The use of two-dimensional electrophoresis to detect mutations induced in mouse spermatogonia by ethylnitrosourea

Two-dimensional electrophoresis should, in theory, be a suitable method for the measurement of induced mutation rates in the germ cells of mice. Not only can the polypeptide products of a large number of genes be resolved on a single gel but the detection of mutations which lead to proteins with altered electrophoretic properties (but not necessarily altered function) is possible. Our attempts to apply two-dimensional electrophoresis to the detection of mutation in vivo have involved three stages: (i) the rapid production of gels of high resolution and reproducibility; (ii) the identification of eight interstrain protein variants and demonstration of their simple genetic basis; and (iii) a pilot experiment using the powerful germ-cell mutagen ethylnitrosourea. It was found that although interstrain protein variants could be detected and shown to be inherited in a codominant manner, induced variants were rarely detected even on high quality gels. Only 2 variants were detected among 67 offspring of male mice treated with 150 mg/kg ethylnitrosourea. This represented a mutation rate of 0.88 X 10(-4) mutations per locus per gamete.     

Localisation of the human N-ras oncogene to chromosome 1cen - p21 by in situ hybridisation.

The N-ras gene is a transforming gene isolated from a variety of human tumour cell lines and is a member of a family of related ras genes. Somatic cell hybrids have previously shown that the N-ras gene is located on chromosome 1. We have confirmed this localisation by in situ hybridisation to metaphase preparations of lymphocytes and localised the gene to the region 1cen - p21. A survey has found 47 reported cases of malignancy involving deletions in the short arm of chromosome 1. Fifteen of the 47 involved a deletion in this region.     

Nonenzymic in vitro isolation of perinatal islets of Langerhans

We have developed a method to circumvent the use of exogenous proteolytic enzymes in the isolation of islets of Langerhans from the perinatal rodent pancreas. Advantage is taken of the propensity of fibroblastlike cells to attach and migrate on polystyrene at low-serum concentrations (5%). In contrast, at this serum level, rat islet epithelial cells tend not to adhere to the substrate. At 3 d of culture, islets are visible at the edges of the explants. With further fibroblast outgrowth the majority of islets are freefloating by 7 d. Simple agitation of the medium and centrifugation yields approximately 50 micrograms of islet tissue per perinatal pancreas. Further purification of the islets can be obtained by subculture. Rat islets can be maintained in this manner for several months in Medium F12 supplemented with 25% horse serum in an atmosphere of 5% CO2 and air at 37 degrees C. Hormone content of the islet tissue remains constant during prolonged subculture and such islets continue to exhibit appropriate insulin and glucagon responses to glucose and theophylline. The morphological integrity of the endocrine cells within the cultured islets was confirmed by immunocytochemistry and ultrastructural study. Nonendocrine cells are not identifiable within the long-term cultured islets.     

Suppression of the transformed phenotype with retention of the viral ‘src’ gene in cell hybrids between rous sarcoma virus-transformed rat cells and untransformed mouse cells

Hybrid cell lines between untransformed mouse 3T3TK-cells and normal rat kidney (NRK) cells transformed by the B77 strain of Rous Sarcoma Virus (RSV) express a non-transformed phenotype, as determined by anchorage-dependent growth and organization of microfilament bundles. Virus rescue experiments and genetic experiments using an RSV mutant temperature-sensitive for maintenance of the transformed phenotype demonstrate that RSV is retained in the non-transformed hybrids. The action of the viral transformation gene ‘src’ therefore appears to be ‘suppressed’ in these hybrids. The suppressed hybrids generate variants in which the expression of the transformed phenotype and the ‘src’ gene is regained. This system should prove to be of value in identifying cellular genes involved in the expression of virally induced transformation.     

Evidence for an indirect effect of radiation on mammalian chromosomes : I. Indirectly-induced chromosome loss from somatic cell hybrids

Mouse cells UV-irradiated with doses of 0–72 J/m² were fused with unirradiated Chinese hamster cells, and the chromosome constitutions of cell hybrids were examined. The number of mouse chromosomes retained by hybrids decreased with UV dose, and, unexpectedly, the number of hamster chromosomes also decreased in a dose-dependent manner. It is suggested that some component contributed by the irradiated mouse parent cell has indirectly induced damage and loss of hamster chromosomes.     

