Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.     

Transport and control of Ca by pigeon erythrocytes. II. Evidence against a simple feedback control of cell Ca and evidence for the involvement of more than one pool

Cells were subjected to a range of ⁴⁵Ca²⁺ influx loads with A23187. We measured cell ⁴⁵Ca²⁺ with time and A23187 dose, and the apparent ⁴⁵Ca²⁺ influxes (≡“J(in,app)”) at Ca²⁺ steady state. We also measured endogeneous exchangeable and total cell Ca²⁺, which were 50 and 17–220 μM respectively. The effects of A23187 and Ca²⁺ on cell ATP, swelling, net Cl⁻ permeability, and cell morphology were measured. These were modest and do not affect our conclusions.J(in,app) 3 × 10⁻⁴ [A23187]^2.9·[Ca²⁺(o)]μmoles/l·min with 92–552 μM [Ca²⁺(o)] (≡ external Ca²⁺ concentration) and 0–7 μM A23187. J(in,app) was increased an order of magnitude by vanadate and is probably much less than the true influx. The least unlikely explanation found for the high [A23187] exponent, 2.9, was that most of the Ca²⁺ crossing the membrane is expelled by the pump before it can move deeper into the cell.Calcium pumping increased rapidly in response to increased influx, but the steady state cell ⁴⁵Ca²⁺ was approximately proportional to J(in,app) rather than approximately constant between 10 and 120 μmoles/l·min with 184 μM [Ca²⁺(o)]. This is not the result expected from a simple feedback mechanism.At high A23187 doses the pump appears fully activated resulting in a linear relation between cell/medium ⁴⁵Ca²⁺ and [A23187]⁻². From the plot we calculated α≡free/total exchangeable Ca²⁺ = 0.38 ± 0.08 (n = 3) and a maximum pump rate, “Pmax” = 78 μmole/l·min. Pmax is underestimated insofar as J(in,app) is less than the true influx.     

Structure and chromosomal arrangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetraploidization

We have determined the structure of one of the leghemoglobin (Lb) genes of Phaseolus vulgaris (kidney bean) and deduced the chromosomal arrangement of leghemoglobin genes by genomic hybridizations with Lb and two other sequences, each specific to the 5' or 3' region of the soybean leghemoglobin loci. By comparing this organization with two other species of legumes, Glycine max (soybean) and G. soja (wild soybean), a phylogeny of leghemoglobin gene loci was traced. The intragenic structure of the kidney bean leghemoglobin gene shows the same intron/exon arrangement as that of soybean leghemoglobin genes and extensive sequence homologies in both coding as well as 5' and 3' non-coding regions. The presence in the kidney bean genome of four leghemoglobin genes suggests that tandem duplications of a single primordial plant globin gene had occurred to generate four leghemoglobin genes in an `Lb-locus' before Glycine and Phaseolus species diverged. Chromosome duplication by tetraploidization in Glycine generated two loci containing four genes each. A large deletion in one of the two four-gene loci in soybean resulted in the generation of the Lbc₂ locus containing two leghemoglobin genes, one functional and another pseudo (LbΨ₂). This pseudogene, unlike that present on the main locus, is represented by only two and a half exons and appears to be truncated. The two other truncated genes (LbT₁ and LbT₂) were probably generated similarly in the genome of Glycine spp. following tetraploidization before the divergence of G. max and G. soja.     

Colonization of congenitally athymic, gnotobiotic mice by Candida albicans

Colony counts, scanning electron microscopy, and light microscopy were used to assess the capacity of Candida albicans to colonize (naturally) and infect the alimentary tract of adult and neonatal (athymic [nu/nu] or heterozygous [+/nu] littermates) germfree BALB/c mice. When exposed to yeast-phase C. albicans, the alimentary tract of adult germfree mice (nu/nu or +/nu) is quickly (within 24 to 48 h) colonized with yeast cells. Neither morbidity nor mortality was evident in any mice that were colonized with a pure culture of C. albicans for 6 months. Yeast cells of C. albicans predominated on mucosal surfaces in the oral cavities and vaginas of adult athymic and heterozygous mice. In both genotypes, C. albicans hyphae were observed in keratinized tissue on the dorsal posterior tongue surface and in the cardial-atrium section of the stomach. Conversely, neonatal athymic or heterozygous mice, born to germfree or C. albicans-colonized mothers, do not become heavily colonized or infected with C. albicans until 11 to 15 days after birth. Although yeast cells adhered to some mucosal surfaces in vivo, neither widespread mucocutaneous candidiasis, i.e., invasion of mucosal surfaces with C. albicans hyphae, nor overwhelming systemic candidiasis was evident in neonatal (nu/nu or +/nu) mice. Thus, even in the absence of functional T-cells and a viable bacterial flora, athymic and heterozygous littermate mice (adult or neonatal BALB/c) that are colonized with a pure culture of C. albicans manifest resistance to extensive mucocutaneous and systemic candidiasis.     

