Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila.

DNA deletion by site-specific chromosome breakage and rejoining occurs extensively during macronuclear development in the ciliate Tetrahymena thermophila. We have sequenced both the micronuclear (germ line) and rearranged macronuclear (somatic) forms of one region from which 1.1 kilobases of micronuclear DNA are reproducibly deleted during macronuclear development. The deletion junctions lie within a pair of 6-base-pair direct repeats. The termini of the deleted sequence are not inverted repeats. The precision of deletion at the nucleotide level was also characterized by hybridization with a synthetic oligonucleotide matching the determined macronuclear (rejoined) junction sequence. This deletion occurs in a remarkably sequence-specific manner. However, a very minor degree of variability in the macronuclear junction sequences was detected and was shown to be inherent in the mechanism of deletion itself. These results suggest that DNA deletion during macronuclear development in T. thermophila may constitute a novel type of DNA recombination and that it can create sequence heterogeneity on the order of a few base pairs at rejoining junctions.     

The limited proteolysis of tumor necrosis factor-α

The limited proteolysis of human recombinant TNF- by trypsin yields two stable products resulting from cleavage after Arg6 and Arg44. In solution these two products remain associated together in a trimer with a Stokes' radius slightly greater than the radius of intact TNF- and, therefore, could not be separated from each other under nondenaturing conditions. This limited digest retains at least 20% of the activity of the original TNF- sample, and has a tertiary structure that is similar to that of the native protein by circular dichroism. On the other hand, incorrectly folded, inactive TNF- undergoes extensive digestion following similar treatment with trypsin. These results indicate that the active form of TNF- has a tight core structure which is maintained afterN-terminal cleavage and removal.     

Lysophosphatidylcholine metabolism and lipoprotein secretion by cultured rat hepatocytes deficient in choline.

A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures.     

Regulation of the distribution of excitation energy in Ochromonas danica,an organism containing a chlorophyll-A/C/carotenoid light harvesting antenna

A chlorophyll a, c-fucoxanthin pigment-protein complex8 functions as the major light harvesting antenna in the Chrysophyte Ochromonas danica. The regulated distribution of excitation energy between the two photosystems was investigated in these organisms and was shown to be strongly wavelength dependent. A light state transition was induced by pre-illumination of cells using light 2 (640 nm) and light 1 (700 nm) of equal absorbed intensity, and detected by reversible changes in the 77 K chlorophyll fluorescence emission spectra. Peaks at 690 nm and 720 nm in the low temperature spectra are most likely associated with PS2 and PS1 respectively. A room temperature fluorescence emission at 680 nm induced by modulated light 2 (500 nm) was strongly quenched in the presence of background light 1 (720 nm). Removal of light 1 led to an increase in fluorescence followed by a slow quenching. The room temperature fluorescence changes were directly correlated with changes in the 77 K emission spectra that indicated a change in the distribution of excitation energy between the two photosystems. It was established that DCMU (1 mol) prevented the state 2. The conversion to state 1 followed a simple photochemical dose dependence and had a half-time of 20 s-1.5 min at 6 W m^-2. In contrast, the conversion to state 2 was independent of light intensity. These data indicate that O. danica undergoes a light state transition in response to the preferential excitation of PS2 or PS1.Abbreviations PS2 photosystem 2 - PS1 photosystem 1 - LHC light harvesting chlorophyll a/b protein - fx fucoxanthin - PQ plastoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea     

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri. We document the expression of a C₃ plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C₄ plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.     

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: I. Fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases

The phase transitions in fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases have been studied by lowangle time-resolved x-ray diffraction under conditions similar to those employed in calorimetry (scan rates 0.05-0.5°C/min and uniform temperature throughout the samples). This approach provides more adequate characterization of the equilibrium transition pathways and allows for close correlations between structural and thermodynamic data. No coexistence of the rippled gel (P_β') and liquid-crystalline (L_α) phases was found in the main transition of DPPC; rather, a loss of correlation in the lamellar structure, observed as broadening of the lamellar reflections, takes place in a narrow temperature range of ~100 mK at the transition midpoint. Formation of a long-living metastable phase, denoted by P_β'(mst), differing from the initial P_β' was observed in cooling direction by both x-ray diffraction and calorimetry. No direct conversion of P_β'(mst) into P_β' occurs for over 24 h but only by way of the phase sequence P_β'(mst) → L_β' → P_β'. According to differential scanning calorimetry (DSC), the enthalpy of the P_β'(mst)-L_α transition is by ~5% lower than that of the P_β'-L_α transition. The effects of ethanol (Rowe, E. S. 1983. Biochemistry. 22:3299-3305; Simon, S. A., and T. J. McIntosh. 1984. Biochim. Biophys. Acta 773:169-172) on the mechanism and reversibility of the DPPC main transition were clearly visualized. At ethanol concentrations inducing formation of interdigitated gel phase, the main transition proceeds through a coexistence of the initial and final phases over a finite temperature range. During the subtransition in DPPC recorded at scan rate 0.3°C/min, a smooth monotonic increase of the lamellar spacing from its subgel (L_c) to its gel (L_β') phase value takes place. The width of the lamellar reflections remains unchanged during this transformation. This provides grounds to propose a “sequential” relaxation mechanism for the subgel-gel transition which is not accompanied by growth of domains of the final phase within the initial one.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

Ischemic flow threshold for striatal dopamine release in rats

To determine the level of cerebral blood flow reduction which causes striatal dopamine release, extracellular dopamine and cerebral blood flow was simultaneously determined using in vivo brain dialysis and a hydrogen clearance method, respectively, in the striatum of spontaneously hypertensive rats, before and during experimental cerebral ischemia. The ischemic flow threshold for neurotransmitter dopamine release was found to be 20% of the resting value or 8–10 ml/100g/min of cerebral blood flow, being similar to those for energy and membrane failures.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Transformation of Tetrahymena thermophila with hypermethylated rRNA genes.

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.