Small mitochondrial DNA molecules of wild abortive cytoplasm in rice are not necessarily associated with CMS

Summary Mitochondrial DNA was isolated from leaf tissue of both the cytoplasmic male sterile line of Indica rice variety V41, which carries wild abortive (WA) cytoplasm, and from the corresponding maintainer line. In addition to the main mitochondrial DNA, four small plasmid-like DNA molecules were detected in both the male sterile and fertile lines. Restriction analysis of total mitochondrial DNA from the male sterile and fertile lines showed DNA fragments unique to each. Our findings suggest that the four small mitochondrial DNA (mtDNA) molecules are conserved when WA cytoplasm is transferred into different nuclear backgrounds. However, there is no simple correlation between the presence/ absence of small mitochondrial DNA molecules and the expression of WA cytoplasmic male sterility (CMS).     

Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance.

A series of replication-competent Moloney murine leukemia virus vectors was constructed in which each vector contained a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat. Two of the resulting viruses, MLV (murine leukemia virus) DHFR*-5 and MLV DHFR*-7, were able to stably transfer methotrexate resistance to infected fibroblast cells upon multiple rounds of virus replication and in the absence of drug selection. Cell lines producing recombinant virus with high titers were established, which indicated that the insert did not grossly interfere with viral replication functions. These vectors should be useful for introducing and expressing foreign genes in vivo in tissues and whole animals in which virus spread is needed for efficient infection.     

Microbial cell responses to a non-ionic surfactant

Summary The effects of a non-ionic surfactant, Pluronic F-68, on growth of microbial cell cultures have been studied. Growth ofSaccharomyces cerevisiae at 30°C or 37°C as measured by viable cell counts was unaffected by culture with pluronic. However, corresponding absorbance measurements forS. cerevisiae incubated with 5–10% pluronic were lower than controls at both temperatures. Absorbance ofE. coli cultures was also significantly reduced by incubation with 5.0–10.0% pluronic at both temperatures although viable counts again revealed no significant inhibition of growth.     

High efficiency gene transfer into murine T cell clones using a retroviral vector

To establish a gene transfer and expression system for murine T cell clones, we have introduced the neomycin phosphotransferase gene encoding resistance to the neomycin analogue, G418, into non-neoplastic inducer T cell clones by using a replication-defective retroviral vector. This method allowed highly efficient gene transfer (20 to 40%) into two inducer T cell clones. The level of viral RNA expression in G418r T cells was 0.1% of poly(A)+ RNA. The infected G418r cells retained physiologic responsiveness to specific antigen as judged by antigen-specific proliferation and production of IL 3.     

Determination of DNA content in the nurse and follicle cells from wild type and mutant Drosophila melanogaster by DNA-Feulgen cytophotometry

DNA replication patterns in the nurse and follicle cells of wild type and a female sterile mutant, fs(1)1304, of Drosophila melanogaster have been studied by DNA-Feulgen cytophotometry, using a cell dispersal technique that allowed the measurement of DNA amounts in individual nuclei from egg chambers of known developmental stages. DNA-Feulgen values associated with various ovarian nuclei from egg chambers at different stages of development were used to assess a base line DNA content for ovarian tissues and to estimate the extent of DNA replication in the nurse cells and follicle cells of growing and mature egg chambers. Our data show that both the nurse and follicle cells undergo multiple cycles of endonuclear DNA replication and that there may be selective amplification as well as underreplication by portions of the genome in these highly polyploid, ovarian cells. Alternative models are proposed to account for the DNA replication patterns observed. Comparisons of DNA-Feulgen levels in wild type ovarian nuclei with those found for the fs(1)1304 mutant and its heterozygote in the balanced stock fs/FM3, show that equivalent DNA levels are present in follicle cell nuclei from all three types of females. Nurse cell nuclei in the homozygous fs stock, however, fail to achieve the same high DNA levels observed in both fs/FM3 and wild type nurse cell nuclei. Although the nuclei of follicle cells in ovaries from fs/fs females appear morphologically like those surrounding egg chambers in wild type ovaries, nurse cell nuclei from mutant females show a more compacted organization of their chromatin than found for nurse cell nuclei from wild type ovaries at similar developmental stages. Our findings suggest that a major effect of the fs(1)1304 mutation may be on the coiling behavior of chromatin and the conformation of DNA-protein moieties in both nurse cell and follicle cell nuclei. These changes in chromatin structure apparently are manifest by perturbations in DNA replication patterns and normal gene function in these biosynthetically active cells.     

Expression of a chimaeric kanamycin resistance gene introduced into the wild soybeanGlycine canescens using a cointegrate Ri plasmid vector

Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl^–1 BAP, 0.05 mgl^–1 IBA and 50 gml^–1 kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.Abbreviations BAP 6-benzylaminopurine - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - IBA indole-butyric acid - PAGE polyacrylamide gel electrophoresis - NPTII neomycin phosphotransferase II - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecylsulphate     

Transfer of a mutant dihydrofolate reductase gene into pre- and postimplantation mouse embryos by a replication-competent retrovirus vector.

In order to explore the potential of retrovirus vectors for efficiently transferring foreign genes into mouse embryos, a replication-competent recombinant Moloney murine leukemia virus (Mo-MLV) vector carrying a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat was used to infect pre- and postimplantation embryos. When preimplantation mouse embryos were infected with the vector, as expected, the provirus integrated into the embryos and the germ line with the same efficiency as that observed with wild-type Mo-MLV, leading to inactivation of the recombinant virus. In contrast, when postimplantation mouse embryos were microinjected with virus-producing cells, between 90 to 100% of the surviving animals proved to be infected with the virus. The recombinant virus spread as efficiently as wild-type Mo-MLV in the infected embryos, resulting in up to three to five proviral copies per genome in heart, thymus, and brain tissues. Substantial expression of mutant DHFR*-coding viral message was found in all somatic tissues analyzed, the amounts correlating with the proviral copy number in the respective organ. These results suggest that replication-competent vectors are useful for efficient transfer and expression of foreign genes into tissues or whole animals when virus spread is needed.