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Abstract. The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm/and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect iramunofluorescence microscopy and by transmission electron microscopy. The mdm/lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm/or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin-or actin-based cytoskeletons. The mdm/ and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.

Abstract. Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature . The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone. The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD. The predicted protein sequence is identical N essential component ofcell division is the distribution of cytoplasmic organelles to daughter cells. This A IL intracellular distribution process ensures a continuance of the cytoplasmic organellar population, since mitochondria and other subcellular structures proliferate by the growth and division of pre-existing organelles (Palade, 1983;Attardi and Schatz, 1988). The mechanisms by which cytoplasmic organelles are distributed during mitosis are poorly understood. Observations in a number of cell types have suggested roles for cytoskeletal components in organellar movement and position in (Ball and Singer, 1982;Hirokawa, 1982;Aufderheide, 1977;Vale, 1987;Wang and Goldman, 1978;Kachar and Reese, 1988), but evidence for the function of these structures in organellar inheritance is lacking . Additionally, structural features of mitochondria and other organelles that are important for organellar distribution have yet to be identified .We have undertaken a genetic approach using Saccharomyces cerewsiae in order to identify molecular mechanisms responsible for the delivery of mitochondria to daughter cells during mitosis . We reported previously (McConnell et al., 1990) the isolation ofseveral mitochondrial distribution and morphology (mdm) mutants which display temperature-sensitive growth and a failure to transfer mitochondria into a growing bud during incubation at the non-permissive temperature . The ntdm mutations are single, nuclear lesions which uncouple mitochondrial movement from bud formation and growth . One ofthese mutants, mdm2, is highly specific for mitochondria : the nuclei and vacuoles are faithfully transmitted to developing buds, secretion, nuclear division, and cytokinesis processed normally, and the structure and function of the actin and tubulin cytoskeletons appear similar to those in wild-type cells.In the present investigation, we report the isolation and characterization of the MDM2 gene. We demonstrate that it encodes the A9 fatty acid desaturase, and further show that a product of the desaturase, oleate, will complement all of the mutant phenotypes of mdm2 cells. These findings indicate a critical role for unsaturated fatty acids in intracellular mitochondrial movement . Materials and Methods Strains and Growth ConditionsYeast strains used in this study (Table 1) were grown in YPD (1% Bactoyeast extract, 2% Bacto-peptone, 2% glucose), YPG (1% Bacto-yeast extract, 2 % Bacto-peptone, 3 % glycerol), or in minimal medium (Sherman e...

The steady-state limiting kinetic parameters Vmax, V/KDA, and V/KO2, together with deuterium isotope effects on these parameters, have been determined for the dopamine beta-monooxygenase (D beta M) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascorbate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of Vmax and V/KDA, and we conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/KO2 have been measured with [2,2-2H2]dopamine as substrate both in the presence and in the absence of added ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)

A full-length cDNA for dopamine beta-monooxygenase (D beta M) from bovine adrenal glands has been cloned and sequenced. The soluble and membrane-derived forms of D beta M have also been sequenced from their N-termini. While the observed sequences for the soluble protein correspond to those previously reported [Joh, T.H., & Hwang, O. (1986) Ann. N.Y. Acad. Sci. 493, 343-350], the heavy subunit of membrane-derived enzyme is found to contain a unique N-terminus. Alignment of this N-terminus with that deduced from cDNA cloning indicates identity at 22 (and possibly 26) out of 27 residues. This comparison leads us to conclude that the membranous form of bovine D beta M retains an uncleaved N-terminal signal peptide as the source of membrane anchoring.

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A real-time speech spectrograph has been developed which is practical for clinical use. It produces and stores a frequency-time-intensity display on a video monltor while the sound is being spoken. The display closely resembles a conventional, broad-band spectrogram in time, frequency and grey scale resolution. Preliminary evaluations have been made to show its usefulness 1. as an aid to the therapist in diagnosis and communication

Heptafluorocycloocta-1,3,5,7-tetraenyl, Hexaf luoroc ycloocta-1 ,3,5 ,I-tetraenedi y I, Heptafluorotricyclo[ 4.2 .O .02i5]octa-3,7-dienyI, and Hexaf luorotricyclo[ 4.2.0. 02*5]octa-3,7-dienediyl Transition-Metal Compounds. Crystal and Molecular Structures of[ Fe( q-C5R5) (q'-heptafluorocycloocta-I ,3,5,7-tetraenyl) (CO),](1 q,5q)-hexafluorocycloocta-1,3,5,7-tetraenedlyl)], and [ Mn( (3~)-heptafluorotricyclo[ 4.2.0 .02i5]octa-3,7-dienyI) (CO),] Cyclooct at et raenes. Synthesis of (R = H, Me), [[Fe(rl-C~Ha)(Co)212(I.Lz-Octafluorocyclooctatetraene (OFCOT; 1) reacts with various transition-metal carbonyl anions to yield the 7'-heptafluorocyclooctatetraenyl complexes [M(q'-CJ?,)] (M = Mn(C0)5 (7), Re(CO)5 (161, Fe(C&5)(CO), (171, Fe(CsMeS)(CO), (24), Ru(CsH5)(CO), (27)), which in most cases are in dynamic equilibrium with small amounts of their heptafluorobicyclo[4.2.0]octatrienyl valence isomers 12,18,25, and 28, respectively. The formation of the bicyclic complex 18 has been shown to occur via initial formation of 17 and subsequent isomerization. The conformational dynamics of the fluorinated rings in these complexes have been examined. In all cases ring inversion with concomitant bond shift isomerization is slow on the NMR time scale. In complex 17 ring inversion has been shown to be slow on the NMR time scale. Complex 17 reacts with EPh3 (E = P, As) to yield the complexes [Fe(CsH6)(q'-C,F,)(CO)(EPhB)] (E = P (291, As (30)) as diastereoisomeric pairs that can be separated and that do not interconvert at room temperature. Kinetic investigations of the conversion of one diastereomer of 30 into the other have yielded the activation parameters E, = 23.0(*0.9) kcal mol-', In A = 26.1 (f1.4), AG* = 24.4 (h0.3) kcal mol-', pH' = 22.4 (f0.9) kcal mol-', and A S = -6.6 (f1.8) eu; these have been shown to represent minimum values for the inversion of the fluorinated ring. Comparison of the AG* value for 30 with that for ring inversion of a hydrocarbon analogue indicates that fluorination of the ring increases PG' for this process by 27.1 kcal mol-'. The fluorinated ring in the monosubstituted complex 17 reacts with a further 1 equiv of [Fe(CsHJ(CO),]-to yield the disubstituted ring compound 22. The valence isomer of OFCOT anti-perfluorotricyclo-[4.2.0.02,5]octa-3,7-diene (9) reacts with metal carbonyl anions to afford the mono-and disubstituted compounds 13, 19,20, and 21. An unusual transformation of the 7'-heptafluorocyclooctatetraenyl ligand to the pentafluorobenzocyclobutenone ligand has been shown to occur during chromatography. Single-crystals X-ray diffraction studies of complexes 17,24,22, and 13 have been carried out. X-ray data were collected on a Syntex P21 autodiffractometer at -110 "C, and structures were refined by the full-matrix least-squares method. For 17, a = 21.390 (5) A, b = 7.507 (1) A, c = 18.767 ( 5 ) A, 0 = 108.63 (2)O, monoclinic, C2/c, Z = 8, R = 0.053, and R, = 0.050 for 2660 reflections with F, 2 40(F,,). For 24, a = 15.307 (4) A, b = 13.952 (3) A, c = 9.058 (2) A, /3 = 92.01 (2)O, monoclinic, P2...