment for this difference reduced the number with elevated cholesterol to 41 (36%, 26 at moderate risk, 15 at high risk). This experience illustrates how small systematic laboratory errors in cholesterol determination can greatly exaggerate the number of persons reported to have clinically important cholesterol elevations. Clinical laboratories should report age and sex adjusted cholesterol reference ranges and provide clients periodic quality assurance reports that their measurements of cholesterol levels are accurate.

Kinetic interaction between fluoxetine and imipramine as a function of elevated serum alpha-1-acid glycoprotein levels.

The effect of elevated serum alpha-1-acid glycoprotein (AAG) levels on the pharmacokinetic interaction between imipramine and fluoxetine has been examined by utilizing a novel strain of transgenic mice which express serum AAG levels several times greater than normal. Before fluoxetine treatment, serum imipramine levels were approximately three times greater in transgenic mice than in control mice. Despite higher serum imipramine levels in transgenic mice, brain drug levels were lower than those found in control mice. Fluoxetine pre-treatment (20 mg kg(-1) for 5 days) resulted in an increase in serum imipramine levels in both groups of mice and the extent of the increase was greater in transgenic mice than in control mice (4.5-fold increase compared with 3.1-fold). Similarly, fluoxetine pre-treatment resulted in an increase in brain levels of imipramine in both groups of mice and the extent of the increase was greater in transgenic mice than in control mice (3.0-fold increase compared with 2.0-fold). Similar trends were observed for levels of desipramine in the serum and brain. Serum imipramine and desipramine levels did not correlate with their respective brain levels in the presence of elevated serum AAG levels before and after pre-treatment. These findings indicate that the extent of increases in imipramine and desipramine serum and brain levels are greater during elevated serum AAG states than during normal AAG states when imipramine is co-administered with fluoxetine.

Genetic control of erythrocyte volume regulation: effect of a single gene (rol) on cation metabolism.

In laboratory mice we previously defined a gene, rol (resistance to osmotic lysis), based on its effect on erythrocyte osmotic fragility. Here we report a physiological characterization of rol gene action utilizing congenic strains developed for the purpose; these two strains have a common genetic background and differ only by the two alleles of rol, susceptible (rols) or resistant (rolr). In comparison to rols/s erythrocytes, rolr/r cells have a reduced mean cell volume, a higher mean corpuscular hemoglobin concentration and hemolytic volume, and respond differently to swelling induced by ion influx. Rolr/r erythrocytes also have reduced cell water and K, which are associated with a threefold higher activity of the Na-K-Cl cotransporter (measured as ouabain-resistant, bumetanide-sensitive 86Rb influx) and 30% higher Na pump activity. Apart from differences in ion transport and water content, the content of 2,3-diphosphoglycerate (2,3-DPG) in rolr/r cells is 15% lower than in rols/s ones. Analyses of membrane structural components revealed no rol-associated differences in their phospholipid or fatty acid content, nor were strain differences evident among the membrane and cytoskeletal proteins and their posttranslational modifications (phosphorylation and fatty acylation). Rol is not the structural gene for either the alpha- or the beta-chain of hemoglobin and has no effect on erythrocyte production or destruction. The concerted effect of rol variation on erythrocyte volume, water and cation content, cation cotransport, and 2,3-DPG levels is similar in many ways to the variation observed among individual humans for the same characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Moderately repeated mouse Y chromosomal sequence families present distinct types of organization and evolutionary change.

Male specific repeated sequences compose approximately 10% of the mouse Y chromosome. This was deduced from studies of genomic phage clones that contain male specific DNA, and three subcloned EcoRI fragments pBC10-0.6, pBC15-1.1, and pBA33-1.8. Southern analyses and in situ hybridization of metaphase chromosomes show these three sequences to be present in 100-200 copies on the Y chromosome and in female DNA as single copies. One of these, pBC10-0.6, was directly demonstrated to be on the X chromosome. All three exhibited male specific transcription. These sequences are found in two distinct organizations. Subclone pBC10-0.6 is uniformly found in a long HpaI repeat unit of 9.7 Kbp. Subclones pBC15-1.1 and pBA33-1.8 are distributed throughout several EcoR1 repeat families in which the male specific fragments are interspersed. These male specific fragments also differ among themselves with regard to the types and frequency of evolutionary changes.

Variable evolutionary stability of Y chromosomal repeated sequences in the genus Mus.

The study reported here is an examination of the organization and evolution of three Y chromosomal repeated sequences, designated pBC10-0.6, pBC15-1.1, and pBA33-1.8, in five closely related species of the genus Mus. The species distributions of major restriction fragment length polymorphisms produced with a panel of restriction enzymes is used to develop the phylogenetic relationships between the five species studied. However, the apparent degree of relatedness among these species varied a great deal with each of the three probes and was also highly dependent on the particular restriction enzyme used. The usefulness for phylogenetic studies of closely associated sequences varying in evolutionary stability is discussed.

Multiple forms of male-specific simple repetitive sequences in the genus Mus.

Previous reports indicate that in laboratory strains of mice, males are distinct from females in possession of repetitive DNA, notably devoid of Eco RI and Hae III sites and rich in the simple tetranucleotides GATA/GACA. We report here that such sequences originated in an ancestor common to laboratory mice, Mus hortulanus, M. spretus, and possibly also M. cookii. Interestingly, other male-specific satellite sequences were detected in M. caroli, M. cookii, M. saxicola, and M. minutoides. This novel satellite is also likely to be composed of simple repetitious sequences, but does not contain GATA and GACA. Thus, the Y chromosome appears to contain a disproportionately large amount of simple repetitious DNA. An attractive explanation for these results is that long tandem arrays of simple repeated sequences are generated at high frequency throughout the genome and that they are retained for a longer time on the Y chromosome due to the absence of homologous pairing at meiosis.