Michael J. Dewey

Genetic control of hematopoietic kinetics revealed by analyses of allophenic mice and stem cell suicide

The pluripotential hematopoietic stem cell is influenced by at least one gene that differs between DBA/2 and C3H/He, and C57BL/6 inbred mouse strains. This gene(s) manifests itself by its effect on susceptibility to killing of spleen colony forming cells (CFU-S) caused by hydroxyurea (HU). In strains DBA/2 and C3H/He 20% of the CFU-S population is normally in S phase whereas practically none from strain C57BL/6 are synthesizing DNA. On the other hand, in C57BL/6 in equilibrium DBA/2 allophenic mice we observed that the proportion of DBA/2 erythrocytes was higher than the proportion of DBA/2 lymphocytes; the fraction of platelets and neutrophils with the DBA/2 genotype fell between the values for erythrocytes and lymphocytes. Control experiments using mice congenic at the Fv-2 locus confirm that in both situations we are examining effects of a gene(s) other than Fv-2. For the effect on the S phase fraction of CFU-S, we refer to the gene(s) as Stk (stem cell kinetics). We suggest that the observed skewing in composition among the various mature blood cell types in C57BL/6 in equilibrium DBA/2 allophenic mice is caused by allelic variants of the Stk gene. Such variation would favor the formation of DBA/2 erythrocytes, platelets, and neutrophils over those of the C57BL/6 genotype.

Influences of inbreeding and genetics on telomere length in mice

Abstract. We measured telomere lengths of blood leukocytes in several inbred and outbred mammalian species, using a telomere-specific fluorescent probe and flow cytometry. Humans, non-human primates, and three outbred populations of Peromyscus mice (Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus) have short telomeres. Two common strains of laboratory mice, C57BL/6J and DBA/2J, have telomeres several times longer than most other mammals surveyed. Moreover, the two inbred laboratory mouse strains display significantly different telomere lengths, suggesting the existence of strain-specific genetic determinants. To further examine the effects of inbreeding, we studied three Peromyscus leucopus inbred lines (GS109, GS16A1, and GS16B), all derived from the outbred P. leucopus stock. Telomeres of all three inbred lines are significantly lengthened relative to outbred P. leucopus, and the three lines display strain-specific significantly different telomere lengths, much like the C57BL/6J and DBA/2J strains of M. musculus. To further characterize the genetic inheritance of telomere length, we carried out several crosses to obtain hybrid F1 mice between parental strains displaying the phenotype of long and short telomeres. In all F1 mice assayed, peripheral blood leukocyte telomere length was intermediate to that of the parents. Additionally, we generated F2 mice from a cross of the (P. leucopus outbred × GS16B)F1. Based on the distribution of telomere length in the F2 population, we determined that more than five loci contribute to telomere length regulation in Peromyscus. We concluded that inbreeding, through unknown mechanisms, results in the elongation of telomeres, and that telomere length for a given species and/or sub-strain is genetically determined by multiple segregating loci.

Comparative genome mapping of the deer mouse (Peromyscus maniculatus) reveals greater similarity to rat (Rattus norvegicus) than to the lab mouse (Mus musculus)

BackgroundDeer mice (Peromyscus maniculatus) and congeneric species are the most common North American mammals. They represent an emerging system for the genetic analyses of the physiological and behavioral bases of habitat adaptation. Phylogenetic evidence suggests a much more ancient divergence of Peromyscus from laboratory mice (Mus) and rats (Rattus) than that separating latter two. Nevertheless, early karyotypic analyses of the three groups suggest Peromyscus to be exhibit greater similarities with Rattus than with Mus.ResultsComparative linkage mapping of an estimated 35% of the deer mouse genome was done with respect to the Rattus and Mus genomes. We particularly focused on regions that span synteny breakpoint regions between the rat and mouse genomes. The linkage analysis revealed the Peromyscus genome to have a higher degree of synteny and gene order conservation with the Rattus genome.ConclusionThese data suggest that: 1. the Rattus and Peromyscus genomes more closely represent ancestral Muroid and rodent genomes than that of Mus. 2. the high level of genome rearrangement observed in Muroid rodents is especially pronounced in Mus. 3. evolution of genome organization can operate independently of more commonly assayed measures of genetic change (e.g. SNP frequency).