Substrate and metal ion binding to carbamate kinase: NMR and EPR studies

Metal ion and substrate binding to carbamate kinase from Streptococcus faecalis was studied by nuclear magnetic resonance (NMR) and electron paramagnetic resonance using Mn²⁺ as the paramagnetic probe. The enzyme binds Mn²⁺ weakly (K_D = 0.45 ± 0.05 mm) with a stoichiometry of one per two subunits. However, in the presence of nucleotides, tighter binding of Mn²⁺ was observed with K_D = 44 ± 4 μm in the presence of ADP and K_D = 23 ± 4 μm with ATP present. Proton relaxation rate enhancement studies were conducted on water molecules interacting with ternary enzyme-Mn²⁺-nucleotide and binary enzyme-Mn²⁺ complexes. Mn²⁺ bound to carbamate kinase enhances the proton relaxation rate of water giving a binary enhancement value of ?_b = 9.3 ± 0.4. When enzyme-Mn²⁺ was titrated with ADP or ATP, a bell-shaped titration curve was obtained typical of many other enzyme-Mn²⁺-nucleotide ternary complexes. Computer fits to the titration data gave ternary enhancement values of ?_t^ADP = 14 ± 1 and ?_t^ATP = 19 ± 1. The dissociation constants for Mn-ADP and Mn-ATP binding to carbamate kinase were also obtained from these data analyses and are K₁ = 2.5 ± 0.5 μm and K₁ = 50 ± 8 μm, respectively. Therefore, these data demonstrate the formation of a ternary enzyme-metal-nucleotide bridge complex at the nucleotide substrate site of carbamate kinase. Distance measurements were conducted by NMR techniques with ¹³C-enriched carbamate and demonstrate that carbamate is 4–8 Å from enzyme-bound Mn²⁺. Thus carbamate binds near the metal-nucleotide substrate site of carbamate kinase.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

Specific Adhesion of Bacteria to Heterocysts of Anabaena spp. and Its Ecological Significance

Two bacterial isolates, Pseudomonas sp. SL10 and Zoogloea sp. SL20, attach to heterocysts of Anabaena spp. with a high degree of selectivity, and this attachment can be expressed quantitatively in terms of adsorption isotherms. Adhesion of Pseudomonas sp. SL10 was restricted to a monolayer and exhibited a type I (Langmuir) isotherm, whereas adhesion of Zoogloea sp. SL20 involved multilayer attachment and exhibited a type II isotherm. The degree of adhesion by the bacteria to heterocysts of different Anabaena species may reflect the distribution and abundance of binding sites on the surface of different heterocysts. Both Pseudomonas sp. SL10 and Zoogloea sp SL20 promoted higher rates of acetylene reduction by Anabaena spp. under oxygenated culture conditions when compared with a cyanobacterial control. At ambient oxygen levels, however, only Zoogloea sp. SL20 stimulated acetylene reduction by Anabaena spp.     

OBSERVATIONS ON THE ULTRASTRUCTURE OF THE MALI GAMETES OF BIDDULPHIA LEVIS EHR.1

The marine centric diatom Biddulphia levis produced uniflagellate fusiform male gametes completely within the parent cell frustule. These gametes lacked both a central pair of microtubules in the flagellar axoneme and chloroplasts but did contain a cone of microtubules which passed posteriorly from the base of the kinetosome along the nuclear envelope. The gametes were released through a specialized pore in the girdle band leaving behind a cytoplasmic mass which contained chloroplasts and other cytoplasmic components. Tubules which resembled the flimmer hairs on the gamete flagellum occurred in cisternae of the cytoplasmic reticulum in the residual cytoplasm and in the nuclear envelope of the gametes. Gametogenesis in B. levis is compared with similar processes in other centric diatoms.