A vital function for mitochondrial DNA in the petite-negative yeastKluyveromyces lactis

Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeastKluyveromyces lactis, specific mutations in the nuclear genesMGI2 andMGI5 encoding theα- andγ-subunits of the mitochondrial F₁-ATPase, allow mtDNA to be lost. In this study we show that wild-typeK. lactis does not survive in the absence of its mitochondrial genome and that the function ofmgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing amgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned theK. lactis homologue of theSaccharomyces cerevisiae mitochondrial genome maintenance geneMGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption ofMGM101 is not lethal in a haploidmgi strain with a specific mutation in a subunit of the mitochondrial F₁-ATPase. These observations suggest that mtDNA inK. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of F₀.     

A gene encoding a truncated large subunit of Rubisco is transcribed and salt-inducible in rice

Using the rice salt-tolerant mutant 20 as material, a cDNA library was constructed and two salt-inducible clones, SIR5.5 and SIR8.1, were isolated by differential screening. Homology analysis revealed that the two clones together constituted a chimeric rbcL which encoded a truncated large subunit of Rubisco with 337 amino-acids, plus 64 amino-acids of unknown origin. The expressions of both the normal and the chimeric locus appeared to be developmentally regulated and salt-inducible in shoots of the salt-tolerant mutant 20 and its original variety 77–170. In roots, their expressions were salt-inducible in the salt-tolerant mutant 20 whereas no, or only premature, forms were present in the salt-treated original variety 77–170. Higher concentrations of salt reduced the expressions of both normal rbcL and the chimeric locus. ABA showed no effect on their expression.     

Isolation,characterization and application of a species-specific repeated sequence from Haynaldia villosa

A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations.     

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca²⁺ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca²⁺ binding site are thereby changed, permitting vectorial dissociation of bound Ca²⁺ against a concentration gradient. A long range linkage between phosphorylation and Ca²⁺ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.     

A Protease Inhibitor of the Serpin Family Is a Major Protein in Carp Perimeningeal Fluid: I. Protein Purification and Characterization

Abstract: Two isoforms of a protease inhibitor of the serpin family (p62) have been purified from bighead carp perimeningeal fluid. Both isoforms migrate with an apparent molecular mass of 62 kDa on reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gels. Both proteins inhibited the activities of bovine trypsin, bovine chymotrypsin, and porcine pancreatic elastase. They also formed complexes with these proteases that were resistant to sodium dodecyl sulfate treatment. p62 exists in the extracts of all tissues examined, including brain, head kidney, kidney, liver, muscle, ovary, pituitary, and spleen. It is also present in serum, ovarian fluid, and milt as well as perimeningeal fluid. The protease inhibitor is a glycoprotein, and its carbohydrate moiety could be removed by endoglycosidase F. Because p62 resembles mammalian α₁-antitrypsin in many aspects, it is likely a fish equivalent of α₁-antitrypsin.     

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.     

Potential role of natural killer cells in controlling tumorigenesis by human T-cell leukemia viruses.

Human T-cell leukemia virus (HTLV) is the etiologic agent of adult T-cell leukemia (ATL), a malignancy of T lymphocytes that is characterized by a long latency period after virus exposure. Intraperitoneal inoculation of severe combined immunodeficient (SCID) mice with HTLV-transformed cell lines and ATL tumor cells was employed to investigate the tumorigenic potential of HTLV type I (HTLV-I)-infected cells. In contrast to inoculation of ATL (RV-ATL) cells into SCID mice, which resulted in the formation of lymphomas, inoculation of HTLV-I- and HTLV-II-transformed cell lines (SLB-I and JLB-II cells, respectively) did not result in tumor formation. Immunosuppression of SCID mice, either by whole-body irradiation or by treatment with an antiserum, anti-asialo GM1 (alpha-AGM1), which transiently abrogates natural killer cell activity in vivo, was necessary to establish the growth of tumors derived from HTLV-transformed cell lines. PCR and flow cytometric studies reveal that HTLV-I-transformed cells are eliminated from the peritoneal cavities of inoculated mice by 3 days postinoculation; in contrast, RV-ATL cells persist and are detected until the mice succumb to lymphoma development. The differing behaviors of HTLV-infected cell lines and ATL tumor cells in SCID mice suggest that ATL cells have a higher tumorigenic potential in vivo than do HTLV-infected cell lines because of their ability to evade natural killer cell-mediated cytolysis.     

Effects of dehydration on the electron transport of Chlorella. An in vivo fluorescence study

Chlorella was used to study the effects of dehydration on photosynthetic activities. The use of unicellular green algae assured that the extent of dehydration was uniform throughout the whole cell population during the course of desiccation. Changes in the activities of the cells were monitored by measurements of fluorescence induction kinetics. It was found that inhibition of most of the photosynthetic activities started at a similar level of cellular water content. They included CO₂ fixation, photochemical activity of Photosystem II and electron transport through Photosystem I. The blockage of electron flow through Photosystem I was complete and the whole transition occurred within a relative short time of dehydration. On the other hand, the suppression of Photosystem II activity was incomplete and the transition took a longer time of dehydration. Upon rehydration, the inhibition of Photosystem II activity was fully reversible when samples were in the middle of the transition, but was not thereafter. The electron transport through Photosystem I was also reversible during the transition, but was only partially afterward.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - F_m maximum fluorescence yield - F₀ non-variable fluorescence level emitted when all PS II centers are open - F_v variable part of fluorescence - PS photosystem - Q_A primary quinone acceptor of Photosystem II