Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.     

Mouse Chromosome 7

Chair of Committee for Mouse Chromosome 7     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences.     

Cytogenetic Definition and Morphogenetic Analysis of Delta, a Gene Affecting Neurogenesis in Drosophila Melanogaster

We have conducted a genetic analysis of a small interval of the third chromosome known to include Delta (Dl), a locus that affects the segregation of the ectoderm into neural and epidermal lineages during embryogenesis and the morphogenesis of some ectodermally derived structures, in Drosophila melanogaster. This analysis has led to the definition of seven independent complementation groups, one of which is Delta, within the interval extending from 91F6-13 to 92A2. Among the extant mutations in these seven loci, only mutations in Dl lead to the so-called neurogenic phenotype: hypertrophy of the nervous system and reduction of the epidermis. Combined cytogenetic and genetic analyses allow us to define absolute proximal (91F5-92A1) and distal (92A2) cytogenetic limits for the Dl locus. We have isolated hypomorphic and amorphic alleles of Dl and find that, for any given allele, there is an inverse correlation between neural hypertrophy and epidermal reduction in embryos and a direct correlation between the severity of embryonic phenotypes in mutant homozygotes and hemizygotes and the imaginal phenotype in heterozygous adults.     

Activated human T cells can present alloantigens but cannot present soluble antigens

Human T cells, when activated by antigen or mitogen, express Ia antigens. We have examined the capacity of activated T cells to stimulate autologous and allogeneic T cells and their ability to present soluble antigen. Interleukin 2-dependent T-cell lines (TCL), free of accessory cells, were used for antigen-presenting cells. These activated T cells were potent stimulators in an autologous mixed lymphocyte reaction (AMLR), more so than autologous irradiated non-T mononuclear cells. Activated T cells were also able to stimulate proliferation of allogeneic T cells in the absence of any other accessory cells, and this stimulation was blocked by anti-Ia antibodies. Resting unstimulated T cells were unable to stimulate autologous or allogeneic responses. Thus, activated T cells were able to present self antigens and alloantigens. However, activated T cells could not present soluble antigens to autologous T cells or to antigen-specific TCL even if exogenous interleukin 1 was added to cultures. The ability of activated T cells to stimulate an AMLR in vitro may reflect an important immunologic amplification mechanism in vivo. The ability of activated T cells to present alloantigens but not soluble antigens suggests an inability to process antigen, and this may provide further insights into the complexities of antigen presentation.     

Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.     

Decreased Exopolysaccharide Synthesis by Anaerobic and Symbiotic Cells of Bradyrhizobium japonicum

Experiments were conducted to determine whether symbiotic bacteroids of Bradyrhizobium japonicum produce exopolysaccharide within soybean (Glycine max [L.] Merr. cv `Lee 74') nodules. B. japonicum strains RT2, a derivative of USDA 110 with resistance to streptomycin and rifampicin, and RT176-1, a mutant deficient in exopolysaccharide synthesis, were used. Although aerobically cultured RT2 produced 1550 micrograms of exopolysaccharide per 10¹⁰ cells, root nodules formed by RT2 contained only 55.7 micrograms of polysaccharide per 10¹⁰ bacteroids, indicating that little exopolysaccharide synthesis occurred within the nodules. The polysaccharide level of RT2 nodules was about equal to that of nodules containing the exopolysaccharide mutant RT176-1 (61.0 micrograms per 10¹⁰ bacteroids). Gas chromatographic analysis showed that the sugar composition of polysaccharide from nodules of RT2 or RT176-1 was almost the same as that of polysaccharide from unnodulated root tissue, but differed strikingly from that of rhizobial exopolysaccharide from aerobic cultures. Thus, the host plant and not the bacteroids was probably the source of most or all of the polysaccharide in the nodule extracts. Also, bacteroids from nodules failed to bind soybean lectin, confirming the absence of an exopolysaccharide capsule.