Detection of immunoglobulin heavy chain IgG3 polymorphism in wild mice with xenogeneic monoclonal antibodies

We have generated four xenogeneic rat antimouse IgG₃ monoclonal antibodies recognizing at least three different antigenic determinants (epitopes) on BALB/c IgG₃ molecules. These antibodies were used in solid-phase blocking radioimmunoassays for detection of the epitopes in sera of 40 inbred strains and 134 wild mice. These antibodies detect genetic polymorphism of IgG₃ isotype among wild mice even though there is no polymorphism found among 40 inbred strains tested (except X-chromosome-linked immunodeficient CBA/N strain which lacks IgG₃ molecules). An IgG₃ variant was also isolated from hybridomas derived from Mus spretus.Abbreviations Igh-C immunoglobulin heavy chain constant region - PVC polyvinyl chloride - RIA radioimmune assay - ELISA enzyme linked immunosorbent assay     

Cyclo(Leu-Gly) has opposite effects on D-2 dopamine receptors in different brain areas

Cyclo(Leu-Gly) (cLG), a diketopiperazine analog of Pro-Leu-Gly-NH2 (MIF), affects a number of physiological and behavioral responses to the endogenous neurotransmitter, dopamine (DA). In the present series of experiments, the effect of in vivo administration of cLG (8 mg/kg) was investigated five days following subcutaneous administration. It was found that cLG administration of cLG (8 mg/kg) was investigated five days following subcutaneous administration. It was found that cLG administration caused a supersensitive behavioral response, measured by increased stereotypic sniffing, to the DA agonist, apomorphine (APO). At the same time, an increase was found in the affinity for dopamine (DA), as measured by dopamine inhibition of 3H-spiroperidol binding to D-2 DA receptors in striatum (nigro-striatal DA tract). In contrast, the same peptide treatment caused a subsensitive physiological response to APO-induced hypothermia, concomitant with a decrease in affinity for dopamine, as measured by DA inhibition of 3H-spiroperidol binding to D-2 DA receptors in hypothalamus (incerto-hypothalamic DA tract). These results suggest that a single neuromodulatory agent, the peptide cLG, can elicit diametrically opposite effects on D-2 DA receptors and on the corresponding physiological endpoints in two different brain areas.     

The karyotype and ultrastructural characteristics of spontaneous preimplantation mouse parthenotes

Karyotypic and light and electron microscopical analyses were made of spontaneous preimplantation mouse parthenotes from the LT/Sv inbred strain. It was found that the activated oocyte and developing embryos were diploid. We believe that diploidization is achieved by the oogonium undergoing a premeiotic mitosis without cytokinesis followed by two meiotic divisions, thus producing diploid parthenotes. The developmental events with respect to membrane specialization, such as junctional complexes, were similar to those observed in fertilized embryos. A unique feature of the developing parthenote was the failure of the mitochondria to change during the morula stage. The mitochondria retained a few irregularly oriented cristae rather than many transversely oriented ones observed in morulae developing from fertilized eggs. The significance of this observation is discussed.     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E₂ (PGE₂), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [³H]Thymidine and [³H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h.PGE₂ (10⁻⁷ M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10⁻⁷ M) inhibited DNA and collagen synthesis. The addition of PGE₂ reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collage synthesis in central bone to levels above control untreated cultures.We conclude that PGE₂ has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

Electrophoresis of proteins and nucleic acids on acrylamide-agarose gels lacking covalent crosslinking

The preparation of acrylamide-agarose gels lacking covalent crosslinking with methylenebisacrylamide is described. These hybrid gels melt at 85 degrees C and, consequently, allow quantitative analysis of tritium-labeled protein after electrophoresis. Recovery of tritium-labeled ribonucleic acids extracted from hybrid gels is 20 to 25% greater than from standard acrylamide-methylenebisacrylamide gels. Standard curves of electrophoretic mobilities as a function of molecular weights of dissociated proteins and ribonucleic acids are compared for acrylamide-agarose gels and acrylamide-methylenebisacrylamide gels.