Genomic imprinting of a placental lactogen gene in Peromyscus

Abstract. The mammalian genome contains over 30 genes whose expression is dependent upon their parent-of-origin. Of these imprinted genes the majority are involved in regulating the rate of fetal growth. In this report we show that in the deer mouse Peromyscus the placental lactogen-1-variant (pPl1-v) gene is paternally expressed throughout fetal development, whereas the linked and closely related pPl1 gene is expressed in a biallelic manner. Neither the more distantly related pPl2A gene, nor the Mus Pl1 gene displays any preferential expression of the paternal allele, suggesting that the acquisition of imprinting of pPl1-v is a relatively recent event in evolution. Although pPl1 expression is temporally mis-regulated in the dysplastic placentae of hybrids between two Peromyscus species, its over-expression cannot account for the aberrant phenotypes of these placentae. We argue that the species-specific imprinting of pPl1-v, encoding a growth factor that regulates nutrient transfer from mothers to their offspring, is consistent with the parent-offspring conflict model that has been proposed to explain the evolution of genomic imprinting.

Stable genotypic composition of blood cells in allophenic mice derived from congenic C57BL 6 strains

Random shifts in blood cell genotypic composition are commonly observed in allophenic mice. This phenomenon was studied in 16 mosaic mice produced from very closely related strains, and no such changes were observed in the mosaic composition of erythrocytes, platelets, and lymphocytes over a period of 14 weeks. Furthermore, the mosaic distribution of a large group (66) of these mosaic mice was markedly biased in favor of those animals containing major contributions of both strains. This contrasts with what is normally found in collections of allophenic mice, in which the mosaic distribution curves are usually much flatter. While most allophenic mice have been produced from inbred strains with many genetic differences our results were obtained with congenic strains. This suggests that both properties, the unstable mosaic composition of blood cells and the flat mosaic distribution curves, are caused by specific genetic differences between cells of the two strains and are not inherent properties of allophenic mice. We propose that genetic differences cause these phenomena by inhibiting the mixing of cells of the two strains. Such might occur for example if, throughout development, cells of the same H-2 haplotype had greater affinity for each other than for ones of disparate H-2 haplotypes.

Quantification of fluoxetine and norfluoxetine serum levels by reversed-phase high-performance liquid chromatography with ultraviolet detection

A rapid and sensitive high-performance liquid chromatography assay method was developed to determine serum fluoxetine and norfluoxetine levels by single extraction of 0.1 ml of serum with sodium hydroxide. The mobile phase (55% acetonitrile-45% distilled water containing 10 mM aqueous triethylamine) was used to separate fluoxetine and norfluoxetine (25-1000 ng/ml, using clomipramine as the internal standard) by ultraviolet detection at 226 nm. The inter- and intra-day variabilities of fluoxetine and norfluoxetine were 13-18%, and the recoveries of both drugs exceeded 89%. This assay method was applied to a pharmacokinetic disposition study of fluoxetine in mice.

Cellular site and mode of Fv-2 gene action

The Fv-2 genotype of erythroid progenitors directly determines whether they will undergo viral-induced transformation. This conclusion was reached from studies of allophenic mice compounded from congenic C57BL/6 strains differing at Fv-2 and an enzyme marker (GPI). Infection of these Fv-2ss in equilibrium Fv-2rr mosaic animals with the polycythemic strain of Friend virus results in the development of Friend disease. Concomitant with disease symptoms is a shift in the mosaic composition of the erythrocytes in favor of those of the susceptible strain. The observed viral-induced shift in the erythrocyte composition is paralleled by a similar change in the mosaic composition of the CFU-E pool but not the primitive (d8) BFU-E pool. Thus, with regard to this particular Fv-2 phenotype (susceptibility to FV-P-induced cellular hyperplasia), Fv-2 manifests itself specifically in the erythroid lineage, either in mature (d3) BFU-E or CFU-E.

Rapid microsample analysis of imipramine and desipramine by reversed-phase high-performance liquid chromatography with ultraviolet detection

A rapid and highly sensitive HPLC assay method was developed to measure small amounts of imipramine and its major metabolite, desipramine. The assay involved simple extraction procedures using clomipramine as the internal standard. The mobile phase consisted of acetonitrile (60%) and 0.01 M triethylamine in distilled water (40%) with the pH adjusted to 3.0. Separations were achieved on a C18 column and the effluent measured for UV absorption at 260 nm. The chromatographic separation was excellent, with no interference from endogenous serum constituents. This assay was suitable for measuring drug concentrations in the range of 10-1000 ng/ml using a 0.1-ml serum sample. The method was applied to a drug disposition study in transgenic mice with increased plasma alpha 1-acid glycoprotein.

Activation of the sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation

The proliferative response of hematopoietic cells is regulated by many factors, including the presence and type of growth factors, the cellular microenvironment, and the physiochemical conditions prevailing in the tissue milieu. A process fundamental to all cells is the regulation of the intracellular acid‐base conditions. One of the mechanisms by which intracellular pH (pHi) is regulated is through the sodium/hydrogen exchanger, a ubiquitous membrane protein which exploits the intra‐ and extracellular sodium ion gradient to drive hydrogen ions out of the cell. However, activation of the exchanger via mitogenic and nonmitogenic signals leads to an increase in pHi which, in turn, may directly or indirectly result in a proliferative response. It has been shown that interaction of fibronectin with its integrin receptor subunits α4 and α5 can result in activation of the Na+/H+ exchanger. In this report, we demonstrate that when mouse bone marrow cells are physically brought together in a preculture system we designate as high cell density culture (HCDC), in a small volume and at the same cellularity as that in the marrow, hematopoietic stem and progenitor cell populations are stimulated with no additional stimulation in the presence of growth factors. Neutralizing antibodies to the growth factors added to HCDC had little, if any, effect on the degree of stimulation. However, when antibodies to fibronectin or the α4 integrin subunit were added to HCDC, inhibition was observed, indicating that the observed hematopoietic stimulation occurred via the fibronectin‐integrin pathway. Addition of 5 μM 5‐(N,N‐hexamethylene) amiloride (5‐HMA), a specific inhibitor of the Na+/H+ exchanger, also resulted in inhibition of in vitro hematopoiesis. Since the exchanger was implicated, we then measured the pHi of normal and HCDC‐treated bone marrow cells in the absence and presence of 5‐HMA by flow cytometry using the fluorescent pH‐sensitive indicator, carboxy SNARF‐1 AM. It was found that cells subjected to HCDC exhibited a higher pHi than normal fresh cells. In each case, the pHi was lowered in the presence of 5‐HMA. Furthermore, addition of antibodies to fibronectin or the α4 integrin subunit to HCDC also reduced the pHi to a similar level to that found for 5‐HMA. Our results demonstrate, for the first time, that a hematopoietic stem and progenitor cell proliferative response can be initiated by activation of the Na+/H+ exchanger, leading to an increase in pHi, via cell‐cell interaction through the fibronectin‐integrin pathway. This pathway could, therefore, be significant not only in normal hematopoietic regulation, but also under pathophysiological conditions. J. Cell. Physiol. 177:109–122, 1998.

Multiple forms of male-specific simple repetitive sequences in the genusMus

SummaryPrevious 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 alsoM. cookii. Interestingly, other male-specific satellite sequences were detected inM. caroli, M. cookii, M. saxicola, andM. 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.